Covalent targeting of e3 ligases

Abstract

To provide a targeted protein degrader for the treatment of diseases such as cancer.SOLUTION: Disclosed herein is a targeted protein degrader exemplified by the following formula, in which a targeted protein binder and an E3 Ubiquitin ligase binder are covalently linked by a binder linker, wherein the E3 Ubiquitin ligase is human RNF4 or human RNF114.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 743,337, filed October 9, 2018, which is incorporated herein by reference in its entirety for all purposes.

[0002] See "Sequence Listing," table, or computer program listing appendix submitted as an ASCII file The sequence listing set forth in file 052103-517001WO_Sequence_Listing_ST25.txt, machine format 18,670 bytes, IBM-PC, MS Windows operating system, created October 4, 2019, is incorporated herein by reference. [Background technology]

[0003] Targeted protein degradation has emerged as a powerful strategy for the treatment of disease. This approach uses bifunctional degraders consisting of protein-targeting ligands linked to E3 ligase recruiters that bind E3 ligases to specific protein substrates, resulting in their ubiquitination and degradation in a proteasome-dependent manner. However, one challenge with this approach is the relatively limited number of E3 ligase recruiters currently available for targeted protein degradation applications. Provided herein are solutions to these and other problems in the art, among others. Summary of the Invention

[0004] In one aspect, the targeted protein degradation agent comprises 1) a target protein binding agent and 2) an E3 ubiquitin ligase binding agent, wherein the E3 ubiquitin ligase is human RNF4 or human RNF114.

[0005] In one aspect, a pharmaceutical composition is provided that includes a compound described herein, including embodiments, and a pharmaceutically acceptable excipient.

[0006] In one aspect, a method of reducing (e.g., reducing compared to a control) the level of a cellular protein is provided, comprising contacting the cellular protein with a targeted protein degradation agent. In embodiments, the targeted protein degradation agent is a compound described herein.

[0007] In one aspect, a method of treating cancer is provided, comprising contacting a cellular protein associated with the cancer with a targeted protein degradation agent (e.g., a compound described herein).

[0008] In one aspect, a method of treating cancer is provided, comprising administering to a subject in need thereof an effective amount of a targeted protein degradation agent as described herein, including embodiments.

[0009] In one aspect, a method is provided for reducing the level of a cellular protein, comprising contacting the cellular protein with a targeted protein degrading agent, thereby forming a targeted protein degrading agent-cellular protein complex, wherein the targeted protein degrading agent comprises (i) a monovalent E3 ubiquitin ligase binding agent, (ii) a monovalent target protein binding agent, and (iii) a binder linker directly attached to the monovalent E3 ubiquitin ligase binding agent and the target protein binding agent.

[0010] In one aspect, a method for identifying cellular proteins contacted by a target protein-binding agent includes: (A) contacting a first sample of cellular proteome or cells with a target protein-binding agent, thereby forming a cellular protein-target protein-binding agent complex; and (B) quantifying both the resulting first sample of cellular proteome or cells from step A and a second sample of cellular proteome or cells not contacted with the target protein-binding agent by a method according to the formula: [ka] (C) contacting the resulting first sample of step B with a compound having a detectable agent; (D) measuring the levels of the first detectable agent and the second detectable agent bound to the selected protein; and (E) identifying the cellular protein in the cellular protein-target protein-binding agent complex by measuring the difference in the levels of the first detectable agent and the second detectable agent bound to the cellular protein, respectively, that can form the cellular protein-target protein-binding agent complex.

[0011] In one aspect, a method of producing an E3 ubiquitin ligase-E3 ubiquitin ligase binder-cellular protein complex comprises: (A) contacting an E3 ubiquitin ligase with an E3 ubiquitin ligase binder, thereby forming an E3 ubiquitin ligase-E3 ubiquitin ligase binder complex; and (B) contacting the E3 ubiquitin ligase-E3 ubiquitin ligase binder complex with a cellular protein, thereby forming an E3 ubiquitin ligase-E3 ubiquitin ligase binder-cellular protein complex.

[0012] In one aspect, a method of producing a cellular protein-E3 ubiquitin ligase binder-E3 ubiquitin ligase complex is provided, the method comprising: (A) contacting a cellular protein with an E3 ubiquitin ligase binder, thereby forming a cellular protein-E3 ubiquitin ligase binder complex; and (B) contacting the cellular protein-E3 ubiquitin ligase binder complex with an E3 ubiquitin ligase, thereby forming a cellular protein-E3 ubiquitin ligase binder-E3 ubiquitin ligase complex.

[0013] In one aspect, a method of inhibiting the formation of a cellular protein-E3 ubiquitin ligase complex is provided, the method comprising contacting an E3 ubiquitin ligase with an E3 ubiquitin ligase-binding agent, thereby inhibiting the formation of a cellular protein-E3 ubiquitin ligase complex. [Brief explanation of the drawings]

[0014] [Figure 1A] Covalent ligand screening for RNF4 using gel-based ABPP. (Figure 1A) Gel-based ABPP labeling of the E3 ligases MDM2, RNF4, and UBE3A. Pure proteins were labeled with IA-rhodamine for 30 minutes at room temperature, followed by SDS / PAGE and visualization by in-gel fluorescence. (Figure 1B) Schematic of gel-based ABPP screening of covalent ligands (50 μM) for IA-rhodamine labeling of pure RNF4, searching for compounds that inhibit probe labeling, resulting in loss of fluorescence. (Figures 1C-1D) Gel-based ABPP screening of cysteine-reactive covalent ligands for IA-rhodamine labeling of RNF4. Covalent ligands were preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling (250 nM) for 1 hour. Proteins were subjected to SDS / PAGE and visualized by in-gel fluorescence. Potential hits from this screen are highlighted. (Figure 1E) Structural and gel-based ABPP confirmation of reproducible RNF4 screening hits, performed as described in (Figures 1C-1D). The gel was also silver stained. [Figure 1B]Covalent ligand screening for RNF4 using gel-based ABPP. (Figure 1A) Gel-based ABPP labeling of the E3 ligases MDM2, RNF4, and UBE3A. Pure proteins were labeled with IA-rhodamine for 30 minutes at room temperature, followed by SDS / PAGE and visualization by in-gel fluorescence. (Figure 1B) Schematic of gel-based ABPP screening of covalent ligands (50 μM) for IA-rhodamine labeling of pure RNF4, searching for compounds that inhibit probe labeling, resulting in loss of fluorescence. (Figures 1C-1D) Gel-based ABPP screening of cysteine-reactive covalent ligands for IA-rhodamine labeling of RNF4. Covalent ligands were preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling (250 nM) for 1 hour. Proteins were subjected to SDS / PAGE and visualized by in-gel fluorescence. Potential hits from this screen are highlighted. (Figure 1E) Structural and gel-based ABPP confirmation of reproducible RNF4 screening hits, performed as described in (Figures 1C-1D). The gel was also silver stained. [Figure 1C]Covalent ligand screening for RNF4 using gel-based ABPP. (Figure 1A) Gel-based ABPP labeling of the E3 ligases MDM2, RNF4, and UBE3A. Pure proteins were labeled with IA-rhodamine for 30 minutes at room temperature, followed by SDS / PAGE and visualization by in-gel fluorescence. (Figure 1B) Schematic of gel-based ABPP screening of covalent ligands (50 μM) for IA-rhodamine labeling of pure RNF4, searching for compounds that inhibit probe labeling, resulting in loss of fluorescence. (Figures 1C-1D) Gel-based ABPP screening of cysteine-reactive covalent ligands for IA-rhodamine labeling of RNF4. Covalent ligands were preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling (250 nM) for 1 hour. Proteins were subjected to SDS / PAGE and visualized by in-gel fluorescence. Potential hits from this screen are highlighted. (Figure 1E) Structural and gel-based ABPP confirmation of reproducible RNF4 screening hits, performed as described in (Figures 1C-1D). The gel was also silver stained. [Figure 1D]Covalent ligand screening for RNF4 using gel-based ABPP. (Figure 1A) Gel-based ABPP labeling of the E3 ligases MDM2, RNF4, and UBE3A. Pure proteins were labeled with IA-rhodamine for 30 minutes at room temperature, followed by SDS / PAGE and visualization by in-gel fluorescence. (Figure 1B) Schematic of gel-based ABPP screening of covalent ligands (50 μM) for IA-rhodamine labeling of pure RNF4, searching for compounds that inhibit probe labeling, resulting in loss of fluorescence. (Figures 1C-1D) Gel-based ABPP screening of cysteine-reactive covalent ligands for IA-rhodamine labeling of RNF4. Covalent ligands were preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling (250 nM) for 1 hour. Proteins were subjected to SDS / PAGE and visualized by in-gel fluorescence. Potential hits from this screen are highlighted. (Figure 1E) Structural and gel-based ABPP confirmation of reproducible RNF4 screening hits, performed as described in (Figures 1C-1D). The gel was also silver stained. [Figure 1E]Covalent ligand screening for RNF4 using gel-based ABPP. (Figure 1A) Gel-based ABPP labeling of the E3 ligases MDM2, RNF4, and UBE3A. Pure proteins were labeled with IA-rhodamine for 30 minutes at room temperature, followed by SDS / PAGE and visualization by in-gel fluorescence. (Figure 1B) Schematic of gel-based ABPP screening of covalent ligands (50 μM) for IA-rhodamine labeling of pure RNF4, searching for compounds that inhibit probe labeling, resulting in loss of fluorescence. (Figures 1C-1D) Gel-based ABPP screening of cysteine-reactive covalent ligands for IA-rhodamine labeling of RNF4. Covalent ligands were preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling (250 nM) for 1 hour. Proteins were subjected to SDS / PAGE and visualized by in-gel fluorescence. Potential hits from this screen are highlighted. (Figure 1E) Structural and gel-based ABPP confirmation of reproducible RNF4 screening hits, performed as described in (Figures 1C-1D). The gel was also silver stained. [Figure 2A]TRH1-23 reacts nonfunctionally with the zinc-coordinated cysteine ​​of RNF4. (Figure 2A) LC-MS / MS analysis of TRH1-23 covalent adducts to RNF4. RNF4 was incubated with TRH1-23 (50 μM) at room temperature for 30 min. RNF4 was digested with trypsin, and the tryptic digest was analyzed by LC-MS / MS to search for TRH1-23-modified adducts. Shown are MS / MS spectra of TRH1-23-modified C132 and C135 RNF4 tryptic peptides. The modified cysteines are highlighted in the peptide sequences. The peptide sequences correspond to residues 118-147 of SEQ ID NO: 1. (Figure 2B) Schematic of TRH1-23 reactivity with C132 or C135 of RNF4. (Figure 2C) TRH1-23 does not inhibit the RNF4 autoubiquitination assay. RNF4 was preincubated with TRH1-23 (100 μM) followed by addition of UBA1, E2 enzyme, and ATP for 40 min at 37 °C. The reaction was quenched and subjected to SDS-PAGE and Western blotting of RNF4. The gel shown in Figure 2C is a representative gel from n = 3. [Figure 2B]TRH1-23 reacts nonfunctionally with the zinc-coordinated cysteine ​​of RNF4. (Figure 2A) LC-MS / MS analysis of TRH1-23 covalent adducts to RNF4. RNF4 was incubated with TRH1-23 (50 μM) at room temperature for 30 min. RNF4 was digested with trypsin, and the tryptic digest was analyzed by LC-MS / MS to search for TRH1-23-modified adducts. Shown are MS / MS spectra of TRH1-23-modified C132 and C135 RNF4 tryptic peptides. The modified cysteines are highlighted in the peptide sequences. The peptide sequences correspond to residues 118-147 of SEQ ID NO: 1. (Figure 2B) Schematic of TRH1-23 reactivity with C132 or C135 of RNF4. (Figure 2C) TRH1-23 does not inhibit the RNF4 autoubiquitination assay. RNF4 was preincubated with TRH1-23 (100 μM) followed by addition of UBA1, E2 enzyme, and ATP for 40 min at 37 °C. The reaction was quenched and subjected to SDS-PAGE and Western blotting of RNF4. The gel shown in Figure 2C is a representative gel from n = 3. [Figure 2C]TRH1-23 reacts nonfunctionally with the zinc-coordinated cysteine ​​of RNF4. (Figure 2A) LC-MS / MS analysis of TRH1-23 covalent adducts to RNF4. RNF4 was incubated with TRH1-23 (50 μM) at room temperature for 30 min. RNF4 was digested with trypsin, and the tryptic digest was analyzed by LC-MS / MS to search for TRH1-23-modified adducts. Shown are MS / MS spectra of TRH1-23-modified C132 and C135 RNF4 tryptic peptides. The modified cysteines are highlighted in the peptide sequences. The peptide sequences correspond to residues 118-147 of SEQ ID NO: 1. (Figure 2B) Schematic of TRH1-23 reactivity with C132 or C135 of RNF4. (Figure 2C) TRH1-23 does not inhibit the RNF4 autoubiquitination assay. RNF4 was preincubated with TRH1-23 (100 μM) followed by addition of UBA1, E2 enzyme, and ATP for 40 min at 37 °C. The reaction was quenched and subjected to SDS-PAGE and Western blotting of RNF4. The gel shown in Figure 2C is a representative gel from n = 3. [Figure 3A] Optimization of RNF4 covalent ligands using gel-based ABPP. (Figures 3A-3B) Analogs of TRH1-23 were tested against IA-rhodamine labeling of RNF4 using gel-based ABPP. (Figure 3C) CCW16 was tested against IA-rhodamine labeling of RNF4 using gel-based ABPP. For (Figures 3A-3C), the covalent ligands were preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling for 1 hour. Proteins were subjected to SDS-PAGE and visualized by in-gel fluorescence. For (Figure 3C), gels were quantified by densitometry to calculate IC50 values. The gel shown in (Figure 3C) is a representative gel from n=3. [Figure 3B-1]Optimization of RNF4 covalent ligands using gel-based ABPP. (Figures 3A-3B) Analogs of TRH1-23 were tested against IA-rhodamine labeling of RNF4 using gel-based ABPP. (Figure 3C) CCW16 was tested against IA-rhodamine labeling of RNF4 using gel-based ABPP. For (Figures 3A-3C), the covalent ligands were preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling for 1 hour. Proteins were subjected to SDS-PAGE and visualized by in-gel fluorescence. For (Figure 3C), gels were quantified by densitometry to calculate IC50 values. The gel shown in (Figure 3C) is a representative gel from n=3. [Figure 3B-2] Optimization of RNF4 covalent ligands using gel-based ABPP. (Figures 3A-3B) Analogs of TRH1-23 were tested against IA-rhodamine labeling of RNF4 using gel-based ABPP. (Figure 3C) CCW16 was tested against IA-rhodamine labeling of RNF4 using gel-based ABPP. For (Figures 3A-3C), the covalent ligands were preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling for 1 hour. Proteins were subjected to SDS-PAGE and visualized by in-gel fluorescence. For (Figure 3C), gels were quantified by densitometry to calculate IC50 values. The gel shown in (Figure 3C) is a representative gel from n=3. [Figure 3C] Optimization of RNF4 covalent ligands using gel-based ABPP. (Figures 3A-3B) Analogs of TRH1-23 were tested against IA-rhodamine labeling of RNF4 using gel-based ABPP. (Figure 3C) CCW16 was tested against IA-rhodamine labeling of RNF4 using gel-based ABPP. For (Figures 3A-3C), the covalent ligands were preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling for 1 hour. Proteins were subjected to SDS-PAGE and visualized by in-gel fluorescence. For (Figure 3C), gels were quantified by densitometry to calculate IC50 values. The gel shown in (Figure 3C) is a representative gel from n=3. [Figure 4A] RNF4 recruiter-based BRD4 degrader. (Figure 4A) Structure of CCW28-3, an RNF4 recruiter-based degrader, bound to the BRD4 inhibitor JQ1. (Figure 4B) Gel-based ABPP analysis of CCW28-3 against pure human RNF4. CCW28-3 was preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling for 1 hour. Proteins were subjected to SDS-PAGE and visualized by in-gel fluorescence. Gels were quantified by densitometry, and IC50 values ​​were calculated. (Figure 4C) CCW28-3 treatment in 231MFP breast cancer cells results in BRD4 degradation. 231MFP breast cancer cells were treated with vehicle DMSO or CCW28-3 for 3 hours. Proteins were subjected to SDS-PAGE and Western blotting of BRD4 and GAPDH loading controls. (Figures 4D, 4E) CCW28-3 treatment in 231MFP breast cancer cells results in proteasome-, E1 inhibitor-, and BRD4 inhibitor-dependent BRD4 degradation. After preincubation with vehicle DMSO or the proteasome inhibitor bortezomib (BTZ) (10 μM), E1 inhibitor TAK-243 (10 μM), or BRD4 inhibitor JQ1 (10 μM) for 30 min, cells were treated with MZ1 (1 μM) or CCW28-3 (1 μM) for 3 h. Proteins were subjected to SDS / PAGE and Western blotting for BRD4 and actin loading controls. (Figure 4F) RNF4 wild-type and knockout HeLa cells were treated with CCW28-3 (10 μM) for 5 h and then subjected to SDS / PAGE and Western blotting for BRD4, RNF4, and GAPDH. Blots (Figures 4B–4F) were quantified by densitometry. Data in (Figures 4B-4F) are from representative gels with n=3. Bar graphs are mean ± sem, n=3-5 / group. Significance is expressed as *p<0.05 compared to vehicle-treated controls, #p<0.05 compared to CCW28-3-treated groups in (B, D-E) and CCW28-3-treated wild-type cells in (Figure 4F). [Figure 4B]RNF4 recruiter-based BRD4 degrader. (Figure 4A) Structure of CCW28-3, an RNF4 recruiter-based degrader, bound to the BRD4 inhibitor JQ1. (Figure 4B) Gel-based ABPP analysis of CCW28-3 against pure human RNF4. CCW28-3 was preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling for 1 hour. Proteins were subjected to SDS-PAGE and visualized by in-gel fluorescence. Gels were quantified by densitometry, and IC50 values ​​were calculated. (Figure 4C) CCW28-3 treatment in 231MFP breast cancer cells results in BRD4 degradation. 231MFP breast cancer cells were treated with vehicle DMSO or CCW28-3 for 3 hours. Proteins were subjected to SDS-PAGE and Western blotting of BRD4 and GAPDH loading controls. (Figures 4D, 4E) CCW28-3 treatment in 231MFP breast cancer cells results in proteasome-, E1 inhibitor-, and BRD4 inhibitor-dependent BRD4 degradation. After preincubation with vehicle DMSO or the proteasome inhibitor bortezomib (BTZ) (10 μM), E1 inhibitor TAK-243 (10 μM), or BRD4 inhibitor JQ1 (10 μM) for 30 min, cells were treated with MZ1 (1 μM) or CCW28-3 (1 μM) for 3 h. Proteins were subjected to SDS / PAGE and Western blotting for BRD4 and actin loading controls. (Figure 4F) RNF4 wild-type and knockout HeLa cells were treated with CCW28-3 (10 μM) for 5 h and then subjected to SDS / PAGE and Western blotting for BRD4, RNF4, and GAPDH. Blots (Figures 4B–4F) were quantified by densitometry. Data in (Figures 4B-4F) are from representative gels with n=3. Bar graphs are mean ± sem, n=3-5 / group. Significance is expressed as *p<0.05 compared to vehicle-treated controls, #p<0.05 compared to CCW28-3-treated groups in (B, D-E) and CCW28-3-treated wild-type cells in (Figure 4F). [Figure 4C]RNF4 recruiter-based BRD4 degrader. (Figure 4A) Structure of CCW28-3, an RNF4 recruiter-based degrader, bound to the BRD4 inhibitor JQ1. (Figure 4B) Gel-based ABPP analysis of CCW28-3 against pure human RNF4. CCW28-3 was preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling for 1 hour. Proteins were subjected to SDS-PAGE and visualized by in-gel fluorescence. Gels were quantified by densitometry, and IC50 values ​​were calculated. (Figure 4C) CCW28-3 treatment in 231MFP breast cancer cells results in BRD4 degradation. 231MFP breast cancer cells were treated with vehicle DMSO or CCW28-3 for 3 hours. Proteins were subjected to SDS-PAGE and Western blotting of BRD4 and GAPDH loading controls. (Figures 4D, 4E) CCW28-3 treatment in 231MFP breast cancer cells results in proteasome-, E1 inhibitor-, and BRD4 inhibitor-dependent BRD4 degradation. After preincubation with vehicle DMSO or the proteasome inhibitor bortezomib (BTZ) (10 μM), E1 inhibitor TAK-243 (10 μM), or BRD4 inhibitor JQ1 (10 μM) for 30 min, cells were treated with MZ1 (1 μM) or CCW28-3 (1 μM) for 3 h. Proteins were subjected to SDS / PAGE and Western blotting for BRD4 and actin loading controls. (Figure 4F) RNF4 wild-type and knockout HeLa cells were treated with CCW28-3 (10 μM) for 5 h and then subjected to SDS / PAGE and Western blotting for BRD4, RNF4, and GAPDH. Blots (Figures 4B–4F) were quantified by densitometry. Data in (Figures 4B-4F) are from representative gels with n=3. Bar graphs are mean ± sem, n=3-5 / group. Significance is expressed as *p<0.05 compared to vehicle-treated controls, #p<0.05 compared to CCW28-3-treated groups in (B, D-E) and CCW28-3-treated wild-type cells in (Figure 4F). [Figure 4D]RNF4 recruiter-based BRD4 degrader. (Figure 4A) Structure of CCW28-3, an RNF4 recruiter-based degrader, bound to the BRD4 inhibitor JQ1. (Figure 4B) Gel-based ABPP analysis of CCW28-3 against pure human RNF4. CCW28-3 was preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling for 1 hour. Proteins were subjected to SDS-PAGE and visualized by in-gel fluorescence. Gels were quantified by densitometry, and IC50 values ​​were calculated. (Figure 4C) CCW28-3 treatment in 231MFP breast cancer cells results in BRD4 degradation. 231MFP breast cancer cells were treated with vehicle DMSO or CCW28-3 for 3 hours. Proteins were subjected to SDS-PAGE and Western blotting of BRD4 and GAPDH loading controls. (Figures 4D, 4E) CCW28-3 treatment in 231MFP breast cancer cells results in proteasome-, E1 inhibitor-, and BRD4 inhibitor-dependent BRD4 degradation. After preincubation with vehicle DMSO or the proteasome inhibitor bortezomib (BTZ) (10 μM), E1 inhibitor TAK-243 (10 μM), or BRD4 inhibitor JQ1 (10 μM) for 30 min, cells were treated with MZ1 (1 μM) or CCW28-3 (1 μM) for 3 h. Proteins were subjected to SDS / PAGE and Western blotting for BRD4 and actin loading controls. (Figure 4F) RNF4 wild-type and knockout HeLa cells were treated with CCW28-3 (10 μM) for 5 h and then subjected to SDS / PAGE and Western blotting for BRD4, RNF4, and GAPDH. Blots (Figures 4B–4F) were quantified by densitometry. Data in (Figures 4B-4F) are from representative gels with n=3. Bar graphs are mean ± sem, n=3-5 / group. Significance is expressed as *p<0.05 compared to vehicle-treated controls, #p<0.05 compared to CCW28-3-treated groups in (B, D-E) and CCW28-3-treated wild-type cells in (Figure 4F). [Figure 4E]RNF4 recruiter-based BRD4 degrader. (Figure 4A) Structure of CCW28-3, an RNF4 recruiter-based degrader, bound to the BRD4 inhibitor JQ1. (Figure 4B) Gel-based ABPP analysis of CCW28-3 against pure human RNF4. CCW28-3 was preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling for 1 hour. Proteins were subjected to SDS-PAGE and visualized by in-gel fluorescence. Gels were quantified by densitometry, and IC50 values ​​were calculated. (Figure 4C) CCW28-3 treatment in 231MFP breast cancer cells results in BRD4 degradation. 231MFP breast cancer cells were treated with vehicle DMSO or CCW28-3 for 3 hours. Proteins were subjected to SDS-PAGE and Western blotting of BRD4 and GAPDH loading controls. (Figures 4D, 4E) CCW28-3 treatment in 231MFP breast cancer cells results in proteasome-, E1 inhibitor-, and BRD4 inhibitor-dependent BRD4 degradation. After preincubation with vehicle DMSO or the proteasome inhibitor bortezomib (BTZ) (10 μM), E1 inhibitor TAK-243 (10 μM), or BRD4 inhibitor JQ1 (10 μM) for 30 min, cells were treated with MZ1 (1 μM) or CCW28-3 (1 μM) for 3 h. Proteins were subjected to SDS / PAGE and Western blotting for BRD4 and actin loading controls. (Figure 4F) RNF4 wild-type and knockout HeLa cells were treated with CCW28-3 (10 μM) for 5 h and then subjected to SDS / PAGE and Western blotting for BRD4, RNF4, and GAPDH. Blots (Figures 4B–4F) were quantified by densitometry. Data in (Figures 4B-4F) are from representative gels with n=3. Bar graphs are mean ± sem, n=3-5 / group. Significance is expressed as *p<0.05 compared to vehicle-treated controls, #p<0.05 compared to CCW28-3-treated groups in (B, D-E) and CCW28-3-treated wild-type cells in (Figure 4F). [Figure 4F]RNF4 recruiter-based BRD4 degrader. (Figure 4A) Structure of CCW28-3, an RNF4 recruiter-based degrader, bound to the BRD4 inhibitor JQ1. (Figure 4B) Gel-based ABPP analysis of CCW28-3 against pure human RNF4. CCW28-3 was preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling for 1 hour. Proteins were subjected to SDS-PAGE and visualized by in-gel fluorescence. Gels were quantified by densitometry, and IC50 values ​​were calculated. (Figure 4C) CCW28-3 treatment in 231MFP breast cancer cells results in BRD4 degradation. 231MFP breast cancer cells were treated with vehicle DMSO or CCW28-3 for 3 hours. Proteins were subjected to SDS-PAGE and Western blotting of BRD4 and GAPDH loading controls. (Figures 4D, 4E) CCW28-3 treatment in 231MFP breast cancer cells results in proteasome-, E1 inhibitor-, and BRD4 inhibitor-dependent BRD4 degradation. After preincubation with vehicle DMSO or the proteasome inhibitor bortezomib (BTZ) (10 μM), E1 inhibitor TAK-243 (10 μM), or BRD4 inhibitor JQ1 (10 μM) for 30 min, cells were treated with MZ1 (1 μM) or CCW28-3 (1 μM) for 3 h. Proteins were subjected to SDS / PAGE and Western blotting for BRD4 and actin loading controls. (Figure 4F) RNF4 wild-type and knockout HeLa cells were treated with CCW28-3 (10 μM) for 5 h and then subjected to SDS / PAGE and Western blotting for BRD4, RNF4, and GAPDH. Blots (Figures 4B–4F) were quantified by densitometry. Data in (Figures 4B-4F) are from representative gels with n=3. Bar graphs are mean ± sem, n=3-5 / group. Significance is expressed as *p<0.05 compared to vehicle-treated controls, #p<0.05 compared to CCW28-3-treated groups in (B, D-E) and CCW28-3-treated wild-type cells in (Figure 4F). [Figure 5]Additional hits against RNF4 and general evaluation of the proteome-wide selectivity of RNF4 hits by gel-based ABPP. Gel-based ABPP analysis of DKM2-76 and TRH1-74 against IA-rhodamine labeling of RNF4. Covalent ligands were preincubated with pure RNF4 protein for 30 minutes, followed by IA-rhodamine labeling (250 nM) for 1 hour. Proteins were subjected to SDS / PAGE and visualized by in-gel fluorescence. Gel-based ABPP screen of RNF4 hits (YP1-44 and TRH1-23) against IA-rhodamine labeling of the 231 MFP breast cancer cell proteome. YP1-44 and TRH1-23 were preincubated with the proteome for 30 minutes, followed by IA-rhodamine labeling (250 nM) for 1 hour. Proteins were subjected to SDS / PAGE and visualized by in-gel fluorescence. [Figure 6] isoTOP-ABPP analysis of CCW28-3 in 231MFP breast cancer cells in situ. 231MFP cells were treated in situ with DMSO vehicle or CCW28-3 (10 μM) for 1 hour, followed by in vitro proteome labeling with IA-alkyne (100 μM) for 1 hour. Isotopically light (DMSO treatment) or heavy (compound treatment) TEV protease-cleavable biotin-azide tags were added by CuAAC for isoTOP-ABPP analysis. Data are from three biological replicates. [Figure 7] CCW28-3 was tested at low concentrations in RNF4 wild-type and knockout Hela cells. RNF4 wild-type and knockout Hela cells were treated with DMSO vehicle or CCW28-3 (5 or 1 μM) for 5 hours and subjected to SDS / PAGE and Western blotting for BRD4 and GAPDH. Blots were quantified by densitometry. Data in ** are from a representative gel of n=3. Bar graphs are mean ± sem, n=3 / group. Significance is expressed as *p<0.05 compared to vehicle-treated controls and #p<0.05 compared to wild-type cells treated with CCW28-3. [Figure 8A]Nimbolide reduces breast cancer cell proliferation or survival. (Figure 8A) Structure of nimbolide. Nimbolide possesses a cyclic enone that is potentially cysteine-reactive. (Figures 8B-8C) Proliferation of 231MFP and HCC38 breast cancer cells in serum-containing medium and serum-free cell survival. Cells were treated with DMSO vehicle or nimbolide, and cell viability was assessed by Hoechst staining 48 hours later. Data shown in (Figures 8B-8C) are mean ± sem, n = 6 per group. Significance is expressed as *p<0.05 compared to vehicle-treated controls. [Figure 8B] Nimbolide reduces breast cancer cell proliferation or survival. (Figure 8A) Structure of nimbolide. Nimbolide possesses a cyclic enone that is potentially cysteine-reactive. (Figures 8B-8C) Proliferation of 231MFP and HCC38 breast cancer cells in serum-containing medium and serum-free cell survival. Cells were treated with DMSO vehicle or nimbolide, and cell viability was assessed by Hoechst staining 48 hours later. Data shown in (Figures 8B-8C) are mean ± sem, n = 6 per group. Significance is expressed as *p<0.05 compared to vehicle-treated controls. [Figure 8C] Nimbolide reduces breast cancer cell proliferation or survival. (Figure 8A) Structure of nimbolide. Nimbolide possesses a cyclic enone that is potentially cysteine-reactive. (Figures 8B-8C) Proliferation of 231MFP and HCC38 breast cancer cells in serum-containing medium and serum-free cell survival. Cells were treated with DMSO vehicle or nimbolide, and cell viability was assessed by Hoechst staining 48 hours later. Data shown in (Figures 8B-8C) are mean ± sem, n = 6 per group. Significance is expressed as *p<0.05 compared to vehicle-treated controls. [Figure 9A]isoTOP-ABPP analysis of nimbolide in the 231MFP breast cancer cell proteome reveals RNF114 as a target. (Figure 9A) Schematic of isoTOP-ABPP of the 231MFP breast cancer cell proteome or cells pretreated with DMSO or nimbolide (10 μM, 30 min in vitro or 1.5 h in situ) followed by in vitro labeling of the proteome with IA-alkyne (100 μM, 1 h), followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC) to add an isotopically light (DMSO treatment) or heavy (nimbolide treatment) TEV protease-cleavable biotin-azide tag. Subsequently, the control and treated proteomes were combined at a 1:1 ratio, probe-labeled proteins were avidin-enriched, digested with trypsin, and probe-modified tryptic peptides were eluted with TEV protease and analyzed by LC-MS / MS to quantify the light-to-heavy probe-modified peptide ratio. (Figure 9B) IsoTOP-ABPP analysis of nimbolide (10 μM) in the 231MFP breast cancer cell proteome analyzed in vitro as described in (Figure 9A). Three targets that showed a light / heavy ratio greater than 5 included C308 of PTOV1, C8 of RNF114, and C123 of PFKP. (Figure 9C) IsoTOP-ABPP analysis of nimbolide (10 μM) in 231MFP breast cancer cells analyzed in situ as described in (Figure 9A). (Figure 9D) RNF114 was stably knocked down in 231MFP cells using short hairpin RNA oligonucleotides. RNF114 knockdown in shRNF114 cells was confirmed by qPCR compared with sh-control cells. Cell proliferation was assessed 48 hours after cell plating by Hoechst staining. (Figure 9E) Sensitivity of 231MFP sh-control and shRNF114 to nimbolide. 231MFP sh-control and shRNF114 cells were treated with DMSO vehicle or nimbolide, and cell proliferation was assessed 48 hours later by Hoechst staining. % proliferation with nimbolide treatment was normalized to the sh-control or shRNF114 control groups, respectively. Data shown in (Figures 9D-9E) are mean ± sem.Data shown in Figures 9B-9E are from n=6 / group. Significance in Figures 9D, 9E is expressed as *p<0.05 compared to the control group. [Figure 9B]isoTOP-ABPP analysis of nimbolide in the 231MFP breast cancer cell proteome reveals RNF114 as a target. (Figure 9A) Schematic of isoTOP-ABPP of the 231MFP breast cancer cell proteome or cells pretreated with DMSO or nimbolide (10 μM, 30 min in vitro or 1.5 h in situ) followed by in vitro labeling of the proteome with IA-alkyne (100 μM, 1 h), followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC) to add an isotopically light (DMSO treatment) or heavy (nimbolide treatment) TEV protease-cleavable biotin-azide tag. Subsequently, the control and treated proteomes were combined at a 1:1 ratio, probe-labeled proteins were avidin-enriched, digested with trypsin, and probe-modified tryptic peptides were eluted with TEV protease and analyzed by LC-MS / MS to quantify the light-to-heavy probe-modified peptide ratio. (Figure 9B) IsoTOP-ABPP analysis of nimbolide (10 μM) in the 231MFP breast cancer cell proteome analyzed in vitro as described in (Figure 9A). Three targets that showed a light / heavy ratio greater than 5 included C308 of PTOV1, C8 of RNF114, and C123 of PFKP. (Figure 9C) IsoTOP-ABPP analysis of nimbolide (10 μM) in 231MFP breast cancer cells analyzed in situ as described in (Figure 9A). (Figure 9D) RNF114 was stably knocked down in 231MFP cells using short hairpin RNA oligonucleotides. RNF114 knockdown in shRNF114 cells was confirmed by qPCR compared with sh-control cells. Cell proliferation was assessed 48 hours after cell plating by Hoechst staining. (Figure 9E) Sensitivity of 231MFP sh-control and shRNF114 to nimbolide. 231MFP sh-control and shRNF114 cells were treated with DMSO vehicle or nimbolide, and cell proliferation was assessed 48 hours later by Hoechst staining. % proliferation with nimbolide treatment was normalized to the sh-control or shRNF114 control groups, respectively. Data shown in (Figures 9D-9E) are mean ± sem.Data shown in Figures 9B-9E are from n=6 / group. Significance in Figures 9D, 9E is expressed as *p<0.05 compared to the control group. [Figure 9C]isoTOP-ABPP analysis of nimbolide in the 231MFP breast cancer cell proteome reveals RNF114 as a target. (Figure 9A) Schematic of isoTOP-ABPP of the 231MFP breast cancer cell proteome or cells pretreated with DMSO or nimbolide (10 μM, 30 min in vitro or 1.5 h in situ) followed by in vitro labeling of the proteome with IA-alkyne (100 μM, 1 h), followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC) to add an isotopically light (DMSO treatment) or heavy (nimbolide treatment) TEV protease-cleavable biotin-azide tag. Subsequently, the control and treated proteomes were combined at a 1:1 ratio, probe-labeled proteins were avidin-enriched, digested with trypsin, and probe-modified tryptic peptides were eluted with TEV protease and analyzed by LC-MS / MS to quantify the light-to-heavy probe-modified peptide ratio. (Figure 9B) IsoTOP-ABPP analysis of nimbolide (10 μM) in the 231MFP breast cancer cell proteome analyzed in vitro as described in (Figure 9A). Three targets that showed a light / heavy ratio greater than 5 included C308 of PTOV1, C8 of RNF114, and C123 of PFKP. (Figure 9C) IsoTOP-ABPP analysis of nimbolide (10 μM) in 231MFP breast cancer cells analyzed in situ as described in (Figure 9A). (Figure 9D) RNF114 was stably knocked down in 231MFP cells using short hairpin RNA oligonucleotides. RNF114 knockdown in shRNF114 cells was confirmed by qPCR compared with sh-control cells. Cell proliferation was assessed 48 hours after cell plating by Hoechst staining. (Figure 9E) Sensitivity of 231MFP sh-control and shRNF114 to nimbolide. 231MFP sh-control and shRNF114 cells were treated with DMSO vehicle or nimbolide, and cell proliferation was assessed 48 hours later by Hoechst staining. % proliferation with nimbolide treatment was normalized to the sh-control or shRNF114 control groups, respectively. Data shown in (Figures 9D-9E) are mean ± sem.Data shown in Figures 9B-9E are from n=6 / group. Significance in Figures 9D, 9E is expressed as *p<0.05 compared to the control group. [Figure 9D]isoTOP-ABPP analysis of nimbolide in the 231MFP breast cancer cell proteome reveals RNF114 as a target. (Figure 9A) Schematic of isoTOP-ABPP of the 231MFP breast cancer cell proteome or cells pretreated with DMSO or nimbolide (10 μM, 30 min in vitro or 1.5 h in situ) followed by in vitro labeling of the proteome with IA-alkyne (100 μM, 1 h), followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC) to add an isotopically light (DMSO treatment) or heavy (nimbolide treatment) TEV protease-cleavable biotin-azide tag. Subsequently, the control and treated proteomes were combined at a 1:1 ratio, probe-labeled proteins were avidin-enriched, digested with trypsin, and probe-modified tryptic peptides were eluted with TEV protease and analyzed by LC-MS / MS to quantify the light-to-heavy probe-modified peptide ratio. (Figure 9B) IsoTOP-ABPP analysis of nimbolide (10 μM) in the 231MFP breast cancer cell proteome analyzed in vitro as described in (Figure 9A). Three targets that showed a light / heavy ratio greater than 5 included C308 of PTOV1, C8 of RNF114, and C123 of PFKP. (Figure 9C) IsoTOP-ABPP analysis of nimbolide (10 μM) in 231MFP breast cancer cells analyzed in situ as described in (Figure 9A). (Figure 9D) RNF114 was stably knocked down in 231MFP cells using short hairpin RNA oligonucleotides. RNF114 knockdown in shRNF114 cells was confirmed by qPCR compared with sh-control cells. Cell proliferation was assessed 48 hours after cell plating by Hoechst staining. (Figure 9E) Sensitivity of 231MFP sh-control and shRNF114 to nimbolide. 231MFP sh-control and shRNF114 cells were treated with DMSO vehicle or nimbolide, and cell proliferation was assessed 48 hours later by Hoechst staining. % proliferation with nimbolide treatment was normalized to the sh-control or shRNF114 control groups, respectively. Data shown in (Figures 9D-9E) are mean ± sem.Data shown in Figures 9B-9E are from n=6 / group. Significance in Figures 9D, 9E is expressed as *p<0.05 compared to the control group. [Figure 9E]isoTOP-ABPP analysis of nimbolide in the 231MFP breast cancer cell proteome reveals RNF114 as a target. (Figure 9A) Schematic of isoTOP-ABPP of the 231MFP breast cancer cell proteome or cells pretreated with DMSO or nimbolide (10 μM, 30 min in vitro or 1.5 h in situ) followed by in vitro labeling of the proteome with IA-alkyne (100 μM, 1 h), followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC) to add an isotopically light (DMSO treatment) or heavy (nimbolide treatment) TEV protease-cleavable biotin-azide tag. Subsequently, the control and treated proteomes were combined at a 1:1 ratio, probe-labeled proteins were avidin-enriched, digested with trypsin, and probe-modified tryptic peptides were eluted with TEV protease and analyzed by LC-MS / MS to quantify the light-to-heavy probe-modified peptide ratio. (Figure 9B) IsoTOP-ABPP analysis of nimbolide (10 μM) in the 231MFP breast cancer cell proteome analyzed in vitro as described in (Figure 9A). Three targets that showed a light / heavy ratio greater than 5 included C308 of PTOV1, C8 of RNF114, and C123 of PFKP. (Figure 9C) IsoTOP-ABPP analysis of nimbolide (10 μM) in 231MFP breast cancer cells analyzed in situ as described in (Figure 9A). (Figure 9D) RNF114 was stably knocked down in 231MFP cells using short hairpin RNA oligonucleotides. RNF114 knockdown in shRNF114 cells was confirmed by qPCR compared with sh-control cells. Cell proliferation was assessed 48 hours after cell plating by Hoechst staining. (Figure 9E) Sensitivity of 231MFP sh-control and shRNF114 to nimbolide. 231MFP sh-control and shRNF114 cells were treated with DMSO vehicle or nimbolide, and cell proliferation was assessed 48 hours later by Hoechst staining. % proliferation with nimbolide treatment was normalized to the sh-control or shRNF114 control groups, respectively. Data shown in (Figures 9D-9E) are mean ± sem.Data shown in Figures 9B-9E are from n=6 / group. Significance in Figures 9D, 9E is expressed as *p<0.05 compared to the control group. [Figure 10A] Nimbolide covalently reacts with C8 of RNF114. (Figure 10A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 10B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 10C) Nimbolide probe labeling of the 231MFP breast cancer cell proteome. The 231MFP proteome was preincubated with DMSO or nimbolide (10 μM, 30 min) and then labeled with a nimbolide-alkyne probe (1 μM, 1 h), followed by CuAAC addition of rhodamine-azide, SDS / PAGE, and in-gel fluorescence analysis. The nimbolide-competing bands correspond to the molecular weights of PTOV1 and RNF114. (Figure 10D) Gel-based ABPP analysis of pure human RNF114 protein labeled with the nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. Nimbolide-alkyne labeling and silver staining of RNF114 are shown for both experiments. (Figure 10E) Pure RNF114 protein was labeled with nimbolide (100 μM, 1 h) and subjected to trypsin digestion and LC-MS / MS analysis. Shown is the nimbolide-modified adduct at C8 of RNF114. The peptide sequence corresponds to residues 7–26 of SEQ ID NO:2. Data shown in (Figures 10C–10D) are from n=3 / group. [Figure 10B]Nimbolide covalently reacts with C8 of RNF114. (Figure 10A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 10B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 10C) Nimbolide probe labeling of the 231MFP breast cancer cell proteome. The 231MFP proteome was preincubated with DMSO or nimbolide (10 μM, 30 min) and then labeled with a nimbolide-alkyne probe (1 μM, 1 h), followed by CuAAC addition of rhodamine-azide, SDS / PAGE, and in-gel fluorescence analysis. The nimbolide-competing bands correspond to the molecular weights of PTOV1 and RNF114. (Figure 10D) Gel-based ABPP analysis of pure human RNF114 protein labeled with the nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. Nimbolide-alkyne labeling and silver staining of RNF114 are shown for both experiments. (Figure 10E) Pure RNF114 protein was labeled with nimbolide (100 μM, 1 h) and subjected to trypsin digestion and LC-MS / MS analysis. Shown is the nimbolide-modified adduct at C8 of RNF114. The peptide sequence corresponds to residues 7–26 of SEQ ID NO:2. Data shown in (Figures 10C–10D) are from n=3 / group. [Figure 10C]Nimbolide covalently reacts with C8 of RNF114. (Figure 10A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 10B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 10C) Nimbolide probe labeling of the 231MFP breast cancer cell proteome. The 231MFP proteome was preincubated with DMSO or nimbolide (10 μM, 30 min) and then labeled with a nimbolide-alkyne probe (1 μM, 1 h), followed by CuAAC addition of rhodamine-azide, SDS / PAGE, and in-gel fluorescence analysis. The nimbolide-competing bands correspond to the molecular weights of PTOV1 and RNF114. (Figure 10D) Gel-based ABPP analysis of pure human RNF114 protein labeled with the nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. Nimbolide-alkyne labeling and silver staining of RNF114 are shown for both experiments. (Figure 10E) Pure RNF114 protein was labeled with nimbolide (100 μM, 1 h) and subjected to trypsin digestion and LC-MS / MS analysis. Shown is the nimbolide-modified adduct at C8 of RNF114. The peptide sequence corresponds to residues 7–26 of SEQ ID NO:2. Data shown in (Figures 10C–10D) are from n=3 / group. [Figure 10D]Nimbolide covalently reacts with C8 of RNF114. (Figure 10A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 10B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 10C) Nimbolide probe labeling of the 231MFP breast cancer cell proteome. The 231MFP proteome was preincubated with DMSO or nimbolide (10 μM, 30 min) and then labeled with a nimbolide-alkyne probe (1 μM, 1 h), followed by CuAAC addition of rhodamine-azide, SDS / PAGE, and in-gel fluorescence analysis. The nimbolide-competing bands correspond to the molecular weights of PTOV1 and RNF114. (Figure 10D) Gel-based ABPP analysis of pure human RNF114 protein labeled with the nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. Nimbolide-alkyne labeling and silver staining of RNF114 are shown for both experiments. (Figure 10E) Pure RNF114 protein was labeled with nimbolide (100 μM, 1 h) and subjected to trypsin digestion and LC-MS / MS analysis. Shown is the nimbolide-modified adduct at C8 of RNF114. The peptide sequence corresponds to residues 7–26 of SEQ ID NO:2. Data shown in (Figures 10C–10D) are from n=3 / group. [Figure 10E]Nimbolide covalently reacts with C8 of RNF114. (Figure 10A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 10B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 10C) Nimbolide probe labeling of the 231MFP breast cancer cell proteome. The 231MFP proteome was preincubated with DMSO or nimbolide (10 μM, 30 min) and then labeled with a nimbolide-alkyne probe (1 μM, 1 h), followed by CuAAC addition of rhodamine-azide, SDS / PAGE, and in-gel fluorescence analysis. The nimbolide-competing bands correspond to the molecular weights of PTOV1 and RNF114. (Figure 10D) Gel-based ABPP analysis of pure human RNF114 protein labeled with the nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. Nimbolide-alkyne labeling and silver staining of RNF114 are shown for both experiments. (Figure 10E) Pure RNF114 protein was labeled with nimbolide (100 μM, 1 h) and subjected to trypsin digestion and LC-MS / MS analysis. Shown is the nimbolide-modified adduct at C8 of RNF114. The peptide sequence corresponds to residues 7–26 of SEQ ID NO:2. Data shown in (Figures 10C–10D) are from n=3 / group. [Figure 11A]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figure 11A) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (left) or p21 (right). DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 11B) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 11C) In vitro incubation of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). D) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. Gels shown in (Figures 11A-11D) are representative images from n=3 / group. Data shown in (Figure 11C) are mean ± sem. Data shown in (Figure 11C) are from n=3 / group. Significance in (Figure 11C) is expressed as *p<0.05 compared to the p21 / RNF114 group in (Figure 11C) or vehicle-treated control group for each time point in (Figure 11D). [Figure 11B]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figure 11A) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (left) or p21 (right). DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 11B) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 11C) In vitro incubation of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). D) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. Gels shown in (Figures 11A-11D) are representative images from n=3 / group. Data shown in (Figure 11C) are mean ± sem. Data shown in (Figure 11C) are from n=3 / group. Significance in (Figure 11C) is expressed as *p<0.05 compared to the p21 / RNF114 group in (Figure 11C) or vehicle-treated control group for each time point in (Figure 11D). [Figure 11C]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figure 11A) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (left) or p21 (right). DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 11B) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 11C) In vitro incubation of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). D) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. Gels shown in (Figures 11A-11D) are representative images from n=3 / group. Data shown in (Figure 11C) are mean ± sem. Data shown in (Figure 11C) are from n=3 / group. Significance in (Figure 11C) is expressed as *p<0.05 compared to the p21 / RNF114 group in (Figure 11C) or vehicle-treated control group for each time point in (Figure 11D). [Figure 11D]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figure 11A) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (left) or p21 (right). DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 11B) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 11C) In vitro incubation of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). D) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. Gels shown in (Figures 11A-11D) are representative images from n=3 / group. Data shown in (Figure 11C) are mean ± sem. Data shown in (Figure 11C) are from n=3 / group. Significance in (Figure 11C) is expressed as *p<0.05 compared to the p21 / RNF114 group in (Figure 11C) or vehicle-treated control group for each time point in (Figure 11D). [Figure 12A]Nimbolide can be used to recruit RNF114 and target BRD4 for proteolysis. (Figure 12A) Pathway for synthesizing the nimbolide-based degraders XH1 and XH2, which consist of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figures 12B, 12C) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) 30 min before and during 12 h of XH1 (Figure 12B) or XH2 (Figure 12C) treatment (100 nM). (Figure 12D) BRD4 levels in 231MFP breast cancer cells treated with MZ1 or XH2 treatment for 12 h. (Figures 12E-12G) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the E3 ligase inhibitors TAK-243 (10 μM) (E), JQ1 (1 μM) (F), or emetine (75 μM) (Figure 12G) for 30 minutes before and during 12 hours of XH2 treatment (100 nM). Long and short BRD4 isoforms, as well as GAPDH loading controls, were visualized by SDS / PAGE and Western blotting and quantified by densitometry (Figures 12B-12G). Gels shown in (B-G) are representative images of n=3 / group. Data shown in (B, C, E-G) are means ± sem. (Figures 12B, 12C, 12E-12G) Significance is expressed as *p<0.05 compared to the vehicle-treated control group and #p<0.05 compared to the XH2-treated group. [Figure 12B]Nimbolide can be used to recruit RNF114 and target BRD4 for proteolysis. (Figure 12A) Pathway for synthesizing the nimbolide-based degraders XH1 and XH2, which consist of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figures 12B, 12C) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) 30 min before and during 12 h of XH1 (Figure 12B) or XH2 (Figure 12C) treatment (100 nM). (Figure 12D) BRD4 levels in 231MFP breast cancer cells treated with MZ1 or XH2 treatment for 12 h. (Figures 12E-12G) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the E3 ligase inhibitors TAK-243 (10 μM) (E), JQ1 (1 μM) (F), or emetine (75 μM) (Figure 12G) for 30 minutes before and during 12 hours of XH2 treatment (100 nM). Long and short BRD4 isoforms, as well as GAPDH loading controls, were visualized by SDS / PAGE and Western blotting and quantified by densitometry (Figures 12B-12G). Gels shown in (B-G) are representative images of n=3 / group. Data shown in (B, C, E-G) are means ± sem. (Figures 12B, 12C, 12E-12G) Significance is expressed as *p<0.05 compared to the vehicle-treated control group and #p<0.05 compared to the XH2-treated group. [Figure 12C]Nimbolide can be used to recruit RNF114 and target BRD4 for proteolysis. (Figure 12A) Pathway for synthesizing the nimbolide-based degraders XH1 and XH2, which consist of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figures 12B, 12C) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) 30 min before and during 12 h of XH1 (Figure 12B) or XH2 (Figure 12C) treatment (100 nM). (Figure 12D) BRD4 levels in 231MFP breast cancer cells treated with MZ1 or XH2 treatment for 12 h. (Figures 12E-12G) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the E3 ligase inhibitors TAK-243 (10 μM) (E), JQ1 (1 μM) (F), or emetine (75 μM) (Figure 12G) for 30 minutes before and during 12 hours of XH2 treatment (100 nM). Long and short BRD4 isoforms, as well as GAPDH loading controls, were visualized by SDS / PAGE and Western blotting and quantified by densitometry (Figures 12B-12G). Gels shown in (B-G) are representative images of n=3 / group. Data shown in (B, C, E-G) are means ± sem. (Figures 12B, 12C, 12E-12G) Significance is expressed as *p<0.05 compared to the vehicle-treated control group and #p<0.05 compared to the XH2-treated group. [Figure 12D]Nimbolide can be used to recruit RNF114 and target BRD4 for proteolysis. (Figure 12A) Pathway for synthesizing the nimbolide-based degraders XH1 and XH2, which consist of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figures 12B, 12C) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) 30 min before and during 12 h of XH1 (Figure 12B) or XH2 (Figure 12C) treatment (100 nM). (Figure 12D) BRD4 levels in 231MFP breast cancer cells treated with MZ1 or XH2 treatment for 12 h. (Figures 12E-12G) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the E3 ligase inhibitors TAK-243 (10 μM) (E), JQ1 (1 μM) (F), or emetine (75 μM) (Figure 12G) for 30 minutes before and during 12 hours of XH2 treatment (100 nM). Long and short BRD4 isoforms, as well as GAPDH loading controls, were visualized by SDS / PAGE and Western blotting and quantified by densitometry (Figures 12B-12G). Gels shown in (B-G) are representative images of n=3 / group. Data shown in (B, C, E-G) are means ± sem. (Figures 12B, 12C, 12E-12G) Significance is expressed as *p<0.05 compared to the vehicle-treated control group and #p<0.05 compared to the XH2-treated group. [Figure 12E]Nimbolide can be used to recruit RNF114 and target BRD4 for proteolysis. (Figure 12A) Pathway for synthesizing the nimbolide-based degraders XH1 and XH2, which consist of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figures 12B, 12C) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) 30 min before and during 12 h of XH1 (Figure 12B) or XH2 (Figure 12C) treatment (100 nM). (Figure 12D) BRD4 levels in 231MFP breast cancer cells treated with MZ1 or XH2 treatment for 12 h. (Figures 12E-12G) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the E3 ligase inhibitors TAK-243 (10 μM) (E), JQ1 (1 μM) (F), or emetine (75 μM) (Figure 12G) for 30 minutes before and during 12 hours of XH2 treatment (100 nM). Long and short BRD4 isoforms, as well as GAPDH loading controls, were visualized by SDS / PAGE and Western blotting and quantified by densitometry (Figures 12B-12G). Gels shown in (B-G) are representative images of n=3 / group. Data shown in (B, C, E-G) are means ± sem. (Figures 12B, 12C, 12E-12G) Significance is expressed as *p<0.05 compared to the vehicle-treated control group and #p<0.05 compared to the XH2-treated group. [Figure 12F]Nimbolide can be used to recruit RNF114 and target BRD4 for proteolysis. (Figure 12A) Pathway for synthesizing the nimbolide-based degraders XH1 and XH2, which consist of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figures 12B, 12C) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) 30 min before and during 12 h of XH1 (Figure 12B) or XH2 (Figure 12C) treatment (100 nM). (Figure 12D) BRD4 levels in 231MFP breast cancer cells treated with MZ1 or XH2 treatment for 12 h. (Figures 12E-12G) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the E3 ligase inhibitors TAK-243 (10 μM) (E), JQ1 (1 μM) (F), or emetine (75 μM) (Figure 12G) for 30 minutes before and during 12 hours of XH2 treatment (100 nM). Long and short BRD4 isoforms, as well as GAPDH loading controls, were visualized by SDS / PAGE and Western blotting and quantified by densitometry (Figures 12B-12G). Gels shown in (B-G) are representative images of n=3 / group. Data shown in (B, C, E-G) are means ± sem. (Figures 12B, 12C, 12E-12G) Significance is expressed as *p<0.05 compared to the vehicle-treated control group and #p<0.05 compared to the XH2-treated group. [Figure 12G]Nimbolide can be used to recruit RNF114 and target BRD4 for proteolysis. (Figure 12A) Pathway for synthesizing the nimbolide-based degraders XH1 and XH2, which consist of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figures 12B, 12C) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) 30 min before and during 12 h of XH1 (Figure 12B) or XH2 (Figure 12C) treatment (100 nM). (Figure 12D) BRD4 levels in 231MFP breast cancer cells treated with MZ1 or XH2 treatment for 12 h. (Figures 12E-12G) BRD4 levels in 231MFP breast cancer cells pretreated with DMSO vehicle or the E3 ligase inhibitors TAK-243 (10 μM) (E), JQ1 (1 μM) (F), or emetine (75 μM) (Figure 12G) for 30 minutes before and during 12 hours of XH2 treatment (100 nM). Long and short BRD4 isoforms, as well as GAPDH loading controls, were visualized by SDS / PAGE and Western blotting and quantified by densitometry (Figures 12B-12G). Gels shown in (B-G) are representative images of n=3 / group. Data shown in (B, C, E-G) are means ± sem. (Figures 12B, 12C, 12E-12G) Significance is expressed as *p<0.05 compared to the vehicle-treated control group and #p<0.05 compared to the XH2-treated group. [Figure 13A]Chemoproteomics-enabled covalent ligand screening to identify synthetically more tractable covalent ligands for RNF114. (Figure 13A) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive moieties highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min followed by 1 h of probe labeling. (Figure 13B) When screening a library of cysteine-reactive covalent ligands for JNS27 labeling of RNF114, EN62 was one of the top hits. Shown is the structure of EN62 with the acrylamide-reactive moiety highlighted in red. Gel-based ABPP analysis of EN62 relative to JNS27 labeling of pure RNF114 is also shown. (Figure 13C) RNF114 autoubiquitination assay with wild-type or C8A mutant RNF114 treated with DMSO or EN62 (50 μM). (Figure 13D) IsoTOP-ABPP analysis of EN62 in the 231MFP breast cancer cell proteome in vitro. DMSO or EN62 (50 μM) was preincubated with the 231MFP proteome for 30 minutes, followed by IA-alkyne labeling (100 μM) for 1 hour, followed by isoTOP-ABPP. (Figure 13E) EN62-induced 231MFP cell viability after 48 hours assessed by Hoechst staining. (Figure 13F) Growth of 231MFP tumor xenografts in CB-17 female SCID mice treated with vehicle (18:1:1 saline:PEG40:ethanol) or EN62 (50 mg / kg ip, once daily, starting 17 days after subcutaneous injection of 231MFP cells). Gels shown in (Figures 13A-13C) are representative images from n=3 / group. Data shown in (Figures 13D, 13F) are mean ± sem, n=3-8 / group. Significance in (Figures 13E-13F) is expressed as *p<0.05 compared to vehicle-treated controls. [Figure 13B] Chemoproteomics-enabled covalent ligand screening to identify synthetically more tractable covalent ligands for RNF114. (Figure 13A) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive moieties highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min followed by 1 h of probe labeling. (Figure 13B) When screening a library of cysteine-reactive covalent ligands for JNS27 labeling of RNF114, EN62 was one of the top hits. Shown is the structure of EN62 with the acrylamide-reactive moiety highlighted in red. Gel-based ABPP analysis of EN62 relative to JNS27 labeling of pure RNF114 is also shown. (Figure 13C) RNF114 autoubiquitination assay with wild-type or C8A mutant RNF114 treated with DMSO or EN62 (50 μM). (Figure 13D) IsoTOP-ABPP analysis of EN62 in the 231MFP breast cancer cell proteome in vitro. DMSO or EN62 (50 μM) was preincubated with the 231MFP proteome for 30 minutes, followed by IA-alkyne labeling (100 μM) for 1 hour, followed by isoTOP-ABPP. (Figure 13E) EN62-induced 231MFP cell viability after 48 hours assessed by Hoechst staining. (Figure 13F) Growth of 231MFP tumor xenografts in CB-17 female SCID mice treated with vehicle (18:1:1 saline:PEG40:ethanol) or EN62 (50 mg / kg ip, once daily, starting 17 days after subcutaneous injection of 231MFP cells). Gels shown in (Figures 13A-13C) are representative images from n=3 / group. Data shown in (Figures 13D, 13F) are mean ± sem, n=3-8 / group. Significance in (Figures 13E-13F) is expressed as *p<0.05 compared to vehicle-treated controls. [Figure 13C]Chemoproteomics-enabled covalent ligand screening to identify synthetically more tractable covalent ligands for RNF114. (Figure 13A) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive moieties highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min followed by 1 h of probe labeling. (Figure 13B) When screening a library of cysteine-reactive covalent ligands for JNS27 labeling of RNF114, EN62 was one of the top hits. Shown is the structure of EN62 with the acrylamide-reactive moiety highlighted in red. Gel-based ABPP analysis of EN62 relative to JNS27 labeling of pure RNF114 is also shown. (Figure 13C) RNF114 autoubiquitination assay with wild-type or C8A mutant RNF114 treated with DMSO or EN62 (50 μM). (Figure 13D) IsoTOP-ABPP analysis of EN62 in the 231MFP breast cancer cell proteome in vitro. DMSO or EN62 (50 μM) was preincubated with the 231MFP proteome for 30 minutes, followed by IA-alkyne labeling (100 μM) for 1 hour, followed by isoTOP-ABPP. (Figure 13E) EN62-induced 231MFP cell viability after 48 hours assessed by Hoechst staining. (Figure 13F) Growth of 231MFP tumor xenografts in CB-17 female SCID mice treated with vehicle (18:1:1 saline:PEG40:ethanol) or EN62 (50 mg / kg ip, once daily, starting 17 days after subcutaneous injection of 231MFP cells). Gels shown in (Figures 13A-13C) are representative images from n=3 / group. Data shown in (Figures 13D, 13F) are mean ± sem, n=3-8 / group. Significance in (Figures 13E-13F) is expressed as *p<0.05 compared to vehicle-treated controls. [Figure 13D] Chemoproteomics-enabled covalent ligand screening to identify synthetically more tractable covalent ligands for RNF114. (Figure 13A) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive moieties highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min followed by 1 h of probe labeling. (Figure 13B) When screening a library of cysteine-reactive covalent ligands for JNS27 labeling of RNF114, EN62 was one of the top hits. Shown is the structure of EN62 with the acrylamide-reactive moiety highlighted in red. Gel-based ABPP analysis of EN62 relative to JNS27 labeling of pure RNF114 is also shown. (Figure 13C) RNF114 autoubiquitination assay with wild-type or C8A mutant RNF114 treated with DMSO or EN62 (50 μM). (Figure 13D) IsoTOP-ABPP analysis of EN62 in the 231MFP breast cancer cell proteome in vitro. DMSO or EN62 (50 μM) was preincubated with the 231MFP proteome for 30 minutes, followed by IA-alkyne labeling (100 μM) for 1 hour, followed by isoTOP-ABPP. (Figure 13E) EN62-induced 231MFP cell viability after 48 hours assessed by Hoechst staining. (Figure 13F) Growth of 231MFP tumor xenografts in CB-17 female SCID mice treated with vehicle (18:1:1 saline:PEG40:ethanol) or EN62 (50 mg / kg ip, once daily, starting 17 days after subcutaneous injection of 231MFP cells). Gels shown in (Figures 13A-13C) are representative images from n=3 / group. Data shown in (Figures 13D, 13F) are mean ± sem, n=3-8 / group. Significance in (Figures 13E-13F) is expressed as *p<0.05 compared to vehicle-treated controls. [Figure 13E]Chemoproteomics-enabled covalent ligand screening to identify synthetically more tractable covalent ligands for RNF114. (Figure 13A) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive moieties highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min followed by 1 h of probe labeling. (Figure 13B) When screening a library of cysteine-reactive covalent ligands for JNS27 labeling of RNF114, EN62 was one of the top hits. Shown is the structure of EN62 with the acrylamide-reactive moiety highlighted in red. Gel-based ABPP analysis of EN62 relative to JNS27 labeling of pure RNF114 is also shown. (Figure 13C) RNF114 autoubiquitination assay with wild-type or C8A mutant RNF114 treated with DMSO or EN62 (50 μM). (Figure 13D) IsoTOP-ABPP analysis of EN62 in the 231MFP breast cancer cell proteome in vitro. DMSO or EN62 (50 μM) was preincubated with the 231MFP proteome for 30 minutes, followed by IA-alkyne labeling (100 μM) for 1 hour, followed by isoTOP-ABPP. (Figure 13E) EN62-induced 231MFP cell viability after 48 hours assessed by Hoechst staining. (Figure 13F) Growth of 231MFP tumor xenografts in CB-17 female SCID mice treated with vehicle (18:1:1 saline:PEG40:ethanol) or EN62 (50 mg / kg ip, once daily, starting 17 days after subcutaneous injection of 231MFP cells). Gels shown in (Figures 13A-13C) are representative images from n=3 / group. Data shown in (Figures 13D, 13F) are mean ± sem, n=3-8 / group. Significance in (Figures 13E-13F) is expressed as *p<0.05 compared to vehicle-treated controls. [Figure 13F] Chemoproteomics-enabled covalent ligand screening to identify synthetically more tractable covalent ligands for RNF114. (Figure 13A) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive moieties highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min followed by 1 h of probe labeling. (Figure 13B) When screening a library of cysteine-reactive covalent ligands for JNS27 labeling of RNF114, EN62 was one of the top hits. Shown is the structure of EN62 with the acrylamide-reactive moiety highlighted in red. Gel-based ABPP analysis of EN62 relative to JNS27 labeling of pure RNF114 is also shown. (Figure 13C) RNF114 autoubiquitination assay with wild-type or C8A mutant RNF114 treated with DMSO or EN62 (50 μM). (Figure 13D) IsoTOP-ABPP analysis of EN62 in the 231MFP breast cancer cell proteome in vitro. DMSO or EN62 (50 μM) was preincubated with the 231MFP proteome for 30 minutes, followed by IA-alkyne labeling (100 μM) for 1 hour, followed by isoTOP-ABPP. (Figure 13E) EN62-induced 231MFP cell viability after 48 hours assessed by Hoechst staining. (Figure 13F) Growth of 231MFP tumor xenografts in CB-17 female SCID mice treated with vehicle (18:1:1 saline:PEG40:ethanol) or EN62 (50 mg / kg ip, once daily, starting 17 days after subcutaneous injection of 231MFP cells). Gels shown in (Figures 13A-13C) are representative images from n=3 / group. Data shown in (Figures 13D, 13F) are mean ± sem, n=3-8 / group. Significance in (Figures 13E-13F) is expressed as *p<0.05 compared to vehicle-treated controls. [Figure 14] Knockdown of PTOV1 and PFKP in 231MFP breast cancer cells. PTOV1 and PFKP were stably knocked down with shRNA oligonucleotides, and expression was confirmed by qPCR. 231MFP sh-control cells used shRNA oligonucleotides targeting GFP. Proliferation of 231MFP sh-control, shPFKP, and shPTOV1 cells was assessed 48 hours later by Hoechst staining. Data shown are mean ± sem, n = 6 per group. Significance is expressed as *p<0.05 compared to sh-control cells. [Figure 15] p53 levels in nimbolide-treated 231MFP breast cancer cells. p53 levels in 231MFP breast cancer cells treated with DMSO vehicle or nimbolide (100 μM) were assessed by Western blotting with GAPDH levels as a loading control. Gels are representative of n=3 / group. Blots were quantified by densitometry and normalized to the loading control. Data shown in the bar graphs are mean ± sem. [Figure 16A-1] Gel-based ABPP screening of cysteine-reactive ligands for RNF114. Cysteine-reactive covalent ligands were screened against JNS27 labeling of pure human RNF114 protein. Covalent ligands (50 μM) were preincubated with RNF114 for 30 min and then labeled with JNS27 for 1 h. Rhodamine-azide was then added to the probe-labeled protein by CuAAC. Proteins were separated by SDS / PAGE and visualized by in-gel fluorescence. Those proteins that showed inhibition of probe labeling were retested in the following dose-response study to identify hits that reproducibly inhibited JNS27 labeling of RNF114. [Figure 16A-2]Gel-based ABPP screening of cysteine-reactive ligands for RNF114. Cysteine-reactive covalent ligands were screened against JNS27 labeling of pure human RNF114 protein. Covalent ligands (50 μM) were preincubated with RNF114 for 30 min and then labeled with JNS27 for 1 h. Rhodamine-azide was then added to the probe-labeled protein by CuAAC. Proteins were separated by SDS / PAGE and visualized by in-gel fluorescence. Those proteins that showed inhibition of probe labeling were retested in the following dose-response study to identify hits that reproducibly inhibited JNS27 labeling of RNF114. [Figure 16B-1] Gel-based ABPP screening of cysteine-reactive ligands for RNF114. Cysteine-reactive covalent ligands were screened against JNS27 labeling of pure human RNF114 protein. Covalent ligands (50 μM) were preincubated with RNF114 for 30 min and then labeled with JNS27 for 1 h. Rhodamine-azide was then added to the probe-labeled protein by CuAAC. Proteins were separated by SDS / PAGE and visualized by in-gel fluorescence. Those proteins that showed inhibition of probe labeling were retested in the following dose-response study to identify hits that reproducibly inhibited JNS27 labeling of RNF114. [Figure 16B-2] Gel-based ABPP screening of cysteine-reactive ligands for RNF114. Cysteine-reactive covalent ligands were screened against JNS27 labeling of pure human RNF114 protein. Covalent ligands (50 μM) were preincubated with RNF114 for 30 min and then labeled with JNS27 for 1 h. Rhodamine-azide was then added to the probe-labeled protein by CuAAC. Proteins were separated by SDS / PAGE and visualized by in-gel fluorescence. Those proteins that showed inhibition of probe labeling were retested in the following dose-response study to identify hits that reproducibly inhibited JNS27 labeling of RNF114. [Figure 16C]Gel-based ABPP screening of cysteine-reactive ligands for RNF114. Cysteine-reactive covalent ligands were screened against JNS27 labeling of pure human RNF114 protein. Covalent ligands (50 μM) were preincubated with RNF114 for 30 min and then labeled with JNS27 for 1 h. Rhodamine-azide was then added to the probe-labeled protein by CuAAC. Proteins were separated by SDS / PAGE and visualized by in-gel fluorescence. Those proteins that showed inhibition of probe labeling were retested in the following dose-response study to identify hits that reproducibly inhibited JNS27 labeling of RNF114. [Figure 17A] Nimbolide reduces breast cancer cell proliferation or survival. (Figures 17A-17B) Growth of 231MFP breast cancer cells in serum-containing medium (Figure 17A) and serum-free cell survival (Figure 17B). Data shown in Figures 17A-17B are mean ± sem, n = 6 biologically independent samples / group. (Figure 17C) Percentage of propidium iodide and annexin V positive (PI+ / annexin V+) cells assessed by flow cytometry after 231MFP cells were treated with DMSO vehicle or nimbolide for 24 or 48 hours. Shown are representative FACS data from n = 3 biologically independent samples / group. Quantification of the percentage of late apoptotic cells, defined as FITC+ / PI+ cells, is shown in Figure 18D. Statistical significance was calculated using an unpaired, two-tailed Student's t-test. Significance is expressed as *P=7.75x10-14 and 1.14x10-13 for 100 and 10 μM, respectively, in Figure 17A, and *P=3.88x10-8 and 1.53x10-7 for 100 and 10 μM, respectively, in Figure 17B, compared to vehicle-treated controls. [Figure 17B]Nimbolide reduces breast cancer cell proliferation or survival. (Figures 17A-17B) Growth of 231MFP breast cancer cells in serum-containing medium (Figure 17A) and serum-free cell survival (Figure 17B). Data shown in Figures 17A-17B are mean ± sem, n = 6 biologically independent samples / group. (Figure 17C) Percentage of propidium iodide and annexin V positive (PI+ / annexin V+) cells assessed by flow cytometry after 231MFP cells were treated with DMSO vehicle or nimbolide for 24 or 48 hours. Shown are representative FACS data from n = 3 biologically independent samples / group. Quantification of the percentage of late apoptotic cells, defined as FITC+ / PI+ cells, is shown in Figure 18D. Statistical significance was calculated using an unpaired, two-tailed Student's t-test. Significance is expressed as *P=7.75x10-14 and 1.14x10-13 for 100 and 10 μM, respectively, in Figure 17A, and *P=3.88x10-8 and 1.53x10-7 for 100 and 10 μM, respectively, in Figure 17B, compared to vehicle-treated controls. [Figure 17C]Nimbolide reduces breast cancer cell proliferation or survival. (Figures 17A-17B) Growth of 231MFP breast cancer cells in serum-containing medium (Figure 17A) and serum-free cell survival (Figure 17B). Data shown in Figures 17A-17B are mean ± sem, n = 6 biologically independent samples / group. (Figure 17C) Percentage of propidium iodide and annexin V positive (PI+ / annexin V+) cells assessed by flow cytometry after 231MFP cells were treated with DMSO vehicle or nimbolide for 24 or 48 hours. Shown are representative FACS data from n = 3 biologically independent samples / group. Quantification of the percentage of late apoptotic cells, defined as FITC+ / PI+ cells, is shown in Figure 18D. Statistical significance was calculated using an unpaired, two-tailed Student's t-test. Significance is expressed as *P=7.75x10-14 and 1.14x10-13 for 100 and 10 μM, respectively, in Figure 17A, and *P=3.88x10-8 and 1.53x10-7 for 100 and 10 μM, respectively, in Figure 17B, compared to vehicle-treated controls. [Figure 18A]Nimbolide reduces breast cancer cell proliferation and survival and induces apoptosis. (Figures 18A-18B) Proliferation of HCC38 breast cancer cells in serum-containing medium (Figure 18A) and serum-free cell survival (Figure 18B). Cells were treated with DMSO vehicle or nimbolide, and cell viability was assessed 48 hours later. (Figure 18C) Shown is the gating strategy for flow cytometry data. Two gating steps were used. The first step involved gates by forward and side scatter. The second gate was based on quadrant separation by cell death staining (PI / annexin). (Figures 18D-18E) Percentage of propidium iodide- and annexin V-positive (PI+ / annexin V+) cells assessed by flow cytometry after treatment of 231MFP (Figure 18D) and HCC38 (Figure 18E) cells with DMSO vehicle or nimbolide for 24 or 48 hours. Representative FACS data from 231MFP cells in Figure 18D and HCC38 cells shown in Figure 17C are shown in the left panel of (Figure 18E). Bar graphs in Figures 18D-18E are the percentage of late apoptotic cells defined as PI+ / Annexin-V+ cells. Data shown in Figures 18A-18B and Figures 18D-18E are mean ± sem, with n = 6 for Figures 18A-18B and n = 3 for Figures 18D-18E from biologically independent samples / groups. Statistical significance was calculated using an unpaired, two-tailed Student's t-test.Significance compared to vehicle-treated controls: *p=1.64x10-13 and 2.05x10-13 for 100 and 10 μM, respectively, in Figure 18A; *p=2.09x10-11 and 7.07x10-12 for 100 and 10 μM, respectively, in Figure 18B; *p=9.05x10-5 and 1.87x10-5 for 10 and 100 μM, respectively, for the 24 hour data in Figure 18D; and *p=1.87x10-5 for 10 and 100 μM, respectively, for the 48 hour data in Figure 18E. For the 24 hour data in Figure 18E, *p=0.00424, 0.0182, and 2.22x10-11 for 1, 10, and 100 μM, respectively; and for the 48 hour data in Figure 18E, *p=6.68x10-6, 4.70x10-7, and 9.00x10-11 for 1, 10, and 100 μM, respectively. [Figure 18B]Nimbolide reduces breast cancer cell proliferation and survival and induces apoptosis. (Figures 18A-18B) Proliferation of HCC38 breast cancer cells in serum-containing medium (Figure 18A) and serum-free cell survival (Figure 18B). Cells were treated with DMSO vehicle or nimbolide, and cell viability was assessed 48 hours later. (Figure 18C) Shown is the gating strategy for flow cytometry data. Two gating steps were used. The first step involved gates by forward and side scatter. The second gate was based on quadrant separation by cell death staining (PI / annexin). (Figures 18D-18E) Percentage of propidium iodide- and annexin V-positive (PI+ / annexin V+) cells assessed by flow cytometry after treatment of 231MFP (Figure 18D) and HCC38 (Figure 18E) cells with DMSO vehicle or nimbolide for 24 or 48 hours. Representative FACS data from 231MFP cells in Figure 18D and HCC38 cells shown in Figure 17C are shown in the left panel of (Figure 18E). Bar graphs in Figures 18D-18E are the percentage of late apoptotic cells defined as PI+ / Annexin-V+ cells. Data shown in Figures 18A-18B and Figures 18D-18E are mean ± sem, with n = 6 for Figures 18A-18B and n = 3 for Figures 18D-18E from biologically independent samples / groups. Statistical significance was calculated using an unpaired, two-tailed Student's t-test.Significance compared to vehicle-treated controls: *p=1.64x10-13 and 2.05x10-13 for 100 and 10 μM, respectively, in Figure 18A; *p=2.09x10-11 and 7.07x10-12 for 100 and 10 μM, respectively, in Figure 18B; *p=9.05x10-5 and 1.87x10-5 for 10 and 100 μM, respectively, for the 24 hour data in Figure 18D; and *p=1.87x10-5 for 10 and 100 μM, respectively, for the 48 hour data in Figure 18E. For the 24 hour data in Figure 18E, *p=0.00424, 0.0182, and 2.22x10-11 for 1, 10, and 100 μM, respectively; and for the 48 hour data in Figure 18E, *p=6.68x10-6, 4.70x10-7, and 9.00x10-11 for 1, 10, and 100 μM, respectively. [Figure 18C]Nimbolide reduces breast cancer cell proliferation and survival and induces apoptosis. (Figures 18A-18B) Proliferation of HCC38 breast cancer cells in serum-containing medium (Figure 18A) and serum-free cell survival (Figure 18B). Cells were treated with DMSO vehicle or nimbolide, and cell viability was assessed 48 hours later. (Figure 18C) Shown is the gating strategy for flow cytometry data. Two gating steps were used. The first step involved gates by forward and side scatter. The second gate was based on quadrant separation by cell death staining (PI / annexin). (Figures 18D-18E) Percentage of propidium iodide- and annexin V-positive (PI+ / annexin V+) cells assessed by flow cytometry after treatment of 231MFP (Figure 18D) and HCC38 (Figure 18E) cells with DMSO vehicle or nimbolide for 24 or 48 hours. Representative FACS data from 231MFP cells in Figure 18D and HCC38 cells shown in Figure 17C are shown in the left panel of (Figure 18E). Bar graphs in Figures 18D-18E are the percentage of late apoptotic cells defined as PI+ / Annexin-V+ cells. Data shown in Figures 18A-18B and Figures 18D-18E are mean ± sem, with n = 6 for Figures 18A-18B and n = 3 for Figures 18D-18E from biologically independent samples / groups. Statistical significance was calculated using an unpaired, two-tailed Student's t-test.Significance compared to vehicle-treated controls: *p=1.64x10-13 and 2.05x10-13 for 100 and 10 μM, respectively, in Figure 18A; *p=2.09x10-11 and 7.07x10-12 for 100 and 10 μM, respectively, in Figure 18B; *p=9.05x10-5 and 1.87x10-5 for 10 and 100 μM, respectively, for the 24 hour data in Figure 18D; and *p=1.87x10-5 for 10 and 100 μM, respectively, for the 48 hour data in Figure 18E. For the 24 hour data in Figure 18E, *p=0.00424, 0.0182, and 2.22x10-11 for 1, 10, and 100 μM, respectively; and for the 48 hour data in Figure 18E, *p=6.68x10-6, 4.70x10-7, and 9.00x10-11 for 1, 10, and 100 μM, respectively. [Figure 18D]Nimbolide reduces breast cancer cell proliferation and survival and induces apoptosis. (Figures 18A-18B) Proliferation of HCC38 breast cancer cells in serum-containing medium (Figure 18A) and serum-free cell survival (Figure 18B). Cells were treated with DMSO vehicle or nimbolide, and cell viability was assessed 48 hours later. (Figure 18C) Shown is the gating strategy for flow cytometry data. Two gating steps were used. The first step involved gates by forward and side scatter. The second gate was based on quadrant separation by cell death staining (PI / annexin). (Figures 18D-18E) Percentage of propidium iodide- and annexin V-positive (PI+ / annexin V+) cells assessed by flow cytometry after treatment of 231MFP (Figure 18D) and HCC38 (Figure 18E) cells with DMSO vehicle or nimbolide for 24 or 48 hours. Representative FACS data from 231MFP cells in Figure 18D and HCC38 cells shown in Figure 17C are shown in the left panel of (Figure 18E). Bar graphs in Figures 18D-18E are the percentage of late apoptotic cells defined as PI+ / Annexin-V+ cells. Data shown in Figures 18A-18B and Figures 18D-18E are mean ± sem, with n = 6 for Figures 18A-18B and n = 3 for Figures 18D-18E from biologically independent samples / groups. Statistical significance was calculated using an unpaired, two-tailed Student's t-test.Significance compared to vehicle-treated controls: *p=1.64x10-13 and 2.05x10-13 for 100 and 10 μM, respectively, in Figure 18A; *p=2.09x10-11 and 7.07x10-12 for 100 and 10 μM, respectively, in Figure 18B; *p=9.05x10-5 and 1.87x10-5 for 10 and 100 μM, respectively, for the 24 hour data in Figure 18D; and *p=1.87x10-5 for 10 and 100 μM, respectively, for the 48 hour data in Figure 18E. For the 24 hour data in Figure 18E, *p=0.00424, 0.0182, and 2.22x10-11 for 1, 10, and 100 μM, respectively; and for the 48 hour data in Figure 18E, *p=6.68x10-6, 4.70x10-7, and 9.00x10-11 for 1, 10, and 100 μM, respectively. [Figure 18E]Nimbolide reduces breast cancer cell proliferation and survival and induces apoptosis. (Figures 18A-18B) Proliferation of HCC38 breast cancer cells in serum-containing medium (Figure 18A) and serum-free cell survival (Figure 18B). Cells were treated with DMSO vehicle or nimbolide, and cell viability was assessed 48 hours later. (Figure 18C) Shown is the gating strategy for flow cytometry data. Two gating steps were used. The first step involved gates by forward and side scatter. The second gate was based on quadrant separation by cell death staining (PI / annexin). (Figures 18D-18E) Percentage of propidium iodide- and annexin V-positive (PI+ / annexin V+) cells assessed by flow cytometry after treatment of 231MFP (Figure 18D) and HCC38 (Figure 18E) cells with DMSO vehicle or nimbolide for 24 or 48 hours. Representative FACS data from 231MFP cells in Figure 18D and HCC38 cells shown in Figure 17C are shown in the left panel of (Figure 18E). Bar graphs in Figures 18D-18E are the percentage of late apoptotic cells defined as PI+ / Annexin-V+ cells. Data shown in Figures 18A-18B and Figures 18D-18E are mean ± sem, with n = 6 for Figures 18A-18B and n = 3 for Figures 18D-18E from biologically independent samples / groups. Statistical significance was calculated using an unpaired, two-tailed Student's t-test.Significance compared to vehicle-treated controls: *p=1.64x10-13 and 2.05x10-13 for 100 and 10 μM, respectively, in Figure 18A; *p=2.09x10-11 and 7.07x10-12 for 100 and 10 μM, respectively, in Figure 18B; *p=9.05x10-5 and 1.87x10-5 for 10 and 100 μM, respectively, for the 24 hour data in Figure 18D; and *p=1.87x10-5 for 10 and 100 μM, respectively, for the 48 hour data in Figure 18E. For the 24 hour data in Figure 18E, *p=0.00424, 0.0182, and 2.22x10-11 for 1, 10, and 100 μM, respectively; and for the 48 hour data in Figure 18E, *p=6.68x10-6, 4.70x10-7, and 9.00x10-11 for 1, 10, and 100 μM, respectively. [Figure 19A]IsoTOP-ABPP analysis of nimbolide in the 231MFP breast cancer cell proteome revealed RNF114 as a target. (Figure 19A) IsoTOP-ABPP analysis of nimbolide (10 μM) in 231MFP breast cancer cells analyzed in situ as described in Figure 19A. The ratio of light to heavy isotope probe-modified peptides is shown in the plot on the left, with the primary target with the highest ratio being C8 of RNF114. Shown on the right are representative MS1 light to heavy peaks for probe-modified peptides with C8 of RNF114. (Figure 19B) RNF114 knockdown by small interfering RNA (siRNA) targeting RNF114 verified by Western blotting of RNF114 compared to the si control 231MFP cells. GAPDH expression is shown as a loading control. The gel shown is a representative gel from n=3 biological replicates / group. (Figure 19C) Proliferation of 231MFP cells after 24 hours in si-control and siRNF114 cells. (Figure 19D) Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Cells were treated with DMSO vehicle or nimbolide for 24 hours, after which proliferation was assessed. Data for the si-control or siRNF114 groups were normalized to the respective DMSO vehicle control for each group. Individual biologically independent sample data are shown, and lines indicate the mean. Data shown in Figure 19C are mean ± sem. Data shown in Figures 19A-19B are from n=3, and in Figures 18D-18E are from n=5 biologically independent samples / group. Statistical significance in Figures 18D-18E was calculated using an unpaired, two-tailed Student's t-test. Significance in Figure 18D is expressed as *P=4.52x10-5 compared to si-control cells. Significance in Figure 18E is expressed as *p=1.90x10-5, 2.72x10-4 and 0.00101 for 10, 6 and 3 μM, respectively, compared to the corresponding nimbolide treatment concentrations from the si-control group. [Figure 19B]IsoTOP-ABPP analysis of nimbolide in the 231MFP breast cancer cell proteome revealed RNF114 as a target. (Figure 19A) IsoTOP-ABPP analysis of nimbolide (10 μM) in 231MFP breast cancer cells analyzed in situ as described in Figure 19A. The ratio of light to heavy isotope probe-modified peptides is shown in the plot on the left, with the primary target with the highest ratio being C8 of RNF114. Shown on the right are representative MS1 light to heavy peaks for probe-modified peptides with C8 of RNF114. (Figure 19B) RNF114 knockdown by small interfering RNA (siRNA) targeting RNF114 verified by Western blotting of RNF114 compared to the si control 231MFP cells. GAPDH expression is shown as a loading control. The gel shown is a representative gel from n=3 biological replicates / group. (Figure 19C) Proliferation of 231MFP cells after 24 hours in si-control and siRNF114 cells. (Figure 19D) Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Cells were treated with DMSO vehicle or nimbolide for 24 hours, after which proliferation was assessed. Data for the si-control or siRNF114 groups were normalized to the respective DMSO vehicle control for each group. Individual biologically independent sample data are shown, and lines indicate the mean. Data shown in Figure 19C are mean ± sem. Data shown in Figures 19A-19B are from n=3, and in Figures 18D-18E are from n=5 biologically independent samples / group. Statistical significance in Figures 18D-18E was calculated using an unpaired, two-tailed Student's t-test. Significance in Figure 18D is expressed as *P=4.52x10-5 compared to si-control cells. Significance in Figure 18E is expressed as *p=1.90x10-5, 2.72x10-4 and 0.00101 for 10, 6 and 3 μM, respectively, compared to the corresponding nimbolide treatment concentrations from the si-control group. [Figure 19C]IsoTOP-ABPP analysis of nimbolide in the 231MFP breast cancer cell proteome revealed RNF114 as a target. (Figure 19A) IsoTOP-ABPP analysis of nimbolide (10 μM) in 231MFP breast cancer cells analyzed in situ as described in Figure 19A. The ratio of light to heavy isotope probe-modified peptides is shown in the plot on the left, with the primary target with the highest ratio being C8 of RNF114. Shown on the right are representative MS1 light to heavy peaks for probe-modified peptides with C8 of RNF114. (Figure 19B) RNF114 knockdown by small interfering RNA (siRNA) targeting RNF114 verified by Western blotting of RNF114 compared to the si control 231MFP cells. GAPDH expression is shown as a loading control. The gel shown is a representative gel from n=3 biological replicates / group. (Figure 19C) Proliferation of 231MFP cells after 24 hours in si-control and siRNF114 cells. (Figure 19D) Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Cells were treated with DMSO vehicle or nimbolide for 24 hours, after which proliferation was assessed. Data for the si-control or siRNF114 groups were normalized to the respective DMSO vehicle control for each group. Individual biologically independent sample data are shown, and lines indicate the mean. Data shown in Figure 19C are mean ± sem. Data shown in Figures 19A-19B are from n=3, and in Figures 18D-18E are from n=5 biologically independent samples / group. Statistical significance in Figures 18D-18E was calculated using an unpaired, two-tailed Student's t-test. Significance in Figure 18D is expressed as *P=4.52x10-5 compared to si-control cells. Significance in Figure 18E is expressed as *p=1.90x10-5, 2.72x10-4 and 0.00101 for 10, 6 and 3 μM, respectively, compared to the corresponding nimbolide treatment concentrations from the si-control group. [Figure 19D]IsoTOP-ABPP analysis of nimbolide in the 231MFP breast cancer cell proteome revealed RNF114 as a target. (Figure 19A) IsoTOP-ABPP analysis of nimbolide (10 μM) in 231MFP breast cancer cells analyzed in situ as described in Figure 19A. The ratio of light to heavy isotope probe-modified peptides is shown in the plot on the left, with the primary target with the highest ratio being C8 of RNF114. Shown on the right are representative MS1 light to heavy peaks for probe-modified peptides with C8 of RNF114. (Figure 19B) RNF114 knockdown by small interfering RNA (siRNA) targeting RNF114 verified by Western blotting of RNF114 compared to the si control 231MFP cells. GAPDH expression is shown as a loading control. The gel shown is a representative gel from n=3 biological replicates / group. (Figure 19C) Proliferation of 231MFP cells after 24 hours in si-control and siRNF114 cells. (Figure 19D) Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Cells were treated with DMSO vehicle or nimbolide for 24 hours, after which proliferation was assessed. Data for the si-control or siRNF114 groups were normalized to the respective DMSO vehicle control for each group. Individual biologically independent sample data are shown, and lines indicate the mean. Data shown in Figure 19C are mean ± sem. Data shown in Figures 19A-19B are from n=3, and in Figures 18D-18E are from n=5 biologically independent samples / group. Statistical significance in Figures 18D-18E was calculated using an unpaired, two-tailed Student's t-test. Significance in Figure 18D is expressed as *P=4.52x10-5 compared to si-control cells. Significance in Figure 18E is expressed as *p=1.90x10-5, 2.72x10-4 and 0.00101 for 10, 6 and 3 μM, respectively, compared to the corresponding nimbolide treatment concentrations from the si-control group. [Figure 20A]Elucidating the role of RNF114 in nimbolide-mediated effects. (Figure 20A) RNF114 knockdown by three independent siRNAs targeting RNF114 was verified by Western blotting of RNF114 compared to si-control 231MFP cells. GAPDH expression is shown as a loading control. (Figure 20B) Proliferation of 231MFP cells after 24 hours in si-control and siRNF114 cells assessed by Hoechst staining. (Figure 20C) Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Cells were treated with DMSO vehicle or nimbolide for 24 hours, after which proliferation was assessed by Hoechst staining. Data for each si-control or siRNF114 group were normalized to the respective DMSO vehicle control for each group. Data from individual biologically independent samples are shown, and lines indicate mean values. (Figure 20D) Gel-based ABPP analysis of nimbolide, JNS27, and iodoacetamide competition for IA-rhodamine labeling of recombinant human RNF114 protein. RNF114 protein was pretreated with DMSO vehicle or nimbolide, JNS27, or iodoacetamide for 30 minutes, after which RNF114 was labeled with IA-rhodamine (100 nM) for 30 minutes. IA-rhodamine labeling of RNF114 was assessed by SDS / PAGE and in-gel fluorescence. Data shown are from n=1 biological replicate. Data shown are from n=3 biologically independent samples / group. Gels shown in Figures 20A and 20D are representative gels from n=3 biologically independent samples / group. Data shown in Figure 20B are mean ± sem, and in the case of Figure 20B, n=5 biologically independent samples / group. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 20B and 20C. Significance in Figure 20B is expressed as *p=4.52x10-5, 0.0429, and 0.00230 for siRNF114-1, siRNF114-2, and siRNF114-3, respectively, compared to si-control cells.The significance in Figure 20C is expressed as *p=1.90x10-5, 0.000272, 0.00101 for siRNF114-1 at 10, 6, and 3 μM, respectively; *p=0.000626, 0.00139, 3.66x10-5 for siRNF114-2 at 10, 6, and 3 μM, respectively; and *p=0.00134, 9.15x10-5, 0.00769 for siRNF114-3 at 10, 6, and 3 μM, respectively, compared to the corresponding nimbolide treatment concentrations in si-control cells. The significance in Figure 20F is expressed as *p=0.0188 compared to vehicle-treated controls. [Figure 20B]Elucidating the role of RNF114 in nimbolide-mediated effects. (Figure 20A) RNF114 knockdown by three independent siRNAs targeting RNF114 was verified by Western blotting of RNF114 compared to si-control 231MFP cells. GAPDH expression is shown as a loading control. (Figure 20B) Proliferation of 231MFP cells after 24 hours in si-control and siRNF114 cells assessed by Hoechst staining. (Figure 20C) Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Cells were treated with DMSO vehicle or nimbolide for 24 hours, after which proliferation was assessed by Hoechst staining. Data for each si-control or siRNF114 group were normalized to the respective DMSO vehicle control for each group. Data from individual biologically independent samples are shown, and lines indicate mean values. (Figure 20D) Gel-based ABPP analysis of nimbolide, JNS27, and iodoacetamide competition for IA-rhodamine labeling of recombinant human RNF114 protein. RNF114 protein was pretreated with DMSO vehicle or nimbolide, JNS27, or iodoacetamide for 30 minutes, after which RNF114 was labeled with IA-rhodamine (100 nM) for 30 minutes. IA-rhodamine labeling of RNF114 was assessed by SDS / PAGE and in-gel fluorescence. Data shown are from n=1 biological replicate. Data shown are from n=3 biologically independent samples / group. Gels shown in Figures 20A and 20D are representative gels from n=3 biologically independent samples / group. Data shown in Figure 20B are mean ± sem, and in the case of Figure 20B, n=5 biologically independent samples / group. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 20B and 20C. Significance in Figure 20B is expressed as *p=4.52x10-5, 0.0429, and 0.00230 for siRNF114-1, siRNF114-2, and siRNF114-3, respectively, compared to si-control cells.The significance in Figure 20C is expressed as *p=1.90x10-5, 0.000272, 0.00101 for siRNF114-1 at 10, 6, and 3 μM, respectively; *p=0.000626, 0.00139, 3.66x10-5 for siRNF114-2 at 10, 6, and 3 μM, respectively; and *p=0.00134, 9.15x10-5, 0.00769 for siRNF114-3 at 10, 6, and 3 μM, respectively, compared to the corresponding nimbolide treatment concentrations in si-control cells. The significance in Figure 20F is expressed as *p=0.0188 compared to vehicle-treated controls. [Figure 20C]Elucidating the role of RNF114 in nimbolide-mediated effects. (Figure 20A) RNF114 knockdown by three independent siRNAs targeting RNF114 was verified by Western blotting of RNF114 compared to si-control 231MFP cells. GAPDH expression is shown as a loading control. (Figure 20B) Proliferation of 231MFP cells after 24 hours in si-control and siRNF114 cells assessed by Hoechst staining. (Figure 20C) Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Cells were treated with DMSO vehicle or nimbolide for 24 hours, after which proliferation was assessed by Hoechst staining. Data for each si-control or siRNF114 group were normalized to the respective DMSO vehicle control for each group. Data from individual biologically independent samples are shown, and lines indicate mean values. (Figure 20D) Gel-based ABPP analysis of nimbolide, JNS27, and iodoacetamide competition for IA-rhodamine labeling of recombinant human RNF114 protein. RNF114 protein was pretreated with DMSO vehicle or nimbolide, JNS27, or iodoacetamide for 30 minutes, after which RNF114 was labeled with IA-rhodamine (100 nM) for 30 minutes. IA-rhodamine labeling of RNF114 was assessed by SDS / PAGE and in-gel fluorescence. Data shown are from n=1 biological replicate. Data shown are from n=3 biologically independent samples / group. Gels shown in Figures 20A and 20D are representative gels from n=3 biologically independent samples / group. Data shown in Figure 20B are mean ± sem, and in the case of Figure 20B, n=5 biologically independent samples / group. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 20B and 20C. Significance in Figure 20B is expressed as *p=4.52x10-5, 0.0429, and 0.00230 for siRNF114-1, siRNF114-2, and siRNF114-3, respectively, compared to si-control cells.The significance in Figure 20C is expressed as *p=1.90x10-5, 0.000272, 0.00101 for siRNF114-1 at 10, 6, and 3 μM, respectively; *p=0.000626, 0.00139, 3.66x10-5 for siRNF114-2 at 10, 6, and 3 μM, respectively; and *p=0.00134, 9.15x10-5, 0.00769 for siRNF114-3 at 10, 6, and 3 μM, respectively, compared to the corresponding nimbolide treatment concentrations in si-control cells. The significance in Figure 20F is expressed as *p=0.0188 compared to vehicle-treated controls. [Figure 20D]Elucidating the role of RNF114 in nimbolide-mediated effects. (Figure 20A) RNF114 knockdown by three independent siRNAs targeting RNF114 was verified by Western blotting of RNF114 compared to si-control 231MFP cells. GAPDH expression is shown as a loading control. (Figure 20B) Proliferation of 231MFP cells after 24 hours in si-control and siRNF114 cells assessed by Hoechst staining. (Figure 20C) Effect of nimbolide on 231MFP si-control and siRNF114 231MFP breast cancer cells. Cells were treated with DMSO vehicle or nimbolide for 24 hours, after which proliferation was assessed by Hoechst staining. Data for each si-control or siRNF114 group were normalized to the respective DMSO vehicle control for each group. Data from individual biologically independent samples are shown, and lines indicate mean values. (Figure 20D) Gel-based ABPP analysis of nimbolide, JNS27, and iodoacetamide competition for IA-rhodamine labeling of recombinant human RNF114 protein. RNF114 protein was pretreated with DMSO vehicle or nimbolide, JNS27, or iodoacetamide for 30 minutes, after which RNF114 was labeled with IA-rhodamine (100 nM) for 30 minutes. IA-rhodamine labeling of RNF114 was assessed by SDS / PAGE and in-gel fluorescence. Data shown are from n=1 biological replicate. Data shown are from n=3 biologically independent samples / group. Gels shown in Figures 20A and 20D are representative gels from n=3 biologically independent samples / group. Data shown in Figure 20B are mean ± sem, and in the case of Figure 20B, n=5 biologically independent samples / group. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 20B and 20C. Significance in Figure 20B is expressed as *p=4.52x10-5, 0.0429, and 0.00230 for siRNF114-1, siRNF114-2, and siRNF114-3, respectively, compared to si-control cells.The significance in Figure 20C is expressed as *p=1.90x10-5, 0.000272, 0.00101 for siRNF114-1 at 10, 6, and 3 μM, respectively; *p=0.000626, 0.00139, 3.66x10-5 for siRNF114-2 at 10, 6, and 3 μM, respectively; and *p=0.00134, 9.15x10-5, 0.00769 for siRNF114-3 at 10, 6, and 3 μM, respectively, compared to the corresponding nimbolide treatment concentrations in si-control cells. The significance in Figure 20F is expressed as *p=0.0188 compared to vehicle-treated controls. [Figure 21A]Nimbolide covalently reacts with C8 of RNF114. (Figure 21A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 21B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 21C) Gel-based ABPP analysis of pure human RNF114 protein labeled with a nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. (Figure 21D) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive sites highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min, followed by labeling of the probe for 1 h. (Figure 21E) Nimbolide-alkyne labeling of Flag-RNF114 in 231MFP cells. 231MFP cells stably expressing Flag-tagged RNF114 were treated with DMSO vehicle or nimbolide-alkyne for 2 h. RNF114 was subsequently enriched from the collected cell lysates, and rhodamine-azide was then added to the probe-labeled proteins by CuAAC. The nimbolide-alkyne labeling was then visualized by SDS / PAGE and in-gel fluorescence. (Figure 21F) Nimbolide-alkyne labeling of endogenous RNF114 in 231MFP cells. 231MFP cells were treated with DMSO vehicle or nimbolide-alkyne (50 μM) for 1.5 hours. Biotin-azide was added to the probe-labeled proteins by CuAAC, and these proteins were then enriched with avidin. The resulting pull-down proteins were analyzed by SDS / PAGE and Western blotting for RNF114.The gels shown in Figures 21C-21F are representative gels from n=3 biologically independent samples / group. [Figure 21B]Nimbolide covalently reacts with C8 of RNF114. (Figure 21A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 21B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 21C) Gel-based ABPP analysis of pure human RNF114 protein labeled with a nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. (Figure 21D) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive sites highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min, followed by labeling of the probe for 1 h. (Figure 21E) Nimbolide-alkyne labeling of Flag-RNF114 in 231MFP cells. 231MFP cells stably expressing Flag-tagged RNF114 were treated with DMSO vehicle or nimbolide-alkyne for 2 h. RNF114 was subsequently enriched from the collected cell lysates, and rhodamine-azide was then added to the probe-labeled proteins by CuAAC. The nimbolide-alkyne labeling was then visualized by SDS / PAGE and in-gel fluorescence. (Figure 21F) Nimbolide-alkyne labeling of endogenous RNF114 in 231MFP cells. 231MFP cells were treated with DMSO vehicle or nimbolide-alkyne (50 μM) for 1.5 hours. Biotin-azide was added to the probe-labeled proteins by CuAAC, and these proteins were then enriched with avidin. The resulting pull-down proteins were analyzed by SDS / PAGE and Western blotting for RNF114.The gels shown in Figures 21C-21F are representative gels from n=3 biologically independent samples / group. [Figure 21C]Nimbolide covalently reacts with C8 of RNF114. (Figure 21A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 21B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 21C) Gel-based ABPP analysis of pure human RNF114 protein labeled with a nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. (Figure 21D) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive sites highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min, followed by labeling of the probe for 1 h. (Figure 21E) Nimbolide-alkyne labeling of Flag-RNF114 in 231MFP cells. 231MFP cells stably expressing Flag-tagged RNF114 were treated with DMSO vehicle or nimbolide-alkyne for 2 h. RNF114 was subsequently enriched from the collected cell lysates, and rhodamine-azide was then added to the probe-labeled proteins by CuAAC. The nimbolide-alkyne labeling was then visualized by SDS / PAGE and in-gel fluorescence. (Figure 21F) Nimbolide-alkyne labeling of endogenous RNF114 in 231MFP cells. 231MFP cells were treated with DMSO vehicle or nimbolide-alkyne (50 μM) for 1.5 hours. Biotin-azide was added to the probe-labeled proteins by CuAAC, and these proteins were then enriched with avidin. The resulting pull-down proteins were analyzed by SDS / PAGE and Western blotting for RNF114.The gels shown in Figures 21C-21F are representative gels from n=3 biologically independent samples / group. [Figure 21D]Nimbolide covalently reacts with C8 of RNF114. (Figure 21A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 21B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 21C) Gel-based ABPP analysis of pure human RNF114 protein labeled with a nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. (Figure 21D) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive sites highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min, followed by labeling of the probe for 1 h. (Figure 21E) Nimbolide-alkyne labeling of Flag-RNF114 in 231MFP cells. 231MFP cells stably expressing Flag-tagged RNF114 were treated with DMSO vehicle or nimbolide-alkyne for 2 h. RNF114 was subsequently enriched from the collected cell lysates, and rhodamine-azide was then added to the probe-labeled proteins by CuAAC. The nimbolide-alkyne labeling was then visualized by SDS / PAGE and in-gel fluorescence. (Figure 21F) Nimbolide-alkyne labeling of endogenous RNF114 in 231MFP cells. 231MFP cells were treated with DMSO vehicle or nimbolide-alkyne (50 μM) for 1.5 hours. Biotin-azide was added to the probe-labeled proteins by CuAAC, and these proteins were then enriched with avidin. The resulting pull-down proteins were analyzed by SDS / PAGE and Western blotting for RNF114.The gels shown in Figures 21C-21F are representative gels from n=3 biologically independent samples / group. [Figure 21E]Nimbolide covalently reacts with C8 of RNF114. (Figure 21A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 21B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 21C) Gel-based ABPP analysis of pure human RNF114 protein labeled with a nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. (Figure 21D) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive sites highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min, followed by labeling of the probe for 1 h. (Figure 21E) Nimbolide-alkyne labeling of Flag-RNF114 in 231MFP cells. 231MFP cells stably expressing Flag-tagged RNF114 were treated with DMSO vehicle or nimbolide-alkyne for 2 h. RNF114 was subsequently enriched from the collected cell lysates, and rhodamine-azide was then added to the probe-labeled proteins by CuAAC. The nimbolide-alkyne labeling was then visualized by SDS / PAGE and in-gel fluorescence. (Figure 21F) Nimbolide-alkyne labeling of endogenous RNF114 in 231MFP cells. 231MFP cells were treated with DMSO vehicle or nimbolide-alkyne (50 μM) for 1.5 hours. Biotin-azide was added to the probe-labeled proteins by CuAAC, and these proteins were then enriched with avidin. The resulting pull-down proteins were analyzed by SDS / PAGE and Western blotting for RNF114.The gels shown in Figures 21C-21F are representative gels from n=3 biologically independent samples / group. [Figure 21F]Nimbolide covalently reacts with C8 of RNF114. (Figure 21A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 21B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 21C) Gel-based ABPP analysis of pure human RNF114 protein labeled with a nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. (Figure 21D) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive sites highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min, followed by labeling of the probe for 1 h. (Figure 21E) Nimbolide-alkyne labeling of Flag-RNF114 in 231MFP cells. 231MFP cells stably expressing Flag-tagged RNF114 were treated with DMSO vehicle or nimbolide-alkyne for 2 h. RNF114 was subsequently enriched from the collected cell lysates, and rhodamine-azide was then added to the probe-labeled proteins by CuAAC. The nimbolide-alkyne labeling was then visualized by SDS / PAGE and in-gel fluorescence. (Figure 21F) Nimbolide-alkyne labeling of endogenous RNF114 in 231MFP cells. 231MFP cells were treated with DMSO vehicle or nimbolide-alkyne (50 μM) for 1.5 hours. Biotin-azide was added to the probe-labeled proteins by CuAAC, and these proteins were then enriched with avidin. The resulting pull-down proteins were analyzed by SDS / PAGE and Western blotting for RNF114.The gels shown in Figures 21C-21F are representative gels from n=3 biologically independent samples / group. [Figure 21G]Nimbolide covalently reacts with C8 of RNF114. (Figure 21A) Nimbolide targets the intrinsically disordered region within RNF114, as assessed by PONDR. (Figure 21B) Synthetic route for alkyne-functionalized nimbolide probes. (Figure 21C) Gel-based ABPP analysis of pure human RNF114 protein labeled with a nimbolide probe. In the top two panels, pure RNF114 protein was preincubated with DMSO vehicle or nimbolide (100 μM, 30 min) and then labeled with a nimbolide probe (10 μM, 1 h) in PBS. In the bottom two panels, pure wild-type and C8A mutant RNF114 proteins were labeled with a nimbolide probe (10 μM, 1 h) in PBS containing 1 mg / ml BSA. (Figure 21D) Gel-based ABPP analysis of nimbolide competition for IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. The structures of the IA-alkyne and JNS27 probes (reactive sites highlighted in red) are shown. Gel-based ABPP analysis of nimbolide (50 μM) competition for JNS27 labeling of wild-type and C8A mutant RNF114 protein is also shown. In these experiments, DMSO or nimbolide was preincubated for 30 min, followed by labeling of the probe for 1 h. (Figure 21E) Nimbolide-alkyne labeling of Flag-RNF114 in 231MFP cells. 231MFP cells stably expressing Flag-tagged RNF114 were treated with DMSO vehicle or nimbolide-alkyne for 2 h. RNF114 was subsequently enriched from the collected cell lysates, and rhodamine-azide was then added to the probe-labeled proteins by CuAAC. The nimbolide-alkyne labeling was then visualized by SDS / PAGE and in-gel fluorescence. (Figure 21F) Nimbolide-alkyne labeling of endogenous RNF114 in 231MFP cells. 231MFP cells were treated with DMSO vehicle or nimbolide-alkyne (50 μM) for 1.5 hours. Biotin-azide was added to the probe-labeled proteins by CuAAC, and these proteins were then enriched with avidin. The resulting pull-down proteins were analyzed by SDS / PAGE and Western blotting for RNF114.The gels shown in Figures 21C-21F are representative gels from n=3 biologically independent samples / group. [Figure 22A]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figures 22A-22B) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (Figure 22A) or p21 (Figure 22B). (Figure 22C) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 22D) In ​​in vitro incubations of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). (Figure 22E) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. (Figure 22F) Tandem mass tag (TMT)-based quantitative proteomics profiling of 231MFP breast cancer cells treated with DMSO vehicle or nimbolide (100 nM) for 12 hours. Proteins significantly elevated (>2-fold) are highlighted in red. Data shown in (Figure 22F) are for 6,397 proteins quantified with two or more unique peptides in n=3 biologically independent samples / groups. (Figure 22G) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with p21(CDKN1A), PEG10, or CTGF), and blotting for Flag-ubiquitin. DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 22H) Expression of p21 (CDKN1A) and p57 (CDKN1C) in siControl and siCDKN1A / siCDKN1C 231MFP cells assessed by Western blotting, with actin as a loading control.(Figure 22I) Proliferation of 231MFP cells in siControl or siCDKN1A / siCDKN1C cells treated with DMSO vehicle or nimbolide (6 μM) for 24 hours. Gels shown in Figures 22A-22E and 22G are representative images from n=3 biologically independent samples / groups. Quantification of blots shown in Figures 22A-22D is in Figures 23A-23D. All three biologically independent samples / groups are shown in Figure 22H. Data shown in Figure 22I are mean ± sem, n=5 biologically independent samples / groups. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 22E and 22I. Significance is expressed as *p=0.000799, 0.0295, 0.00962, and 0.0135 at 1, 2, 4, and 8 hours, respectively, compared to the vehicle-treated control group at each time point in Figure 22E, and as p=5.65x10-8 and 0.0173, respectively, compared to the vehicle-treated siControl and siCDKN1A / siCDKN1C groups in Figure 221. Significance expressed as #p=6.70x10-5 compared to nimbolide-treated siControl cells in Figure 221. [Figure 22B]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figures 22A-22B) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (Figure 22A) or p21 (Figure 22B). (Figure 22C) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 22D) In ​​in vitro incubations of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). (Figure 22E) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. (Figure 22F) Tandem mass tag (TMT)-based quantitative proteomics profiling of 231MFP breast cancer cells treated with DMSO vehicle or nimbolide (100 nM) for 12 hours. Proteins significantly elevated (>2-fold) are highlighted in red. Data shown in (Figure 22F) are for 6,397 proteins quantified with two or more unique peptides in n=3 biologically independent samples / groups. (Figure 22G) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with p21(CDKN1A), PEG10, or CTGF), and blotting for Flag-ubiquitin. DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 22H) Expression of p21 (CDKN1A) and p57 (CDKN1C) in siControl and siCDKN1A / siCDKN1C 231MFP cells assessed by Western blotting, with actin as a loading control.(Figure 22I) Proliferation of 231MFP cells in siControl or siCDKN1A / siCDKN1C cells treated with DMSO vehicle or nimbolide (6 μM) for 24 hours. Gels shown in Figures 22A-22E and 22G are representative images from n=3 biologically independent samples / groups. Quantification of blots shown in Figures 22A-22D is in Figures 23A-23D. All three biologically independent samples / groups are shown in Figure 22H. Data shown in Figure 22I are mean ± sem, n=5 biologically independent samples / groups. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 22E and 22I. Significance is expressed as *p=0.000799, 0.0295, 0.00962, and 0.0135 at 1, 2, 4, and 8 hours, respectively, compared to the vehicle-treated control group at each time point in Figure 22E, and as p=5.65x10-8 and 0.0173, respectively, compared to the vehicle-treated siControl and siCDKN1A / siCDKN1C groups in Figure 221. Significance expressed as #p=6.70x10-5 compared to nimbolide-treated siControl cells in Figure 221. [Figure 22C]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figures 22A-22B) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (Figure 22A) or p21 (Figure 22B). (Figure 22C) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 22D) In ​​in vitro incubations of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). (Figure 22E) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. (Figure 22F) Tandem mass tag (TMT)-based quantitative proteomics profiling of 231MFP breast cancer cells treated with DMSO vehicle or nimbolide (100 nM) for 12 hours. Proteins significantly elevated (>2-fold) are highlighted in red. Data shown in (Figure 22F) are for 6,397 proteins quantified with two or more unique peptides in n=3 biologically independent samples / groups. (Figure 22G) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with p21(CDKN1A), PEG10, or CTGF), and blotting for Flag-ubiquitin. DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 22H) Expression of p21 (CDKN1A) and p57 (CDKN1C) in siControl and siCDKN1A / siCDKN1C 231MFP cells assessed by Western blotting, with actin as a loading control.(Figure 22I) Proliferation of 231MFP cells in siControl or siCDKN1A / siCDKN1C cells treated with DMSO vehicle or nimbolide (6 μM) for 24 hours. Gels shown in Figures 22A-22E and 22G are representative images from n=3 biologically independent samples / groups. Quantification of blots shown in Figures 22A-22D is in Figures 23A-23D. All three biologically independent samples / groups are shown in Figure 22H. Data shown in Figure 22I are mean ± sem, n=5 biologically independent samples / groups. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 22E and 22I. Significance is expressed as *p=0.000799, 0.0295, 0.00962, and 0.0135 at 1, 2, 4, and 8 hours, respectively, compared to the vehicle-treated control group at each time point in Figure 22E, and as p=5.65x10-8 and 0.0173, respectively, compared to the vehicle-treated siControl and siCDKN1A / siCDKN1C groups in Figure 221. Significance expressed as #p=6.70x10-5 compared to nimbolide-treated siControl cells in Figure 221. [Figure 22D]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figures 22A-22B) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (Figure 22A) or p21 (Figure 22B). (Figure 22C) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 22D) In ​​in vitro incubations of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). (Figure 22E) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. (Figure 22F) Tandem mass tag (TMT)-based quantitative proteomics profiling of 231MFP breast cancer cells treated with DMSO vehicle or nimbolide (100 nM) for 12 hours. Proteins significantly elevated (>2-fold) are highlighted in red. Data shown in (Figure 22F) are for 6,397 proteins quantified with two or more unique peptides in n=3 biologically independent samples / groups. (Figure 22G) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with p21(CDKN1A), PEG10, or CTGF), and blotting for Flag-ubiquitin. DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 22H) Expression of p21 (CDKN1A) and p57 (CDKN1C) in siControl and siCDKN1A / siCDKN1C 231MFP cells assessed by Western blotting, with actin as a loading control.(Figure 22I) Proliferation of 231MFP cells in siControl or siCDKN1A / siCDKN1C cells treated with DMSO vehicle or nimbolide (6 μM) for 24 hours. Gels shown in Figures 22A-22E and 22G are representative images from n=3 biologically independent samples / groups. Quantification of blots shown in Figures 22A-22D is in Figures 23A-23D. All three biologically independent samples / groups are shown in Figure 22H. Data shown in Figure 22I are mean ± sem, n=5 biologically independent samples / groups. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 22E and 22I. Significance is expressed as *p=0.000799, 0.0295, 0.00962, and 0.0135 at 1, 2, 4, and 8 hours, respectively, compared to the vehicle-treated control group at each time point in Figure 22E, and as p=5.65x10-8 and 0.0173, respectively, compared to the vehicle-treated siControl and siCDKN1A / siCDKN1C groups in Figure 221. Significance expressed as #p=6.70x10-5 compared to nimbolide-treated siControl cells in Figure 221. [Figure 22E]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figures 22A-22B) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (Figure 22A) or p21 (Figure 22B). (Figure 22C) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 22D) In ​​in vitro incubations of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). (Figure 22E) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. (Figure 22F) Tandem mass tag (TMT)-based quantitative proteomics profiling of 231MFP breast cancer cells treated with DMSO vehicle or nimbolide (100 nM) for 12 hours. Proteins significantly elevated (>2-fold) are highlighted in red. Data shown in (Figure 22F) are for 6,397 proteins quantified with two or more unique peptides in n=3 biologically independent samples / groups. (Figure 22G) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with p21(CDKN1A), PEG10, or CTGF), and blotting for Flag-ubiquitin. DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 22H) Expression of p21 (CDKN1A) and p57 (CDKN1C) in siControl and siCDKN1A / siCDKN1C 231MFP cells assessed by Western blotting, with actin as a loading control.(Figure 22I) Proliferation of 231MFP cells in siControl or siCDKN1A / siCDKN1C cells treated with DMSO vehicle or nimbolide (6 μM) for 24 hours. Gels shown in Figures 22A-22E and 22G are representative images from n=3 biologically independent samples / groups. Quantification of blots shown in Figures 22A-22D is in Figures 23A-23D. All three biologically independent samples / groups are shown in Figure 22H. Data shown in Figure 22I are mean ± sem, n=5 biologically independent samples / groups. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 22E and 22I. Significance is expressed as *p=0.000799, 0.0295, 0.00962, and 0.0135 at 1, 2, 4, and 8 hours, respectively, compared to the vehicle-treated control group at each time point in Figure 22E, and as p=5.65x10-8 and 0.0173, respectively, compared to the vehicle-treated siControl and siCDKN1A / siCDKN1C groups in Figure 221. Significance expressed as #p=6.70x10-5 compared to nimbolide-treated siControl cells in Figure 221. [Figure 22F]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figures 22A-22B) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (Figure 22A) or p21 (Figure 22B). (Figure 22C) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 22D) In ​​in vitro incubations of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). (Figure 22E) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. (Figure 22F) Tandem mass tag (TMT)-based quantitative proteomics profiling of 231MFP breast cancer cells treated with DMSO vehicle or nimbolide (100 nM) for 12 hours. Proteins significantly elevated (>2-fold) are highlighted in red. Data shown in (Figure 22F) are for 6,397 proteins quantified with two or more unique peptides in n=3 biologically independent samples / groups. (Figure 22G) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with p21(CDKN1A), PEG10, or CTGF), and blotting for Flag-ubiquitin. DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 22H) Expression of p21 (CDKN1A) and p57 (CDKN1C) in siControl and siCDKN1A / siCDKN1C 231MFP cells assessed by Western blotting, with actin as a loading control.(Figure 22I) Proliferation of 231MFP cells in siControl or siCDKN1A / siCDKN1C cells treated with DMSO vehicle or nimbolide (6 μM) for 24 hours. Gels shown in Figures 22A-22E and 22G are representative images from n=3 biologically independent samples / groups. Quantification of blots shown in Figures 22A-22D is in Figures 23A-23D. All three biologically independent samples / groups are shown in Figure 22H. Data shown in Figure 22I are mean ± sem, n=5 biologically independent samples / groups. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 22E and 22I. Significance is expressed as *p=0.000799, 0.0295, 0.00962, and 0.0135 at 1, 2, 4, and 8 hours, respectively, compared to the vehicle-treated control group at each time point in Figure 22E, and as p=5.65x10-8 and 0.0173, respectively, compared to the vehicle-treated siControl and siCDKN1A / siCDKN1C groups in Figure 221. Significance expressed as #p=6.70x10-5 compared to nimbolide-treated siControl cells in Figure 221. [Figure 22G]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figures 22A-22B) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (Figure 22A) or p21 (Figure 22B). (Figure 22C) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 22D) In ​​in vitro incubations of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). (Figure 22E) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. (Figure 22F) Tandem mass tag (TMT)-based quantitative proteomics profiling of 231MFP breast cancer cells treated with DMSO vehicle or nimbolide (100 nM) for 12 hours. Proteins significantly elevated (>2-fold) are highlighted in red. Data shown in (Figure 22F) are for 6,397 proteins quantified with two or more unique peptides in n=3 biologically independent samples / groups. (Figure 22G) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with p21(CDKN1A), PEG10, or CTGF), and blotting for Flag-ubiquitin. DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 22H) Expression of p21 (CDKN1A) and p57 (CDKN1C) in siControl and siCDKN1A / siCDKN1C 231MFP cells assessed by Western blotting, with actin as a loading control.(Figure 22I) Proliferation of 231MFP cells in siControl or siCDKN1A / siCDKN1C cells treated with DMSO vehicle or nimbolide (6 μM) for 24 hours. Gels shown in Figures 22A-22E and 22G are representative images from n=3 biologically independent samples / groups. Quantification of blots shown in Figures 22A-22D is in Figures 23A-23D. All three biologically independent samples / groups are shown in Figure 22H. Data shown in Figure 22I are mean ± sem, n=5 biologically independent samples / groups. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 22E and 22I. Significance is expressed as *p=0.000799, 0.0295, 0.00962, and 0.0135 at 1, 2, 4, and 8 hours, respectively, compared to the vehicle-treated control group at each time point in Figure 22E, and as p=5.65x10-8 and 0.0173, respectively, compared to the vehicle-treated siControl and siCDKN1A / siCDKN1C groups in Figure 221. Significance expressed as #p=6.70x10-5 compared to nimbolide-treated siControl cells in Figure 221. [Figure 22H]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figures 22A-22B) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (Figure 22A) or p21 (Figure 22B). (Figure 22C) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 22D) In ​​in vitro incubations of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). (Figure 22E) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. (Figure 22F) Tandem mass tag (TMT)-based quantitative proteomics profiling of 231MFP breast cancer cells treated with DMSO vehicle or nimbolide (100 nM) for 12 hours. Proteins significantly elevated (>2-fold) are highlighted in red. Data shown in (Figure 22F) are for 6,397 proteins quantified with two or more unique peptides in n=3 biologically independent samples / groups. (Figure 22G) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with p21(CDKN1A), PEG10, or CTGF), and blotting for Flag-ubiquitin. DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 22H) Expression of p21 (CDKN1A) and p57 (CDKN1C) in siControl and siCDKN1A / siCDKN1C 231MFP cells assessed by Western blotting, with actin as a loading control.(Figure 22I) Proliferation of 231MFP cells in siControl or siCDKN1A / siCDKN1C cells treated with DMSO vehicle or nimbolide (6 μM) for 24 hours. Gels shown in Figures 22A-22E and 22G are representative images from n=3 biologically independent samples / groups. Quantification of blots shown in Figures 22A-22D is in Figures 23A-23D. All three biologically independent samples / groups are shown in Figure 22H. Data shown in Figure 22I are mean ± sem, n=5 biologically independent samples / groups. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 22E and 22I. Significance is expressed as *p=0.000799, 0.0295, 0.00962, and 0.0135 at 1, 2, 4, and 8 hours, respectively, compared to the vehicle-treated control group at each time point in Figure 22E, and as p=5.65x10-8 and 0.0173, respectively, compared to the vehicle-treated siControl and siCDKN1A / siCDKN1C groups in Figure 221. Significance expressed as #p=6.70x10-5 compared to nimbolide-treated siControl cells in Figure 221. [Figure 22I]Nimbolide inhibits RNF114 activity by interfering with substrate recognition. (Figures 22A-22B) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with or without p21), and blotting for Flag-ubiquitin (Figure 22A) or p21 (Figure 22B). (Figure 22C) RNF114 autoubiquitination assay using wild-type or C8A mutant RNF114 treated with DMSO or nimbolide (100 μM). (Figure 22D) In ​​in vitro incubations of pure RNF114 and p21 proteins, Flag-RNF114 pull-down and p21 enrichment were inhibited by nimbolide (100 μM). (Figure 22E) 231MFP cells were treated with nimbolide (100 μM). Shown are p21 levels in DMSO control or nimbolide-treated cells. (Figure 22F) Tandem mass tag (TMT)-based quantitative proteomics profiling of 231MFP breast cancer cells treated with DMSO vehicle or nimbolide (100 nM) for 12 hours. Proteins significantly elevated (>2-fold) are highlighted in red. Data shown in (Figure 22F) are for 6,397 proteins quantified with two or more unique peptides in n=3 biologically independent samples / groups. (Figure 22G) RNF114 ubiquitination assay using pure GST-Ube1, GST-UBE2D1, and RNF114 proteins, Flag-ubiquitin, and ATP (with p21(CDKN1A), PEG10, or CTGF), and blotting for Flag-ubiquitin. DMSO or nimbolide (100 μM) was preincubated with RNF114, followed by the addition of E1 and E2 enzymes, Flag-ubiquitin, and ATP to initiate the reaction. (Figure 22H) Expression of p21 (CDKN1A) and p57 (CDKN1C) in siControl and siCDKN1A / siCDKN1C 231MFP cells assessed by Western blotting, with actin as a loading control.(Figure 22I) Proliferation of 231MFP cells in siControl or siCDKN1A / siCDKN1C cells treated with DMSO vehicle or nimbolide (6 μM) for 24 hours. Gels shown in Figures 22A-22E and 22G are representative images from n=3 biologically independent samples / groups. Quantification of blots shown in Figures 22A-22D is in Figures 23A-23D. All three biologically independent samples / groups are shown in Figure 22H. Data shown in Figure 22I are mean ± sem, n=5 biologically independent samples / groups. Statistical significance was calculated using an unpaired two-tailed Student's t-test in Figures 22E and 22I. Significance is expressed as *p=0.000799, 0.0295, 0.00962, and 0.0135 at 1, 2, 4, and 8 hours, respectively, compared to the vehicle-treated control group at each time point in Figure 22E, and as p=5.65x10-8 and 0.0173, respectively, compared to the vehicle-treated siControl and siCDKN1A / siCDKN1C groups in Figure 221. Significance expressed as #p=6.70x10-5 compared to nimbolide-treated siControl cells in Figure 221. [Figure 23A]Nimbolide can be used to recruit RNF114 for targeted proteolysis of BRD4. (Figure 23A) Pathway for synthesizing XH2, a nimbolide-based degrader consisting of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figure 23B) Gel-based ABPP analysis of XH2 against RNF114. RNF114 was preincubated with DMSO vehicle or XH2 for 30 min, followed by JNS27 labeling (50 μM) for 1 h, followed by rhodamine-azide addition with CuAAC, SDS / PAGE, and in-gel fluorescence analysis. (Figures 23C-23D) BRD4 expression in 231MFP breast cancer cells treated with XH2 (Figure 23C) versus MZ1 (Figure 23D) for 12 h. (Figures 23E-23F) BRD4 expression in 231MFP breast cancer cells. Cells were pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) (Figure 23E) or the E1 ubiquitin-activating enzyme inhibitor TAK-243 (10 μM) (Figure 23F) 30 minutes before and during 12 hours of MZ1 (1 μM) or XH2 (100 nM) treatment. (Figure 24G) Expression of RNF114 and loading control GAPDH in RNF114 wild-type (WT) and knockout (KO) HAP1 cells. (Figure 23H) Expression of RNF114 and BRD4 in RNF114 wild-type (WT) or knockout (KO) HAP1 cells treated with DMSO vehicle, MZ1 (1 μM), or XH2 (100 nM) for 12 hours. (Figure 23I) Tandem mass tag (TMT)-based quantitative proteomic profiling of 231MFP breast cancer cells treated with DMSO vehicle or XH2 (100 nM) for 12 hours. Long and short BRD4 isoforms in Figures 23C-23H were visualized by SDS / PAGE and Western blotting, quantified by densitometry, and normalized to GAPDH loading control. Gels shown in Figures 23B-23H are representative images from n=3 biologically independent samples / groups, and quantification of blots in Figures 23C-23F and 23H is shown in Figures 25C-25F and 25I.The data shown in Figure 23I is for 5797 proteins quantified with two or more unique peptides in triplicate. The statistical significance of Figure 23I is described in the Methods section. [Figure 23B]Nimbolide can be used to recruit RNF114 for targeted proteolysis of BRD4. (Figure 23A) Pathway for synthesizing XH2, a nimbolide-based degrader consisting of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figure 23B) Gel-based ABPP analysis of XH2 against RNF114. RNF114 was preincubated with DMSO vehicle or XH2 for 30 min, followed by JNS27 labeling (50 μM) for 1 h, followed by rhodamine-azide addition with CuAAC, SDS / PAGE, and in-gel fluorescence analysis. (Figures 23C-23D) BRD4 expression in 231MFP breast cancer cells treated with XH2 (Figure 23C) versus MZ1 (Figure 23D) for 12 h. (Figures 23E-23F) BRD4 expression in 231MFP breast cancer cells. Cells were pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) (Figure 23E) or the E1 ubiquitin-activating enzyme inhibitor TAK-243 (10 μM) (Figure 23F) 30 minutes before and during 12 hours of MZ1 (1 μM) or XH2 (100 nM) treatment. (Figure 24G) Expression of RNF114 and loading control GAPDH in RNF114 wild-type (WT) and knockout (KO) HAP1 cells. (Figure 23H) Expression of RNF114 and BRD4 in RNF114 wild-type (WT) or knockout (KO) HAP1 cells treated with DMSO vehicle, MZ1 (1 μM), or XH2 (100 nM) for 12 hours. (Figure 23I) Tandem mass tag (TMT)-based quantitative proteomic profiling of 231MFP breast cancer cells treated with DMSO vehicle or XH2 (100 nM) for 12 hours. Long and short BRD4 isoforms in Figures 23C-23H were visualized by SDS / PAGE and Western blotting, quantified by densitometry, and normalized to GAPDH loading control. Gels shown in Figures 23B-23H are representative images from n=3 biologically independent samples / groups, and quantification of blots in Figures 23C-23F and 23H is shown in Figures 25C-25F and 25I.The data shown in Figure 23I is for 5797 proteins quantified with two or more unique peptides in triplicate. The statistical significance of Figure 23I is described in the Methods section. [Figure 23C]Nimbolide can be used to recruit RNF114 for targeted proteolysis of BRD4. (Figure 23A) Pathway for synthesizing XH2, a nimbolide-based degrader consisting of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figure 23B) Gel-based ABPP analysis of XH2 against RNF114. RNF114 was preincubated with DMSO vehicle or XH2 for 30 min, followed by JNS27 labeling (50 μM) for 1 h, followed by rhodamine-azide addition with CuAAC, SDS / PAGE, and in-gel fluorescence analysis. (Figures 23C-23D) BRD4 expression in 231MFP breast cancer cells treated with XH2 (Figure 23C) versus MZ1 (Figure 23D) for 12 h. (Figures 23E-23F) BRD4 expression in 231MFP breast cancer cells. Cells were pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) (Figure 23E) or the E1 ubiquitin-activating enzyme inhibitor TAK-243 (10 μM) (Figure 23F) 30 minutes before and during 12 hours of MZ1 (1 μM) or XH2 (100 nM) treatment. (Figure 24G) Expression of RNF114 and loading control GAPDH in RNF114 wild-type (WT) and knockout (KO) HAP1 cells. (Figure 23H) Expression of RNF114 and BRD4 in RNF114 wild-type (WT) or knockout (KO) HAP1 cells treated with DMSO vehicle, MZ1 (1 μM), or XH2 (100 nM) for 12 hours. (Figure 23I) Tandem mass tag (TMT)-based quantitative proteomic profiling of 231MFP breast cancer cells treated with DMSO vehicle or XH2 (100 nM) for 12 hours. Long and short BRD4 isoforms in Figures 23C-23H were visualized by SDS / PAGE and Western blotting, quantified by densitometry, and normalized to GAPDH loading control. Gels shown in Figures 23B-23H are representative images from n=3 biologically independent samples / groups, and quantification of blots in Figures 23C-23F and 23H is shown in Figures 25C-25F and 25I.The data shown in Figure 23I is for 5797 proteins quantified with two or more unique peptides in triplicate. The statistical significance of Figure 23I is described in the Methods section. [Figure 23D]Nimbolide can be used to recruit RNF114 for targeted proteolysis of BRD4. (Figure 23A) Pathway for synthesizing XH2, a nimbolide-based degrader consisting of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figure 23B) Gel-based ABPP analysis of XH2 against RNF114. RNF114 was preincubated with DMSO vehicle or XH2 for 30 min, followed by JNS27 labeling (50 μM) for 1 h, followed by rhodamine-azide addition with CuAAC, SDS / PAGE, and in-gel fluorescence analysis. (Figures 23C-23D) BRD4 expression in 231MFP breast cancer cells treated with XH2 (Figure 23C) versus MZ1 (Figure 23D) for 12 h. (Figures 23E-23F) BRD4 expression in 231MFP breast cancer cells. Cells were pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) (Figure 23E) or the E1 ubiquitin-activating enzyme inhibitor TAK-243 (10 μM) (Figure 23F) 30 minutes before and during 12 hours of MZ1 (1 μM) or XH2 (100 nM) treatment. (Figure 24G) Expression of RNF114 and loading control GAPDH in RNF114 wild-type (WT) and knockout (KO) HAP1 cells. (Figure 23H) Expression of RNF114 and BRD4 in RNF114 wild-type (WT) or knockout (KO) HAP1 cells treated with DMSO vehicle, MZ1 (1 μM), or XH2 (100 nM) for 12 hours. (Figure 23I) Tandem mass tag (TMT)-based quantitative proteomic profiling of 231MFP breast cancer cells treated with DMSO vehicle or XH2 (100 nM) for 12 hours. Long and short BRD4 isoforms in Figures 23C-23H were visualized by SDS / PAGE and Western blotting, quantified by densitometry, and normalized to GAPDH loading control. Gels shown in Figures 23B-23H are representative images from n=3 biologically independent samples / groups, and quantification of blots in Figures 23C-23F and 23H is shown in Figures 25C-25F and 25I.The data shown in Figure 23I is for 5797 proteins quantified with two or more unique peptides in triplicate. The statistical significance of Figure 23I is described in the Methods section. [Figure 23E]Nimbolide can be used to recruit RNF114 for targeted proteolysis of BRD4. (Figure 23A) Pathway for synthesizing XH2, a nimbolide-based degrader consisting of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figure 23B) Gel-based ABPP analysis of XH2 against RNF114. RNF114 was preincubated with DMSO vehicle or XH2 for 30 min, followed by JNS27 labeling (50 μM) for 1 h, followed by rhodamine-azide addition with CuAAC, SDS / PAGE, and in-gel fluorescence analysis. (Figures 23C-23D) BRD4 expression in 231MFP breast cancer cells treated with XH2 (Figure 23C) versus MZ1 (Figure 23D) for 12 h. (Figures 23E-23F) BRD4 expression in 231MFP breast cancer cells. Cells were pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) (Figure 23E) or the E1 ubiquitin-activating enzyme inhibitor TAK-243 (10 μM) (Figure 23F) 30 minutes before and during 12 hours of MZ1 (1 μM) or XH2 (100 nM) treatment. (Figure 24G) Expression of RNF114 and loading control GAPDH in RNF114 wild-type (WT) and knockout (KO) HAP1 cells. (Figure 23H) Expression of RNF114 and BRD4 in RNF114 wild-type (WT) or knockout (KO) HAP1 cells treated with DMSO vehicle, MZ1 (1 μM), or XH2 (100 nM) for 12 hours. (Figure 23I) Tandem mass tag (TMT)-based quantitative proteomic profiling of 231MFP breast cancer cells treated with DMSO vehicle or XH2 (100 nM) for 12 hours. Long and short BRD4 isoforms in Figures 23C-23H were visualized by SDS / PAGE and Western blotting, quantified by densitometry, and normalized to GAPDH loading control. Gels shown in Figures 23B-23H are representative images from n=3 biologically independent samples / groups, and quantification of blots in Figures 23C-23F and 23H is shown in Figures 25C-25F and 25I.The data shown in Figure 23I is for 5797 proteins quantified with two or more unique peptides in triplicate. The statistical significance of Figure 23I is described in the Methods section. [Figure 23F]Nimbolide can be used to recruit RNF114 for targeted proteolysis of BRD4. (Figure 23A) Pathway for synthesizing XH2, a nimbolide-based degrader consisting of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figure 23B) Gel-based ABPP analysis of XH2 against RNF114. RNF114 was preincubated with DMSO vehicle or XH2 for 30 min, followed by JNS27 labeling (50 μM) for 1 h, followed by rhodamine-azide addition with CuAAC, SDS / PAGE, and in-gel fluorescence analysis. (Figures 23C-23D) BRD4 expression in 231MFP breast cancer cells treated with XH2 (Figure 23C) versus MZ1 (Figure 23D) for 12 h. (Figures 23E-23F) BRD4 expression in 231MFP breast cancer cells. Cells were pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) (Figure 23E) or the E1 ubiquitin-activating enzyme inhibitor TAK-243 (10 μM) (Figure 23F) 30 minutes before and during 12 hours of MZ1 (1 μM) or XH2 (100 nM) treatment. (Figure 24G) Expression of RNF114 and loading control GAPDH in RNF114 wild-type (WT) and knockout (KO) HAP1 cells. (Figure 23H) Expression of RNF114 and BRD4 in RNF114 wild-type (WT) or knockout (KO) HAP1 cells treated with DMSO vehicle, MZ1 (1 μM), or XH2 (100 nM) for 12 hours. (Figure 23I) Tandem mass tag (TMT)-based quantitative proteomic profiling of 231MFP breast cancer cells treated with DMSO vehicle or XH2 (100 nM) for 12 hours. Long and short BRD4 isoforms in Figures 23C-23H were visualized by SDS / PAGE and Western blotting, quantified by densitometry, and normalized to GAPDH loading control. Gels shown in Figures 23B-23H are representative images from n=3 biologically independent samples / groups, and quantification of blots in Figures 23C-23F and 23H is shown in Figures 25C-25F and 25I.The data shown in Figure 23I is for 5797 proteins quantified with two or more unique peptides in triplicate. The statistical significance of Figure 23I is described in the Methods section. [Figure 23G]Nimbolide can be used to recruit RNF114 for targeted proteolysis of BRD4. (Figure 23A) Pathway for synthesizing XH2, a nimbolide-based degrader consisting of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figure 23B) Gel-based ABPP analysis of XH2 against RNF114. RNF114 was preincubated with DMSO vehicle or XH2 for 30 min, followed by JNS27 labeling (50 μM) for 1 h, followed by rhodamine-azide addition with CuAAC, SDS / PAGE, and in-gel fluorescence analysis. (Figures 23C-23D) BRD4 expression in 231MFP breast cancer cells treated with XH2 (Figure 23C) versus MZ1 (Figure 23D) for 12 h. (Figures 23E-23F) BRD4 expression in 231MFP breast cancer cells. Cells were pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) (Figure 23E) or the E1 ubiquitin-activating enzyme inhibitor TAK-243 (10 μM) (Figure 23F) 30 minutes before and during 12 hours of MZ1 (1 μM) or XH2 (100 nM) treatment. (Figure 24G) Expression of RNF114 and loading control GAPDH in RNF114 wild-type (WT) and knockout (KO) HAP1 cells. (Figure 23H) Expression of RNF114 and BRD4 in RNF114 wild-type (WT) or knockout (KO) HAP1 cells treated with DMSO vehicle, MZ1 (1 μM), or XH2 (100 nM) for 12 hours. (Figure 23I) Tandem mass tag (TMT)-based quantitative proteomic profiling of 231MFP breast cancer cells treated with DMSO vehicle or XH2 (100 nM) for 12 hours. Long and short BRD4 isoforms in Figures 23C-23H were visualized by SDS / PAGE and Western blotting, quantified by densitometry, and normalized to GAPDH loading control. Gels shown in Figures 23B-23H are representative images from n=3 biologically independent samples / groups, and quantification of blots in Figures 23C-23F and 23H is shown in Figures 25C-25F and 25I.The data shown in Figure 23I is for 5797 proteins quantified with two or more unique peptides in triplicate. The statistical significance of Figure 23I is described in the Methods section. [Figure 23H]Nimbolide can be used to recruit RNF114 for targeted proteolysis of BRD4. (Figure 23A) Pathway for synthesizing XH2, a nimbolide-based degrader consisting of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figure 23B) Gel-based ABPP analysis of XH2 against RNF114. RNF114 was preincubated with DMSO vehicle or XH2 for 30 min, followed by JNS27 labeling (50 μM) for 1 h, followed by rhodamine-azide addition with CuAAC, SDS / PAGE, and in-gel fluorescence analysis. (Figures 23C-23D) BRD4 expression in 231MFP breast cancer cells treated with XH2 (Figure 23C) versus MZ1 (Figure 23D) for 12 h. (Figures 23E-23F) BRD4 expression in 231MFP breast cancer cells. Cells were pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) (Figure 23E) or the E1 ubiquitin-activating enzyme inhibitor TAK-243 (10 μM) (Figure 23F) 30 minutes before and during 12 hours of MZ1 (1 μM) or XH2 (100 nM) treatment. (Figure 24G) Expression of RNF114 and loading control GAPDH in RNF114 wild-type (WT) and knockout (KO) HAP1 cells. (Figure 23H) Expression of RNF114 and BRD4 in RNF114 wild-type (WT) or knockout (KO) HAP1 cells treated with DMSO vehicle, MZ1 (1 μM), or XH2 (100 nM) for 12 hours. (Figure 23I) Tandem mass tag (TMT)-based quantitative proteomic profiling of 231MFP breast cancer cells treated with DMSO vehicle or XH2 (100 nM) for 12 hours. Long and short BRD4 isoforms in Figures 23C-23H were visualized by SDS / PAGE and Western blotting, quantified by densitometry, and normalized to GAPDH loading control. Gels shown in Figures 23B-23H are representative images from n=3 biologically independent samples / groups, and quantification of blots in Figures 23C-23F and 23H is shown in Figures 25C-25F and 25I.The data shown in Figure 23I is for 5797 proteins quantified with two or more unique peptides in triplicate. The statistical significance of Figure 23I is described in the Methods section. [Figure 23I]Nimbolide can be used to recruit RNF114 for targeted proteolysis of BRD4. (Figure 23A) Pathway for synthesizing XH2, a nimbolide-based degrader consisting of nimbolide as an RNF114 recruiter, a linker, and the BRD4 inhibitor JQ1. (Figure 23B) Gel-based ABPP analysis of XH2 against RNF114. RNF114 was preincubated with DMSO vehicle or XH2 for 30 min, followed by JNS27 labeling (50 μM) for 1 h, followed by rhodamine-azide addition with CuAAC, SDS / PAGE, and in-gel fluorescence analysis. (Figures 23C-23D) BRD4 expression in 231MFP breast cancer cells treated with XH2 (Figure 23C) versus MZ1 (Figure 23D) for 12 h. (Figures 23E-23F) BRD4 expression in 231MFP breast cancer cells. Cells were pretreated with DMSO vehicle or the proteasome inhibitor bortezomib (BTZ) (1 μM) (Figure 23E) or the E1 ubiquitin-activating enzyme inhibitor TAK-243 (10 μM) (Figure 23F) 30 minutes before and during 12 hours of MZ1 (1 μM) or XH2 (100 nM) treatment. (Figure 24G) Expression of RNF114 and loading control GAPDH in RNF114 wild-type (WT) and knockout (KO) HAP1 cells. (Figure 23H) Expression of RNF114 and BRD4 in RNF114 wild-type (WT) or knockout (KO) HAP1 cells treated with DMSO vehicle, MZ1 (1 μM), or XH2 (100 nM) for 12 hours. (Figure 23I) Tandem mass tag (TMT)-based quantitative proteomic profiling of 231MFP breast cancer cells treated with DMSO vehicle or XH2 (100 nM) for 12 hours. Long and short BRD4 isoforms in Figures 23C-23H were visualized by SDS / PAGE and Western blotting, quantified by densitometry, and normalized to GAPDH loading control. Gels shown in Figures 23B-23H are representative images from n=3 biologically independent samples / groups, and quantification of blots in Figures 23C-23F and 23H is shown in Figures 25C-25F and 25I.The data shown in Figure 23I is for 5797 proteins quantified with two or more unique peptides in triplicate. The statistical significance of Figure 23I is described in the Methods section. DETAILED DESCRIPTION OF THE INVENTION

[0015] I. Definition The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulas set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0016] Where substituents are specified by their conventional chemical formula written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0017] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight-chain (i.e., unbranched) or branched carbon chain (or carbons), or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and can include monovalent, divalent, and polyvalent radicals. An alkyl can contain the number of carbons specified (e.g., C-C 10means 1 to 10 carbons). An alkyl is a non-cyclized chain. An unsaturated alkyl group is one having one or more double or triple bonds. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-). The alkyl portion can be an alkenyl portion. The alkyl portion can be an alkynyl portion. The alkyl portion can be fully saturated. An alkenyl can contain more than one double bond and / or one or more triple bonds in addition to one or more double bonds. An alkynyl can contain more than one triple bond and / or one or more double bonds in addition to one or more triple bonds. The term "alkylene," by itself or as part of another substituent, means a divalent radical derived from an alkyl, unless otherwise specified. The term "alkenylene," by itself or as part of another substituent, means a divalent radical derived from an alkene, unless otherwise specified.

[0018] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise specified, a stable linear or branched chain containing at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), or a combination thereof, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatom(s) (e.g., O, N, S, Si, or P) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. A heteroalkyl is a non-cyclized chain. A heteroalkyl moiety can contain one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety can contain two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety can contain three optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety can contain four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety can contain five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety can contain up to eight optionally different heteroatoms (e.g., O, N, S, Si, or P). The term "heteroalkenyl," alone or in combination with another term, means, unless otherwise specified, a heteroalkyl containing at least one double bond. A heteroalkenyl can optionally contain, in addition to one or more double bonds, more than one double bond and / or one or more triple bonds. The term "heteroalkynyl," alone or in combination with another term, means, unless otherwise specified, a heteroalkyl containing at least one triple bond. A heteroalkynyl can optionally contain, in addition to one or more triple bonds, more than one triple bond and / or one or more double bonds.

[0019] Similarly, the term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from a heteroalkyl. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-. When "heteroalkyl" is recited followed by a specific heteroalkyl group, e.g., -NR'R'', etc., it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, the specific heteroalkyl group is recited for clarity. Thus, the term "heteroalkyl" should not be construed herein to exclude specific heteroalkyl groups, e.g., -NR'R'', etc.

[0020] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl," respectively. Cycloalkyls and heterocycloalkyls are not aromatic. Additionally, for heterocycloalkyls, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. "Cycloalkylene" and "heterocycloalkylene," alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0021] In embodiments, the term "cycloalkyl" refers to a monocyclic, bicyclic, or polycyclic cycloalkyl ring system. In embodiments, a monocyclic ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms; such groups can be saturated or unsaturated, but are not aromatic. In embodiments, a cycloalkyl group is fully saturated. In embodiments, a bridged monocyclic ring is one in which two non-adjacent carbon atoms of the monocyclic ring are joined by an alkylene bridge (i.e., of the form (CH)) between 1 to 3 additional carbon atoms. w where w is 1, 2, or 3. In embodiments, a bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring. In embodiments, a polycyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.

[0022] In embodiments, a cycloalkyl is a cycloalkenyl. The term "cycloalkenyl" is used according to its plain and ordinary meaning. In embodiments, a cycloalkenyl is a monocyclic, bicyclic, or polycyclic cycloalkenyl ring system. In embodiments, a monocyclic cycloalkenyl ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, and such group is unsaturated (i.e., contains at least one cyclic carbon-carbon double bond), but is not aromatic. In embodiments, a bicyclic cycloalkenyl ring is a bridged monocyclic ring or a fused bicyclic ring. In embodiments, a bridged monocyclic ring is a ring in which two non-adjacent carbon atoms of a monocyclic ring are joined by an alkylene bridge (i.e., of the form (CH)) between 1 to 3 additional carbon atoms. w where w is 1, 2, or 3. In embodiments, a bridged or fused bicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkenyl ring. In embodiments, a polycyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.

[0023] In embodiments, heterocycloalkyl is heterocyclyl. As used herein, the term "heterocyclyl" refers to a monocyclic, bicyclic, or polycyclic heterocycle. A heterocyclyl monocyclic heterocycle is a 3-, 4-, 5-, 6-, or 7-membered ring in which the ring is saturated or unsaturated but not aromatic and contains at least one heteroatom independently selected from the group consisting of O, N, and S. A 3- or 4-membered ring contains one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring can contain zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 6- or 7-membered ring can contain zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle. A heterocyclyl bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system, and a polycyclic heterocyclyl is connected to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring.

[0024] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0025] The term "acyl," unless otherwise specified, means -C(O)R, where R is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0026] The term "aryl," unless otherwise specified, refers to a polyvalent unsaturated aromatic hydrocarbon substituent, which may be a single ring or multiple rings (preferably 1 to 3 rings) fused together (i.e., fused-ring aryl) or covalently linked together. A fused-ring aryl refers to multiple rings fused together, at least one of which is an aryl ring. The term "heteroaryl" refers to an aryl group (or ring) containing at least one heteroatom, such as N, O, or S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl groups (i.e., multiple rings fused together, at least one of which is an aromatic heterocycle). Heteroaryl groups can be attached to the remainder of the molecule through a carbon atom or a heteroatom. "Arylene" and "heteroarylene," alone or as part of another substituent, refer to divalent radicals derived from aryl and heteroaryl, respectively. Heteroaryl group substituents may be -O-bonded to a ring heteroatom nitrogen.

[0027] Spirocyclic rings are two or more rings in which adjacent rings are connected via a single atom. The individual rings in a spirocyclic ring can be the same or different. The individual rings in a spirocyclic ring can be substituted or unsubstituted, and can have different substituents from the other individual rings in a set of spirocyclic rings. The possible substituents of the individual rings in a spirocyclic ring are the possible substituents of the same ring (e.g., the substituents of a cycloalkyl ring or heterocycloalkyl ring) when they are not part of a spirocyclic ring. When referring to a spirocyclic ring system, a heterocyclic spirocyclic ring means a spirocyclic ring in which at least one ring is a heterocyclic ring, and each ring can be a different ring. When referring to a spirocyclic ring system, a substituted spirocyclic ring means that at least one ring is substituted, and each substituent can be optionally different.

[0028] symbol" [ka] " indicates the point of attachment of the chemical moiety to the remainder of the molecule or chemical formula.

[0029] The term "oxo" as used herein means an oxygen that is double bonded to a carbon atom.

[0030] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl," "heterocycloalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0031] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) range in number from 0 to (2m'+1), where m' is the total number of carbon atoms in such radical, and include, but are not limited to, -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', It can be one or more of a variety of groups selected from: -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R'')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -NR'NR''R''', -ONR'R'', -NR'C(O)NR''NR'''R'''', -CN, -NO2, -NR'S02R'', -NR'C(O)R'', -NR'C(O)-OR'', -NR'OR''. R, R', R'', R''', and R'''' each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1 to 3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy group, or arylalkyl group. When a compound described herein includes more than one R group, e.g., when more than one of these groups is present, each R group is independently selected for each R', R'', R''', and R'''' group. When R' and R'' are attached to the same nitrogen atom, they may be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring.From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is intended to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF and -CHCF) and acyl (e.g., -C(O)CH, -C(O)CF, -C(O)CHOCH, etc.).

[0032] Similar to the substituents described for the alkyl radical, the substituents for the aryl and heteroaryl groups vary and include, for example, —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′′, —OC(O)R′, —C(O)R′, —COR′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′-C(O)NR″R′′′, —NR″C(O)R′, —NR-C(NR′R″R′′)═NR′″, —NR-C(NR′R″)═NR′″, —S(O)R′, —S(O)R′, —S(O)NR′R″, —NRSOR′, —NR′ and R'" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound described herein includes more than one R group, for example, when more than one of R', R", R"', and R"" groups is present, the R groups are each independently selected for each R', R", R'", and R"" group.

[0033] Substituents for a ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) can be shown as substituents on the ring (commonly referred to as floating substituents) rather than on a specific atom of the ring. In such cases, the substituent may be attached to any of the ring atoms (according to the rules of chemical valence), and in the case of a fused or spirocyclic ring, a substituent shown as attached to one member of the fused or spirocyclic ring (a floating substituent on a single ring) can be a substituent on either the fused or spirocyclic ring (a floating substituent on a polycyclic ring). When a substituent is attached to a ring rather than to a specific atom (a floating substituent), and the substituent subscript is an integer greater than 1, multiple substituents may be on the same atom, the same ring, different atoms, different fused rings, or different spirocyclic rings, and each substituent may optionally be different. When the point of attachment of a ring to the rest of the molecule is not limited to a single atom (a floating substituent), the point of attachment may be any atom of the ring, or, in the case of a fused or spirocyclic ring, any atom of the fused or spirocyclic ring, according to the rules of chemical valence. When a ring, fused ring, or spirocyclic ring contains one or more ring heteroatoms and the ring, fused ring, or spirocyclic ring is shown with another floating substituent (including, but not limited to, the point of attachment to the rest of the molecule), the floating substituent may be bonded to the heteroatom. When a ring heteroatom is shown bonded to one or more hydrogens in a structure or formula with a floating substituent (e.g., a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen), it will be understood that when the heteroatom is bonded to the floating substituent, the substituent replaces the hydrogen, according to the rules of chemical valence.

[0034] Two or more substituents may optionally be bonded to form an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group. Such so-called ring-forming substituents are typically, but not necessarily, found to be bonded to a cyclic base structure. In one embodiment, the ring-forming substituents are bonded to adjacent members of the base structure. For example, two ring-forming substituents bonded to adjacent members of a cyclic base structure form a fused ring structure. In another embodiment, the ring-forming substituents are bonded to a single member of the base structure. For example, two ring-forming substituents bonded to a single member of a cyclic base structure form a spirocyclic structure. In yet another embodiment, the ring-forming substituents are bonded to non-adjacent members of the base structure.

[0035] As used herein, the term "heteroatom" or "ring heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0036] As used herein, a "substituent" means a group selected from the following moieties: (A) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO 2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2 Cl, -OCH2Br, -OCHI, -OCH2F, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the following: (i) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO 2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2 Cl, -OCH2Br, -OCHI, -OCH2F, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the following: (a) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO 2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2 Cl, -OCH2Br, -OCHI, -OCH2F, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one group selected from the following: oxo, halogen, —CCl3, —CBr3, —CF3, —CI3, CHCl2, —CHBr2, —CHF2, —CHI2, —CH2Cl, —CH2Br, —CH2F, —CH2I, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —SO3H, —SO4H, —SON2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, —NHC(O)NH2, —NHSO2H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl3, —OCF3, —OCBr3, —OCI3 , -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5-10 membered heteroaryl, 5-9 membered heteroaryl, or 5-6 membered heteroaryl).

[0037] In embodiments, as used herein, a "substituent" means a group selected from the following moieties: (A) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO 2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2 Cl, -OCH2Br, -OCHI, -OCH2F, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (B) alkyl (e.g., C1-C 20 , C1-C 12 , C1-C8, C1-C6, C1-C4, or C1-C2), heteroalkyl (e.g., 2-20 membered, 2-12 membered, 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), cycloalkyl (e.g., C3-C 10 , C3-C8, C3-C6, C4-C6, or C5-C6), heterocycloalkyl (e.g., 3-10 membered, 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), aryl (e.g., C6-C 12 , C6-C 10, or phenyl), or heteroaryl (e.g., 5-12 membered, 5-10 membered, 5-9 membered, or 5-6 membered): (i) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO 2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2 Cl, -OCH2Br, -OCHI, -OCH2F, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (ii) alkyl (e.g., C1-C 20 , C1-C 12 , C1-C8, C1-C6, C1-C4, or C1-C2), heteroalkyl (e.g., 2-20 membered, 2-12 membered, 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), cycloalkyl (e.g., C3-C 10, C3-C8, C3-C6, C4-C6, or C5-C6), heterocycloalkyl (e.g., 3-10 membered, 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), aryl (e.g., C6-C 12 , C6-C 10 , or phenyl), or heteroaryl (e.g., 5-12 membered, 5-10 membered, 5-9 membered, or 5-6 membered): (a) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO 2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2 Cl, -OCH2Br, -OCHI, -OCH2F, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (b) alkyl (e.g., C1-C2) substituted with at least one substituent selected from the following: 20 , C1-C 12, C1-C8, C1-C6, C1-C4, or C1-C2), heteroalkyl (e.g., 2-20 membered, 2-12 membered, 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), cycloalkyl (e.g., C3-C 10 , C3-C8, C3-C6, C4-C6, or C5-C6), heterocycloalkyl (e.g., 3-10 membered, 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), aryl (e.g., C6-C 12 , C6-C 10 , or phenyl), or heteroaryl (e.g., 5-12 membered, 5-10 membered, 5-9 membered, or 5-6 membered): oxo, halogen, —CCl3, —CBr3, —CF3, —CI3, CHCl2, —CHBr2, —CHF2, —CHI2, —CH2Cl, —CH2Br, —CH2F, —CH2I, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —SO3H, —SO4H, —SON2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, —NHC(O)NH2, —NHSO2H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl3, —OCF3, —OCBr3, —OCI3, —OCHCl2, —OCH Br2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (eg, 5-10 membered heteroaryl, 5-9 membered heteroaryl, or 5-6 membered heteroaryl).

[0038] As used herein, a "size-limited substituent" or "size-limited substituent group" means a group selected from all of the substituents described above for "substituent," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C-C 20 each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 20-membered heteroalkyl; each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl; each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 8-membered heterocycloalkyl; and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C8 10 aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 10-membered heteroaryl.

[0039] As used herein, a "lower substituent" or "lower substituent group" means a group selected from all of the substituents described above for "substituent," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C8 10 aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 9-membered heteroaryl.

[0040] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl is a substituted or unsubstituted C-C 20each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 20-membered heteroalkyl; each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl; each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 8-membered heterocycloalkyl; and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C8 10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5-10 membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C-C 20 each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2- to 20-membered heteroalkylene; each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene; each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3- to 8-membered heterocycloalkylene; and each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C8 10 arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5- to 10-membered heteroarylene.

[0041] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-7 membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C7 10In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2-8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3-7 membered heterocycloalkylene, and each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C8 10 and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5- to 9-membered heteroarylene. In some embodiments, the compound is a species described in the Examples section, Figures, or Tables below.

[0042] In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and / or substituted or unsubstituted heteroarylene) is unsubstituted (e.g., unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, and / or unsubstituted heteroarylene, respectively). In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and / or substituted or unsubstituted heteroarylene) is substituted (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene, respectively).

[0043] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent, and when a substituted moiety is substituted with multiple substituents, each substituent can optionally be different. In embodiments, when a substituted moiety is substituted with multiple substituents, each substituent is different.

[0044] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one size-limiting substituent, and when a substituted moiety is substituted with multiple substituents, each size-limiting substituent can optionally be different. In embodiments, when a substituted moiety is substituted with multiple size-limiting substituents, each size-limiting substituent is different.

[0045] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one lower substituent, and when a substituted moiety is substituted with multiple lower substituents, each lower substituent may optionally be different. In embodiments, when a substituted moiety is substituted with multiple lower substituents, each lower substituent is different.

[0046] In embodiments, a substituted moiety (e.g., a substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent, size-limiting substituent, or lower substituent; when a substituted moiety is substituted with multiple groups selected from substituents, size-limiting substituents, and lower substituents, each substituent, size-limiting substituent, and / or lower substituent can optionally be different. In embodiments, when a substituted moiety is substituted with multiple groups selected from substituents, size-limiting substituents, and lower substituents, each substituent, size-limiting substituent, and / or lower substituent can be different.

[0047] Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds, and may be defined in terms of absolute stereochemistry as (R)- or (S)-, or with respect to amino acids, as (D)- or (L)-. Enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include compounds known in the art to be too unstable to synthesize and / or isolate. The present disclosure is intended to include compounds in racemic and optically pure form. Optically active (R)- and (S)- or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other geometrically asymmetric centers, unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers.

[0048] As used herein, the term "isomers" refers to compounds that have the same number and kind of atoms, and therefore the same molecular weight, but differ with regard to the structural arrangement or configuration of the atoms.

[0049] The term "tautomer," as used herein, refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. It will be apparent to one of ordinary skill in the art that certain compounds of the present disclosure may exist in tautomeric forms, and all such tautomeric forms of the compounds are within the scope of the disclosure.

[0050] Unless otherwise specified, structures depicted herein are also intended to include all stereochemical forms of the structure, i.e., R and S configurations for each asymmetric center. Accordingly, single stereochemical isomers, as well as enantiomeric and diastereomeric mixtures of the present compounds, are within the scope of this disclosure. Unless otherwise specified, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, the replacement of hydrogen by deuterium or tritium, or 13 C or 14 Compounds having the present structure except for the replacement of a carbon by a C-enriched carbon are within the scope of this disclosure. Compounds of this disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, compounds may contain, for example, tritium ( 3 H), iodine-125( 125 I), or carbon-14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0051] It should be noted that throughout this application, alternatives, e.g., each amino acid position containing more than one possible amino acid, are described in terms of a Markush group. It is specifically contemplated that each member of a Markush group should be considered separately, thereby including alternative embodiments, and that a Markush group should not be read as a single unit.

[0052] As used herein, the terms "bioconjugate reactive moiety" and "bioconjugate reactive group" refer to moieties or groups capable of forming a bioconjugate (e.g., a covalent linker) as a result of association between atoms or molecules of the bioconjugate reactive group. The association can be direct or indirect. For example, provided herein, conjugates between a first bioconjugate reactive group (e.g., -NH, -COOH, -N-hydroxysuccinimide, or -maleimide) and a second bioconjugate reactive group (e.g., a sulfhydryl, a sulfur-containing amino acid, an amine, an amine side chain containing amino acid, or a carboxylate) can be direct, e.g., via a covalent bond or linker (e.g., a first linker of a second linker), or indirect, e.g., via a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effect), hydrophobic interactions). In embodiments, bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e., the association of two bioconjugate reactive groups), including, but not limited to, nucleophilic substitution (e.g., reaction of amines and alcohols with acyl halides, active esters), electrophilic substitution (e.g., enamine reaction), and addition across carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed, for example, in March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982.In embodiments, a first bioconjugate reactive group (e.g., a maleimide moiety) is covalently linked to a second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, a first bioconjugate reactive group (e.g., a haloacetyl moiety) is covalently linked to a second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, a first bioconjugate reactive group (e.g., a pyridyl moiety) is covalently linked to a second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, a first bioconjugate reactive group (e.g., an -N-hydroxysuccinimide moiety) is covalently linked to a second bioconjugate reactive group (e.g., an amine). In embodiments, a first bioconjugate reactive group (e.g., a maleimide moiety) is covalently linked to a second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, a first bioconjugate reactive group (eg, a -sulfo-N-hydroxysuccinimide moiety) is covalently attached to a second bioconjugate reactive group (eg, an amine).

[0053] Examples of useful bioconjugate reactive moieties for use in the bioconjugate chemistry herein include: (a) carboxyl groups and various derivatives thereof, including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters; (b) hydroxyl groups that can be converted to esters, ethers, aldehydes, and the like; (c) haloalkyl groups in which the halide can be subsequently replaced with a nucleophilic group, such as an amine, carboxylate anion, thiol anion, carbanion, or alkoxide ion, thereby covalently attaching a new group at the site of the halogen atom; (d) dienophile groups that can participate in Diels-Alder reactions, such as, for example, maleimide or maleimide groups; and (e) subsequent derivatization via the formation of carbonyl derivatives, such as, for example, imines, hydrazones, semicarbazones, or oximes, or via mechanisms such as Grignard addition or alkyllithium addition. (f) sulfonyl halide groups, for example, which can then be reacted with amines to form sulfonamides; (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, bound to metals such as gold, or reacted with maleimides; (h) amine or sulfhydryl groups (e.g., present in cysteine), which can be acylated, alkylated, or oxidized; (i) alkenes, which can undergo, for example, cycloaddition, acylation, Michael addition, etc.; (j) amines and sulfhydryl groups, for example, which can undergo cycloaddition, acylation, Michael addition, etc.; (k) epoxides that can react with hydroxyl compounds, (l) phosphoramidites and other standard functional groups useful in nucleic acid synthesis, (l) metal-silicon oxide linkages, (m) metals attached to reactive phosphorus groups (e.g., phosphines) to form, for example, phosphodiester bonds, (n) azides attached to alkynes using copper-catalyzed cycloaddition click chemistry, (o) biotin conjugates that can react with avidin or streptavidin to form avidin-biotin or streptavidin-biotin complexes.Bioconjugate reactive groups can be selected so that they do not contribute to or interfere with the chemical stability of the conjugates described herein. Alternatively, reactive functional groups can be protected from participating in crosslinking reactions by the presence of protecting groups. In embodiments, bioconjugates include molecular entities resulting from the reaction of an unsaturated bond, such as a maleimide, with a sulfhydryl group.

[0054] "Analog" or "analogue" is used according to its plain and ordinary meaning within chemistry and biology to refer to a compound that is structurally similar to another compound (i.e., a so-called "reference" compound) but differs in composition, e.g., the replacement of one atom with an atom of a different element, or the presence of a particular functional group, or the replacement of one functional group with another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Thus, an analog is a compound that is similar or equivalent in function and appearance to the reference compound, but differs in structure or origin.

[0055] As used herein, the terms "a" or "an" mean one or more. Additionally, as used herein, the phrase "substituted with a[n]" means that the specified group can be substituted with one or more of any or all of the specified substituents. For example, when a group such as an alkyl group or heteroaryl group is "substituted with an unsubstituted C-C 20 When "substituted with alkyl or unsubstituted 2-20 membered heteroalkyl," the group is also substituted with one or more unsubstituted C-C 20 The R group may include alkyl and / or one or more unsubstituted 2- to 20-membered heteroalkyl groups. Furthermore, when a moiety is substituted with an R substituent, the group may be referred to as "R-substituted." When a moiety is R-substituted, the moiety is substituted with at least one R substituent, and each R substituent is optionally different. When a particular R group occurs in a description of a chemical species (such as formula (I)), Roman alphabet symbols may be used to distinguish between each occurrence of that particular R group. For example, multiple R 13 When substituents are present, each R13 The substituents are R 13A , R 13B , R 13C , R 13D etc., and R 13A , R 13B , R 13C , R 13D etc. are R 13 are defined within the definition of and are optionally different.

[0056] A "detectable agent" or "detectable moiety" is a substance, compound, element, molecule, or composition that is detectable by appropriate means, such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. For example, useful detectable agents include: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188Re 189 Re 194 Ir 198 I 199 I 211 And 211 Pb、 212 Hello 212 Pb、 213 Hello 223 Ra 225 Ac、Cr、V、Mn、Fe、Co、Ni、Cu、La、Ce、Pr、Nd、Pm、Sm、Eu、Gd、Tb、Dy、Ho、Er、Tm、Yb、Lu、 32P, fluorophores (such as fluorescent dyes), electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO nanoparticle aggregates, single-crystal iron oxide nanoparticles, single-crystal iron oxide, nanoparticle contrast agents, liposomes, or other delivery vehicles containing gadolinium chelate ("Gd-chelate") molecules, gadolinium, radioisotopes, radionuclides (e.g., carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g., fluorine-18 labeled), any gamma-emitting radionuclide, positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biological colloids , microbubbles (e.g., microbubble shells comprising albumin, galactose, lipids, and / or polymers; microbubble gas cores comprising air, heavy gas(es), perfluorocarbon, nitrogen, octafluoropropane, perflexane lipid microspheres, perflutren, etc.), iodinated contrast agents (e.g., iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins, or other entities that can be made detectable, for example, by incorporating a radioactive label into a peptide or antibody that specifically reacts with a target peptide. The detectable moiety is a monovalent detectable agent or a detectable agent that can form a bond with another composition.

[0057] Radioactive substances (e.g., radioisotopes) that may be used as imaging and / or labeling agents according to embodiments of the present disclosure include: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 77 As, 86 Y, 90 These include, but are not limited to, Y. 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99m Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154-1581 Gd, 161 Tb, 166 Dy, 166 Ho, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Paramagnetic ions that can be used as additional imaging agents in accordance with embodiments of the present disclosure include, but are not limited to, ions of transition metals and lanthanide metals (e.g., metals with atomic numbers 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.

[0058] The description of the compound of the present disclosure is limited by the principles of chemical bonding known to those skilled in the art.Therefore, when group can be substituted with one or more of several substituents, such substitution is selected to comply with the principles of chemical bonding and to bring about a compound that is not inherently unstable and / or is known to those skilled in the art to be likely to be unstable under ambient conditions, such as aqueous, neutral and some known physiological conditions.For example, heterocycloalkyl or heteroaryl is bonded to the rest of the molecule through ring heteroatom according to the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0059] Those of skill in the art will understand that when a variable (e.g., a moiety or linker) of a compound or compound genus (e.g., a genus described herein) is described by the name or formula of a stand-alone compound with all valences satisfied, the unsatisfied valences of that variable will be determined by the context in which the variable is used. For example, when a variable of a compound described herein is connected (e.g., bonded) to the remainder of the compound via a single bond, that variable will be understood to represent the monovalent form of the stand-alone compound (i.e., capable of forming a single bond due to unsatisfied valences). (For example, if a variable is named "methane" in one embodiment, but that variable is known to be connected to the remainder of the compound by a single bond, those of skill in the art will understand that that variable is actually the monovalent form of methane, i.e., methyl or -CH3.) Similarly, linker variables (e.g., L as described herein) will be understood to represent the monovalent form of the stand-alone compound (i.e., capable of forming a single bond due to unsatisfied valences). 1 , L 2 or L 3 ), one of skill in the art will understand that the variable is a divalent form of the stand-alone compound (e.g., if a variable is assigned to "PEG" or "polyethylene glycol" in one embodiment, but that variable is connected to the remainder of the compound by two separate bonds, one of skill in the art will understand that the variable is a divalent (i.e., capable of forming two bonds via two unsatisfied valences) form of PEG, rather than the stand-alone compound PEG.

[0060] The term "pharmaceutically acceptable salts" is intended to include salts of active compounds prepared using relatively non-toxic acids or bases, depending on the specific substituents found in the compounds described herein.When a compound of the present disclosure contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts.When a compound of the present disclosure contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, etc. Also included are amino acid salts such as arginate salts, and organic acid salts such as glucuronic acid or galacturonic acid (see, e.g., Berge et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. Thus, the compounds of the present disclosure can exist as salts with pharmaceutically acceptable acids, etc. The present disclosure includes such salts.Non-limiting examples of such salts include hydrochloride, hydrobromide, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, propionate, tartrate (e.g., (+)-tartrate, (-)-tartrate, or a mixture thereof, including a racemic mixture), succinate, benzoate, and salts with amino acids, such as glutamic acid, and quaternary ammonium salts (e.g., methyl iodide, ethyl iodide, and the like). These salts can be prepared by methods known to those skilled in the art. The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0061] In addition to salt forms, the present disclosure provides compounds in prodrug form. Prodrugs of the compounds described herein are compounds that readily undergo chemical changes under physiological conditions to yield compounds of the present disclosure. Prodrugs of the compounds described herein can be converted in vivo after administration. In addition, prodrugs can be converted to compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment, for example, when contacted with a suitable enzyme or chemical reagent.

[0062] Certain compounds of the present disclosure can exist in non-solvated form and solvated form, including hydrated form.In general, solvated form is equivalent to non-solvated form and is included within the scope of the present disclosure.Certain compounds of the present disclosure can exist in multiple crystalline forms or amorphous forms.In general, all physical forms are equivalent for the use contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0063] "Pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that aid in the administration and absorption of active agents by a subject and can be included in the compositions of the present disclosure without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, saline (such as Ringer's solution), alcohol, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such preparations can be sterilized and, if desired, mixed with auxiliary substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not adversely react with the compounds of the present disclosure. Those of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0064] The term "preparation" is intended to include the combination of an active compound with an encapsulating material as a carrier to provide a capsule, in which the active ingredient, with or without other carriers, is surrounded by the carrier and thus associated with it.Similarly, cachets and lozenges are included.Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0065] As used herein, the term "about" refers to a range of values ​​that includes the specified value and that one of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about refers to within a standard deviation using measurements generally accepted in the art. In embodiments, about refers to a range that covers + / - 10% of the specified value. In embodiments, about includes the specified value.

[0066] "Contacting" is used according to its plain and ordinary meaning to refer to a process that allows at least two different species (e.g., chemical compounds, including biomolecules or cells) to come into sufficient proximity to react, interact, or physically contact. However, it should be understood that the resulting reaction product may be generated directly from the reaction between the added reagents or from an intermediate derived from one or more of the added reagents that may be produced in the reaction mixture. The term "contacting" can include allowing two species to react, interact, or physically contact, and the two species may be a compound described herein and a protein or enzyme. In some embodiments, contacting includes allowing a compound described herein to interact with a protein or enzyme involved in a signal transduction pathway.

[0067] As defined herein, the terms "activation," "activate," "activating," "activator," and the like, with respect to protein-inhibitor interactions, refer to positively affecting (e.g., increasing) the activity or function of a protein compared to the activity or function of the protein in the absence of the activator. In embodiments, activation refers to positively affecting (e.g., increasing) the concentration or level of a protein compared to the concentration or level of the protein in the absence of the activator. These terms may refer to activating, activating, sensitizing, or upregulating signal transduction or enzymatic activity or the amount of a protein that is decreased in a disease. Thus, activation may include, at least in part, partially or fully increasing stimulation, increasing signal transduction or enzymatic activity, or the amount of a protein associated with a disease (e.g., a protein that is decreased in a disease compared to an unaffected control), or enabling activation, or activating, sensitizing, or upregulating signal transduction or enzymatic activity, or the amount of a protein. Activation may include, at least in part, partially or fully increasing stimulation, increasing signal transduction or enzymatic activity, or the amount of a protein, or enabling activation, or activating, sensitizing, or upregulating.

[0068] Terms such as "agonist," "activator," "up-regulator," and the like refer to a substance that can detectably increase the expression or activity of a given gene or protein. An agonist can increase expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control in the absence of the agonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more higher than the expression or activity in the absence of the agonist.

[0069] As defined herein, the terms "inhibition," "inhibit," "inhibiting," and the like, with respect to a protein-inhibitor interaction, refer to adversely affecting (e.g., reducing) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibition refers to negatively affecting (e.g., decreasing) the concentration or level of a protein compared to the concentration or level of the protein in the absence of the inhibitor. In embodiments, inhibition refers to the alleviation of a disease or disease symptom. In embodiments, inhibition refers to the reduction of the activity of a specific protein target. Thus, inhibition includes at least partially or completely blocking a stimulus, reducing, preventing, or delaying activation of signaling or enzymatic activity or the amount of a protein, or inactivating, desensitizing, or downregulating. In embodiments, inhibition refers to the reduction of the activity of a target protein due to a direct interaction (e.g., an inhibitor binds to the target protein). In embodiments, inhibition refers to the reduction of the activity of a target protein due to an indirect interaction (e.g., an inhibitor binds to a protein that activates the target protein, thereby preventing activation of the target protein).

[0070] The terms "inhibitor," "suppressor," "antagonist," or "down-regulator" refer interchangeably to a substance that can detectably reduce the expression or activity of a given gene or protein. Antagonists can reduce expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control in the absence of the antagonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more lower than the expression or activity in the absence of the antagonist.

[0071] The terms "RNF4" and "RING finger protein 4" refer to E3 ligase proteins (including homologs, isoforms, and functional fragments thereof). The terms include any recombinant or naturally occurring form of RNF4 or a variant thereof that maintains RNF4 activity (e.g., within the range of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity compared to wild-type RNF4). In embodiments, the RNF4 protein encoded by the RNF4 gene has an amino acid sequence set forth in or corresponding to Entrez 6047, UniProt P78317, RefSeq(protein) NP_002929, RefSeq(protein) NP_001171939, or RefSeq(protein) NP_001171938. In embodiments, the amino acid or nucleic acid sequence is a sequence known at the time of filing this application. In embodiments, the RNF4 is human RNF4, such as from a human cancer that results in RNF4. In embodiments, the RNF4 has the following sequence: MSTRKRRGGAINSRQAQKRTREATSTPEISLEAEPIELVETAGDEIVDLTCESLEPVVVD LTHNDSVVIVDERRRPRRNARRLPQDHADSCVVSSDDEELSRDRDVYVTTHTPRNARDEG ATGLRPSGTVSCPICMDGYSEIVQNGRLIVSTECGHVFCSQCLRDSLKNANTCPTCRKKI NHKRYHPIYI (SEQ ID NO: 1).

[0072] The terms "RNF114" and "RING finger protein 114" and "ZNF228" and "ZNF313" refer to E3 ligase proteins (including homologs, isoforms, and functional fragments thereof). The terms include any recombinant or naturally occurring form of RNF4 or a variant thereof that maintains RNF114 activity (e.g., within the range of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity compared to wild-type RNF114). In embodiments, the RNF114 protein encoded by the RNF4 gene has an amino acid sequence set forth in or corresponding to Entrez 55905, UniProt Q9Y508, or RefSeq(protein) NP_061153. In embodiments, the amino acid or nucleic acid sequence is a sequence known at the time of filing this application. In embodiments, the RNF114 is human RNF114, such as a human cancer that results in RNF114. In embodiments, the RNF114 has the following sequence: MAAQQRDCGGAAQLAGPAAEAADPLGRFTCPVCLEVYEKPVQVPCGHVFCSACLQECLKPK KPVCGVCRSALAPGVRAVELERQIESTETSCHGCRKNFFLSKIRSHVATCSKYQNYIMEG VKATIKDASLQPRNVPNRYTFPCPYCPEKNFDQEGLVEHCKLFHSTDTKSVVCPICASMP WGDPNYRSANFREHIQRRHRFSYDTFVDYDVDEEDMMNQVLQRSIIDQ (SEQ ID NO: 2).

[0073] The terms "BRD4" and "bromodomain-containing protein 4" refer to a protein that associates with chromosomes during mitosis and plays an important role in cell division and the transmission of epigenetic memory through transcriptional regulation. In embodiments, the BRD4 protein encoded by the BRD4 gene has an amino acid sequence described in or corresponding to Entrez 23476, UniProt O60885, RefSeq(protein)NP_001317313.1, RefSeq(protein)NP_055114.1, or RefSeq(protein)NP_490597.1. In embodiments, the amino acid sequence or nucleic acid sequence is a sequence known at the time of filing of this application. In embodiments, BRD4 has the following sequence: MSAESGPGTRLRNLPVMGDGLETSQMSTTQAQAQPQPANAASTNPPPPETSNPNKPKRQT NQLQYLLRVVLKTLWKHQFAWPFQQPVDAVKLNLPDYYKIIKTPMDMGTIKKRLENNYYW NAQECIQDFNTMFTNCYIYNKPGDDIVLMAEALEKLFLQKINELPTEETEIMIVQAKGRG RGRKETGTAKPGVSTVPNTTQASTPPQTQTPQPNPPPVQATPHPFPAVTPDLIVQTPVMT VVPPQPLQTPPPVPPQPQPPPAPAPQPVQSHPPIIAATPQPVKTKKGVKRKADTTTPTTI DPIHEPPSLPPEPKTTKLGQRRESSRPVKPPKKDVPDSQQHPAPEKSSKVSEQLKCCSGI LKEMFAKKHAAYAWPFYKPVDVEALGLHDYCDIIKHPMDMSTIKSKLEAREYRDAQEFGA DVRLMFSNCYKYNPPDHEVVAMARKLQDVFEMRFAKMPDEPEEPVVAVSSPAVPPPTKVV APPSSSDSSSDSSSDSDSSTDDSEEERAQRLAELQEQLKAVHEQLAALSQPQQNKPKKKE KDKKEKKKEKHKRKEEVEENKKSKAKEPPPKKTKKNNSSNSNVSKKEPAPMKSKPPPTYE SEEEDKCKPMSYEEKRQLSLDINKLPGEKLGRVVHIIQSREPSLKNSNPDEIEIDFETLK PSTLRELERYVTSCLRKKRKPQAEKVDVIAGSSKMKGFSSSESESSSESSSSDSEDSETE MAPKSKKKGHPGREQKKHHHHHHQQMQQAPAPVPQQPPPPPQQPPPPPPPQQQQQPPPPP PPPSMPQQAAPAMKSSPPPFIATQVPVLEPQLPGSVFDPIGHFTQPILHLPQPELPPHLP QPPEHSTPPHLNQHAVVSPPALHNALPQQPSRPSNRAAALPPKPARPPAVSPALTQTPLL PQPPMAQPPQVLLEDEEPPAPPLTSMQMQLYLQQLQKVQPPTPLLPSVKVQSQPPPPLPP PPHPSVQQQLQQQPPPPPPPQPQPPPQQQHQPPPRPVHLQPMQFSTHIQQPPPPQGQQPP HPPPGQQPPPPQPAKPQQVIQHHHSPRHHKSDPYSTGHLREAPSPLMIHSPQMSQFQSLT HQSPPQQNVQPKKQELRAASVVQPQPLVVVKEEKIHSPIIRSEPFSPSLRPEPPKHPESI KAPVHLPQRPEMKPVDVGRPVIRPPEQNAPPPGAPDKDKQKQEPKTPVAPKKDLKIKNMG SWASLVQKHPTTPSSTAKSSSDSFEQFRRAAREKEEREKALKAQAEHAEKEKERLRQERM RSREDEDALEQARRAHEEARRRQEQQQQQRQEQQQQQQQQAAAVAAAATPQAQSSQPQS MLDQQRELARKREQERRRREAMAATIDMNFQSDLLSIFEENLF (SEQ ID NO: 3).

[0074] The term "expression" includes any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting proteins (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0075] The term "modulator" refers to a composition that increases or decreases the level of a target molecule, or the function of a target molecule, or the physical state of a molecular target compared to the absence of the modulator. The term "modulate" is used according to its plain and ordinary meaning and refers to the act of changing or varying one or more properties. "Modulation" refers to the process of changing or varying one or more properties.

[0076] In the context of a substance or activity or function of a substance that is associated with a disease, the term "associated" or "related to" means that the disease is caused (in whole or in part) or the symptoms of the disease are caused (in whole or in part) by the substance or activity or function of the substance.

[0077] As used herein, the term "abnormal" refers to something that is different from normal. When used to describe enzyme activity or protein function, abnormal refers to activity or function that is greater than or less than the average of normal control or normal non-disease control samples. Abnormal activity can refer to the amount of activity that causes disease, and restoring the abnormal activity to a normal or non-disease-related amount (e.g., by administering a compound or using the methods described herein) results in the alleviation of the disease or one or more symptoms.

[0078] As used herein, the term "signaling pathway" refers to a series of interactions between cellular and optionally extracellular components (e.g., proteins, nucleic acids, small molecules, ions, lipids) that can transmit a change in one component to one or more other components, which can then transmit changes to additional components, which are optionally propagated to other signaling pathway components.

[0079] In this disclosure, "comprises," "comprising," "containing," "having," and the like can have the meaning ascribed to them in U.S. patent law and can mean "includes," "including," etc. "Consisting essentially of" or "consists essentially" likewise have the meaning ascribed to them in U.S. patent law, and the term is open-ended and allows for the presence of more than what is recited, but does not include prior art embodiments, so long as the basic or novel characteristics of what is recited are not changed by the presence of more than what is recited.

[0080] The term "disease" or "condition" refers to an existing state or state of health of a patient or subject that can be treated with the compounds or methods provided herein. The disease can be cancer. The disease can be an autoimmune disease. The disease can be an inflammatory disease. The disease can be an infectious disease. In some further examples, "cancer" refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., such as solid and lymphoid cancers, kidney cancer, breast cancer, lung cancer, bladder cancer, colon cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, glioma, esophageal cancer, and liver cancer, such as hepatocellular carcinoma, lymphomas, such as B-acute lymphoblastic lymphoma, non-Hodgkin's lymphoma (e.g., Burkitt's, small cell, and large cell lymphoma), Hodgkin's lymphoma, leukemia (including AML, ALL, and CML), or multiple myeloma.

[0081] As used herein, the term "cancer" refers to all types of cancer, neoplasm, or malignant tumor found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that can be treated with the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease, and non-Hodgkin's lymphoma. Exemplary cancers that can be treated with the compounds or methods provided herein include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, gastric cancer, and uterine cancer. Further examples include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, cutaneous melanoma of the skin, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, squamous cell carcinoma of the head and neck, invasive carcinoma of the breast, lung adenocarcinoma, squamous cell carcinoma of the lung, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulanoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine or exocrine pancreatic neoplasms, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, or prostate cancer.

[0082] The term "leukemia" refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by distorted proliferation and development of white blood cells and their precursor cells in the blood and bone marrow. Leukemias are generally classified clinically based on (1) the duration and character of the disease (acute or chronic), (2) the type of cell involved (bone marrow (myeloid), lymph (lymphoid), or monocytic), and (3) the increased or non-increased number of abnormal cells in the blood (leukemic or non-leukemic). Exemplary leukemias that may be treated with the compounds or methods provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, and non-leukemic leukemia. leukemia), leukemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, fetal leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoblastic leukemia, These include lymphocytic leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, small myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Leder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, and anaplastic cell leukemia.

[0083] As used herein, the term "lymphoma" refers to a group of cancers that affect hematopoietic and lymphatic tissues. It develops primarily in lymphocytes, which are blood cells found in lymph nodes, spleen, thymus, and bone marrow. The two main types of lymphoma are non-Hodgkin's lymphoma and Hodgkin's disease. Hodgkin's disease accounts for approximately 15% of all diagnosed lymphomas. It is a cancer associated with Reed-Sternberg malignant B lymphocytes. Non-Hodgkin's lymphoma (NHL) can be classified based on the rate at which the cancer grows and the type of cells involved. NHL includes aggressive (high-grade) and indolent (low-grade) NHL. Based on the cells involved, NHL includes B-cell and T-cell NHL. Exemplary B-cell lymphomas that can be treated with the compounds or methods provided herein include, but are not limited to, small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranodal (MALT) lymphoma, nodal (monocytic B-cell) lymphoma, splenic lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, immunoblastic large cell lymphoma, or precursor B-lymphoblastic lymphoma. Exemplary T-cell lymphomas that can be treated with the compounds or methods provided herein include, but are not limited to, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma, mycosis fungoides, and precursor T-lymphoblastic lymphoma.

[0084] The term "sarcoma" generally refers to a tumor composed of a substance like embryonic connective tissue and generally made up of tightly packed cells embedded in a fibrous or homogeneous substance. Sarcomas that may be treated with the compounds or methods provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, green sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, and fibroblastic sarcoma. These include sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal osteosarcoma, reticulocytic sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, or telangiectatic sarcoma.

[0085] The term "melanoma" is intended to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that may be treated with the compounds or methods provided herein include, for example, acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, or superficial spreading melanoma.

[0086] The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.Exemplary carcinomas that may be treated with the compounds or methods provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, lobular cell carcinoma, acinic cell carcinoma, adenocell carcinoma, adenoid cystic carcinoma, carcinoma adenomatous carcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioloalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, armor carcinoma, skin carcinoma, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, and compact carcinoma. durum, embryonal carcinoma, encephalomyocellular carcinoma, epidermoid carcinoma, epidermoid carcinoma, tonsillar carcinoma, exophytic carcinoma, ulcer carcinoma, fibrous carcinoma, gelatinous carcinoma, colloid adenocarcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, glassy carcinoma, adrenal carcinoma, infantile embryonal carcinoma, carcinoma in situ, carcinoma in situ, Krompecher's carcinoma, Kulchitzky cell carcinoma, large cell carcinoma, lenticular carcinoma, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, melanoma, soft carcinoma, mucinous carcinoma, mucus-secreting carcinoma muciparum, mucocytic carcinoma, mucoepidermoid carcinoma, carcinoma mucosum, mucosal carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, bony carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, pasty carcinoma, renal cell carcinoma of the kidney, storage cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, spheroid cell carcinoma, spindle cell carcinoma, porotic carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, telangiectatic carcinoma, telangiectatic-like carcinoma, transitional cell carcinoma, nodular carcinoma, carcinoma tuberosum, nodular carcinoma, verrucous carcinoma, and choriocarcinoma.

[0087] As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or part of the body. "Metastatic cancer" is also referred to as "Stage IV cancer."

[0088] The term "treating" or "treatment" refers to any indication of success in treating or ameliorating an injury, disease, pathology, or condition, including any objective or subjective parameter, such as remission, remission; reducing symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; making the end point of degeneration less debilitating; or improving the patient's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric examination, and / or psychiatric evaluation. The term "treating" and its conjugations can include prevention of an injury, pathology, condition, or disease. In embodiments, treating is preventing. In embodiments, treating does not include preventing. As used herein (and as is well understood in the art), "treating" or "treatment" broadly includes any approach to obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results may include, but are not limited to, partial or total, detectable or undetectable alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of disease, stabilization of disease conditions (i.e., not worsening), prevention of disease infection or spread, delay or slowing of disease progression, improvement or relief of disease conditions, reduction in disease recurrence, and remission. In other words, "treatment" as used herein includes any cure, amelioration, or prevention of disease. Treatment may prevent disease from occurring, inhibit the spread of disease, relieve symptoms of disease (e.g., eye pain, seeing halos around lights, bloodshot eyes, very high intraocular pressure), completely or partially eliminate the underlying cause of disease, shorten the duration of disease, or a combination thereof. "Treating" and "treatment" as used herein include prophylactic treatment. Treatment methods include administering a therapeutically effective amount of an active agent to a subject. The administering step may consist of a single administration or may include a series of administrations.The length of the treatment period depends on various factors, such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the composition used for treatment, or a combination thereof. It will also be understood that the effective dosage of the agent used for treatment or prevention may increase or decrease during a particular treatment or prevention regimen. Changes in dosage can be effected or determined by standard diagnostic assays known in the art. In some cases, long-term administration may be required. For example, the composition is administered to the subject in an amount and for a duration sufficient to treat the patient. In embodiments, the treating or treatment is not a preventative treatment.

[0089] The term "preventing" refers to a reduction in the occurrence of disease symptoms in a patient. As noted above, prevention can be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely develop without treatment.

[0090] "Patient" or "subject in need thereof" refers to an organism suffering from or susceptible to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human.

[0091] An "effective amount" is an amount sufficient for a compound to achieve a specified purpose (e.g., achieve an effect in a subject to which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signal transduction pathway, or alleviate one or more symptoms of a disease or condition) compared to the absence of the compound. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or alleviation of a symptom or symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Alleviation" of a symptom(s) (and grammatical equivalents of this phrase) means a reduction in the severity or frequency of the symptom(s), or the elimination of the symptom(s). A "prophylactically effective amount" of a drug is an amount of drug that, when administered to a subject, will produce the intended preventative effect, e.g., an amount of drug that prevents or delays the onset (or recurrence) of an injury, disease, lesion, or condition, or reduces the likelihood of the onset (or recurrence) of an injury, disease, lesion, or condition, or its symptoms. A complete preventative effect does not necessarily occur with the administration of a single dose, but may occur only after the administration of a series of doses. Therefore, a prophylactically effective amount can be administered in one or more doses. As used herein, "activity-reducing amount" refers to the amount of antagonist required to reduce the activity of an enzyme compared to the absence of the antagonist. As used herein, "function-disrupting amount" refers to the amount of antagonist required to disrupt the function of an enzyme or protein compared to the absence of the antagonist. The exact amount depends on the purpose of treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0092] For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. The target concentration will be the concentration of active compound(s) that can achieve the methods described herein, measured using methods described herein or known in the art. As is well known in the art, the therapeutically effective amount for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration found to be effective in animals. The dosage in humans can be adjusted by monitoring the effectiveness of the compound and adjusting the dosage upward or downward, as described above. Adjusting the dosage to achieve maximum efficacy in humans based on the above and other methods is well within the capabilities of one skilled in the art.

[0093] The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent sufficient to improve a disorder, as described above. For example, for a given parameter, a therapeutically effective amount will exhibit an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic effectiveness can also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount can be at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effective than a control.

[0094] Dosage may vary depending on the patient's requirements and the compound used. In the context of the present disclosure, the dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the capabilities of one skilled in the art. Generally, treatment is initiated with a smaller dosage that is less than the optimal dose of the compound. Thereafter, the dosage is increased by small increments until the optimal effect is reached under the circumstances. Dosage amount and interval can be individually adjusted to provide a level of the administered compound that is effective for the specific clinical indication being treated. This will provide a treatment regimen that is appropriate for the severity of the individual's condition.

[0095] As used herein, the term "administering" refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a sustained-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. In embodiments, administration does not include administration of any active agent other than the listed active agents.

[0096] "Co-administration" means that the compositions described herein are administered simultaneously with, immediately before, or immediately after the administration of one or more additional therapeutic agents. The compounds provided herein can be administered alone or simultaneously to a patient. Co-administration is intended to include simultaneous or sequential administration of compounds, individually or in combination (more than one compound). Thus, the preparations may be combined with other active substances, if desired (e.g., to reduce metabolic degradation). The compositions of the present disclosure can be delivered transdermally by topical routes or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0097] As used herein, "cell" refers to a cell that performs metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a specific dye, the ability to produce progeny, or, in the case of gametes, the ability to combine with a second gamete to produce viable progeny. A "stem cell" is a cell characterized by the ability to self-renew through mitotic cell division and the potential to differentiate into tissues or organs. Among mammalian stem cells, embryonic stem cells (ES cells) and somatic stem cells (e.g., HSCs) can be distinguished. Embryonic stem cells exist in blastocysts and give rise to embryonic tissues, while somatic stem cells exist in adult tissues for the purpose of tissue regeneration and repair.

[0098] "Control" or "control experiment" is used according to its plain and ordinary meaning, and refers to an experiment in which an experimental subject or reagent is administered, such as in a parallel experiment, except that it omits the experimental subject, reagent, or variable. In some cases, a control is used as a standard of comparison in evaluating experimental effects. In some embodiments, a control is a measurement of protein activity in the absence of a compound described herein (including the embodiments and examples).

[0099] "Anti-cancer agent" and "anticancer agent" are used according to their plain and ordinary meaning and refer to a composition, compound, drug, antagonist, inhibitor, modulator, peptide, protein, nucleic acid, or molecule that has anti-neoplastic properties or the ability to inhibit cell growth or proliferation. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is an agent identified herein that is useful in methods of treating cancer. In some embodiments, the anti-cancer agent is an agent approved by the FDA or a similar regulatory agency in a country other than the United States to treat cancer. Examples of anticancer agents include antiandrogens (e.g., casodex, flutamide, MDV3100, or ARN-509), MEK (e.g., MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g., XL518, CI-1040, PD035901, selumetinib / AZD6244, GSK1120212 / trametinib, GDC-0973, ARRY-162, ARRY-300), and steroid inhibitors (e.g., steroids, anti-cancer drugs, anti-cancer drugs, anti-inflammatory ... , AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY869766), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, amide, chlorambucil, meifaran), ethyleneimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin), triazenes (decarbazine)), antimetabolites (e.g., 5-azathioprine, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., fluorouracil, floxouridine, cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin, etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel,docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g., cisplatin, oxaloplatin, carboplatin), anthracenediones (e.g., mitoxantrone), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), adrenocortical suppressants (e.g., mitotane, aminoglutethimide), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), mitogen-activated protein kinase signaling inhibitors (e.g., U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY43-9006, wortmannin, or LY294002), mTOR inhibitors, antibodies (e.g., Rituxan), 5-aza-2'-deoxycytidine, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec.RTM.), geldanamycin, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), bortezomib, trastuzumab, anastrozole; angiogenesis inhibitors; antiandrogens,Antiestrogens; Antisense oligonucleotides; Apoptosis gene regulators; Apoptosis regulators; Arginine deaminase; BCR / ABL antagonists; Beta-lactam derivatives; bFGF inhibitors; Bicalutamide; Camptothecin derivatives; Casein kinase inhibitors (ICOS); Clomiphene analogs; Cytarabindacliximab; Dexamethasone; Estrogen agonists; Estrogen antagonists; Etanidazole; Etoposide phosphate; Exemestane; Fadrozole; Finasteride; Fludarabine; Fluorodaunornithine hydrochloride; Gadolinium texaphyrin; Gallium nitrate; Gelatinase inhibitors; Gemcitabine; Glutathione inhibitors; Hepsulfame; Immunostimulant peptides peptide); insulin-like growth factor 1 receptor inhibitors; interferon agonists; interferons; interleukins; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; matrilysin inhibitors; matrix metalloproteinase inhibitors; MIF inhibitors; mifepristone; mismatched double-stranded RNA; monoclonal antibodies; mycobacterial cell wall extracts; nitric oxide modulators; oxaliplatin; panomyphen; pentrazole; phosphatase inhibitors; plasminogen activator inhibitors; platinum complexes; platinum compounds; prednisone; proteasome inhibitors; protein A-based immunomodulators; protein kinase C inhibitors; ta Protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; ras farnesyl-protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; ribozymes; signal transduction inhibitors; signal transduction modulators; single-chain antigen-binding proteins; stem cell inhibitors; stem cell division inhibitors; stromelysin inhibitors; synthetic glycosaminoglycans; tamoxifen methiodide; telomerase inhibitors; thyroid-stimulating hormone; translation inhibitors; tyrosine kinase inhibitors; urokinase receptor antagonists; steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone agonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids (e.g.,prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethrylstilbestrol, ethinyl estradiol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogens (e.g., flutamide), immunostimulants (e.g., bacillus calmette-guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-Pseudomonas exotoxin conjugate, etc.), radioimmunotherapy (e.g., 111 In, 90 Y, or 131anti-CD20 monoclonal antibodies conjugated to I), triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR) targeted therapies or treatments (e.g., Iressa™), erlotinib, Erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™), panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib / BIBW2992, CI-1033 / canertinib, neratinib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib / PF299804, OSI-420 / desmethylerlotinib, AZD8931, AEE788, peritoneal steroids tinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, pyrrolobenzodiazepines (e.g., tomaymycin), carboplatin, CC-1065 and CC-1065 analogs (including amino-CBI), nitrogen mustards (such as chlorambucil and melphalan), dolastatins and dolastatin analogs (including auristatins: e.g., monomethylauristatin E), anthraquinone, These include icrin antibiotics (such as doxorubicin and daunorubicin), duocarmycins and duocarmycin analogs, enediynes (such as neocarzinostatin and calicheamicin), leptomycin derivatives, maytansinoids and maytansinoid analogs (e.g., mertansine), methotrexate, mitomycin C, taxoids, vinca alkaloids (such as vinblastine and vincristine), epothilones (e.g., epothilone B), camptothecin and its clinical analogs topotecan and irinotecan, among others.Not limited to these.

[0100] "Specific," "specifically," "specificity," and the like, of a compound refer to the ability of the compound to cause a particular effect, such as inhibition, on a particular molecular target with minimal or no effect on other proteins in the cell.

[0101] The term "electrophilic chemical moiety" or "electrophilic moiety" is used according to its plain and ordinary chemical meaning to refer to a chemical group (eg, a monovalent chemical group) that is electrophilic.

[0102] The term "irreversible covalent bond" is used according to its plain and ordinary meaning in the art to refer to an association obtained between atoms or molecules that have a low probability of dissociation (e.g., an electrophilic chemical moiety and a nucleophilic moiety). In embodiments, an irreversible covalent bond does not readily dissociate under normal biological conditions. In embodiments, an irreversible covalent bond is formed through a chemical reaction between two species (e.g., an electrophilic chemical moiety and a nucleophilic moiety).

[0103] As used herein, the term "capable of binding" refers to a moiety (e.g., a compound described herein) that can measurably bind to a target (e.g., an E3 ubiquitin ligase binder can form a covalent bond with a cysteine ​​of an E3 ubiquitin ligase). In embodiments where a moiety is capable of binding to a target, the moiety can bind with a Kd of less than about 10 μM, 5 μM, 1 μM, 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 1 nM, or about 0.1 nM.

[0104] As used herein, the term "targeted protein degrader" refers to an agent (e.g., a compound or composition) that can induce proteolysis on a target protein (e.g., a protein of interest). Typically, targeted protein degraders recruit E3 ligases to specific protein targets, allowing the targets to be ubiquitinated and degraded in a proteasome-dependent manner. Because functional inhibition of the target is not required for the efficacy of the degrader, this strategy can potentially target and degrade any protein in the proteome for which a ligand is present (e.g., a targeted protein binder or a targeted protein degrader).

[0105] The term "target protein binding agent" refers to a moiety that can bind to a protein (e.g., a target protein). In embodiments, a target protein binding agent is a molecule or substance that forms a complex with a protein (e.g., a target protein). In embodiments, a target protein binding agent is a monovalent form of a molecule or substance that forms a complex with a protein (e.g., a target protein).

[0106] The term "binder linker" refers to a covalent linker that connects a target protein binder and an E3 ubiquitin ligase binder.

[0107] As used herein, the term "E3 ubiquitin ligase-binding agent" refers to a monovalent agent (e.g., a monovalent compound described herein) that can measurably bind to an E3 ubiquitin ligase (E3 ligase) (e.g., RNF4 or RNF114). For example, an E3 ubiquitin ligase-binding agent is a moiety or monovalent form of a compound having the following formula: [ka] (I) In the formula, R 2 , z2, R 1 , z1, L 3 , and R 4 is as described herein, [ka] (II) In the formula, R 7 , z7, R 5 , R 2 , z2, L 3 , R 4 , R 6 , R 1 and z1 is as described herein, or [ka] (III) In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 is described herein.

[0108] As used herein, the term "covalent cysteine ​​modifier moiety" refers to a monovalent electrophilic moiety capable of measurably binding to a cysteine ​​amino acid. In embodiments, the covalent cysteine ​​modifier moiety binds via an irreversible covalent bond. In embodiments, the covalent cysteine ​​modifier moiety can bind with a Kd of less than about 10 μM, 5 μM, 1 μM, 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 1 nM, or about 0.1 nM.

[0109] II. Compounds In one aspect, a targeted protein degradation agent is provided, comprising 1) a target protein binding agent and 2) an E3 ubiquitin ligase binding agent, wherein the E3 ubiquitin ligase is human RNF4 or human RNF114. In embodiments, the E3 ubiquitin ligase is human RNF4. In embodiments, the E3 ubiquitin ligase is human RNF114. In embodiments, the E3 ubiquitin ligase binding agent can form a covalent bond with a cysteine ​​of the E3 ubiquitin ligase. In embodiments, the target protein binding agent and the E3 ubiquitin ligase binding agent are covalently linked by a binder linker.

[0110] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (I) is a part of a compound having

[0111] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (I)

[0112] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (I) is a monovalent form.

[0113] R 1 are independently halogen, -CX 1 3. -CHX 1 2. -CH2X 1 , -CN, -OR 1D , -C(O)R 1C , -C(O)-OR 1C , -C(O)NR 1A R 1B , -N(O) m1 , -SO n1 R 1D , -SOv1 NR 1A R 1B , -NR 1A R 1B , -NHC(O)NR 1A R 1B , -NR 1A SO2R 1D , -NR 1A C(O)R 1C , -NR 1A C(O)OR 1C , -NR 1A OR 1C , -OCX 1 3. -OCH2X 1 , -OCHX 1 2, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to a binder linker, and two R 1 The substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl. R 2 are independently halogen, -CX 2 3. -CHX 2 2. -CH2X 2 , -CN, -OR 2D , -C(O)R 2C , -C(O)-OR 2C , -C(O)NR 2A R 2B , -N(O) m2 , -SO n2 R 2D , -SO v2 NR 2A R 2B , -NR 2A R 2B , -NHC(O)NR 2A R 2B , -NR 2A SO2R 2D , -NR 2A C(O)R 2C , -NR 2A C(O)OR 2C , -NR2A OR 2C , -OCX 2 3. -OCH2X 2 , -OCHX 2 2, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to a binder linker, and two R 2 The substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl. 3 is a bond, -N(R 3 )-, -C(O)-, -C(O)N(R 3 )-, -N(R 3 )C(O)—, —N(H)—, —C(O)N(H)—, —N(H)C(O)—, —C(O)O—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. R 3 are independently hydrogen, oxo, halogen, -CX 3 3. -CHX 3 2. -CH2X 3 , -CN, -OR 3D , -C(O)R 3C , -C(O)-OR 3C , -C(O)NR 3A R 3B , -N(O) m3 , -SO n3 R 3D , -SO v3 NR 3A R 3B , -NR 3A R 3B , -NHC(O)NR 3A R 3B , -NR 3A SO2R 3D , -NR 3A C(O)R 3C , -NR 3AC(O)OR 3C , -NR 3A OR 3C , -OCX 3 3. -OCH2X 3 , -OCHX 3 2, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to a binder linker. 4 are independently hydrogen, oxo, halogen, -CX 4 3. -CHX 4 2. -CH2X 4 , -CN, -OR 4D , -C(O)R 4C , -C(O)-OR 4C , -C(O)NR 4A R 4B , -N(O) m4 , -SO n4 R 4D , -SO v4 NR 4A R 4B , -NR 4A R 4B , -NHC(O)NR 4A R 4B , -NR 4A SO2R 4D , -NR 4A C(O)R 4C , -NR 4A C(O)OR 4C , -NR 4A OR 4C , -OCX 4 3. -OCH2X 4 , -OCHX 4 2, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or E. E is an electrophilic moiety. z1 is an integer from 0 to 4. z2 is an integer from 0 to 5. R 1A , R 1B , R 1C , R1D , R 2A , R 2B , R 2C , R 2D , R 3A , R 3B , R 3C , R 3D , R 4A , R 4B , R 4C , and R 4D are each independently hydrogen, -CX, -CN, -COOH, -CONH, -CHX, -CHX, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R 1A Substituents and R 1B The substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl, and R 2A Substituents and R 2B The substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl, and R 3A Substituents and R 3B The substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl, and R 4A Substituents and R 4B The substituents may optionally be combined to form a substituted or unsubstituted heterocycloalkyl or a substituted or unsubstituted heteroaryl. Each X, X 1 , X 2 , X 3 , and X 4 are independently -F, -Cl, -Br, or -I. n1, n2, n3, and n4 are independently integers from 0 to 4. m1, m2, m3, m4, v1, v2, v3, and v4 are independently 1 or 2. Only one R 1 , or one R 2 , or one R 3is the bond to the binder linker. In embodiments, R 1 is the bond to the binder linker. In embodiments, R 2 is the bond to the binder linker. In embodiments, R 3 is the bond to the binder linker. In embodiments, one R 1 are independently a bond to a binder linker. In embodiments, one R 2 are independently a bond to a binder linker. In embodiments, one R 3 are independently bonds to the binder linker. 1 , or one R 2 , or one R 3 is the bond to the binder linker.

[0114] In embodiments, R 3 are independently hydrogen, halogen, -CX 3 3. -CHX 3 2. -CH2X 3 , -CN, -OR 3D , -C(O)R 3C , -C(O)-OR 3C , -C(O)NR 3A R 3B , -N(O) m3 , -SO n3 R 3D , -SO v3 NR 3A R 3B , -NR 3A R 3B , -NHC(O)NR 3A R 3B , -NR 3A SO2R 3D , -NR 3A C(O)R 3C , -NR 3A C(O)OR 3C , -NR 3A OR 3C , -OCX 3 3. -OCH2X 3 , -OCHX 32, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to a binder linker. 3A , R 3B , R 3C , R 3D , X 3 , n3, m3, and v3 are as described herein, including the embodiments.

[0115] In embodiments, R 4 are independently hydrogen, halogen, -CX 4 3. -CHX 4 2. -CH2X 4 , -CN, -OR 4D , -C(O)R 4C , -C(O)-OR 4C , -C(O)NR 4A R 4B , -N(O) m4 , -SO n4 R 4D , -SO v4 NR 4A R 4B , -NR 4A R 4B , -NHC(O)NR 4A R 4B , -NR 4A SO2R 4D , -NR 4A C(O)R 4C , -NR 4A C(O)OR 4C , -NR 4A OR 4C , -OCX 4 3. -OCH2X 4 , -OCHX 4 2, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or E. E is an electrophilic moiety. R 4A , R 4B , R4C , R 4D , X 4 , n4, m4, and v4 are as described herein, including the embodiments.

[0116] In embodiments, R 1 are independently halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl 3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to a binder linker, and two R 1 The substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl. 2are independently halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl 3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to a binder linker, and two R 2 The substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl. 3 is a bond, -N(R 3 )-, -C(O)-, -C(O)N(R 3 )-, -N(R 3 )C(O)—, —N(H)—, —C(O)N(H)—, —N(H)C(O)—, —C(O)O—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. In embodiments, R 3are independently hydrogen, oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O) OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCHI, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. 4 are independently hydrogen, oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or E. E is an electrophilic moiety. z1 is an integer from 0 to 4. z2 is an integer from 0 to 5. Only one R 1 or one R 2 is the bond to the binder linker. In embodiments, R 1is the bond to the binder linker. In embodiments, R 2 is the bond to the binder linker.

[0117] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (II) is a part of a compound having

[0118] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (II)

[0119] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (II) is a monovalent form.

[0120] R 1 , R 2 , L 3 , z1, z2 and R 4 is as described herein.

[0121] R 5 and R 6are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, - R is OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to a binder linker. 7 are independently oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OC Cl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to a binder linker, and two R 7The substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl. The symbol z7 is an integer from 0 to 10. In embodiments, only one R 1 , R 2 , R 5 , R 6 or R 7 is independently a bond to a binder linker. In embodiments, R 1 is independently a bond to a binder linker. In embodiments, R 2 is independently a bond to a binder linker. In embodiments, R 3 is independently a bond to a binder linker. In embodiments, R 5 is independently a bond to a binder linker. In embodiments, R 6 is independently a bond to a binder linker. In embodiments, R 7 is independently a bond to a binder linker.

[0122] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (III) is the part of the compound having R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is as described herein. 8 , R 9 and R 10are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, - OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to a binder linker. 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 or R 10 is the bond to the binder linker, [ka] is a single or double bond.

[0123] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (III) is a monovalent compound having R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10is as described herein.

[0124] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (III) is the monovalent form of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 is as described herein.

[0125] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (IIIa) is a monovalent form of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 is as described herein.

[0126] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (III-1) is a monovalent form of R 1 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , z1 and z7 are as described herein. 2ware independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCH F2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C 10 , or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered, or 5-6 membered), or a bond to a binder linker. The symbol z3 is an integer from 0 to 3. Only one R 1 , R 2w , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 or R 10 is the bond to the binder linker, [ka] is a single or double bond.

[0127] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] (IIIa-1) is a monovalent form of R 1 , R 2w , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , z1, z3 and z7 are as described herein.

[0128] In embodiments, R 3 is independently a bond to a binder linker. In embodiments, R 8 is independently a bond to a binder linker. In embodiments, R 9 is independently a bond to a binder linker. In embodiments, R 10 is independently a bond to a binder linker.

[0129] In embodiments, R 2w are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, R 21 substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), R 21 substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), R 21 substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), R 21substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), R substituted or unsubstituted aryl (e.g., C-C 10 or phenyl), or R 21 A substituted or unsubstituted heteroaryl (eg, 5-10 membered, 5-9 membered, or 5-6 membered) or a bond to a binder linker.

[0130] In embodiments, R 2w are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NH NH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substitution (e.g., at least one substituent), size substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted alkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heteroalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted cycloalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heterocycloalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted aryl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heteroaryl, or a bond to a binder linker.

[0131] In embodiments, the substitution R 2w(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) are substituted with at least one substituent, size-limited substituent, or lower substituent, and the substituted R 2w When R is substituted with multiple groups selected from substituents, size-limiting substituents, and lower substituents, each substituent, size-limiting substituent, and / or lower substituent may optionally be different. 2w When substituted, R is substituted with at least one substituent. 2w When substituted, R is substituted with at least one size-limited substituent. 2w When substituted, it is substituted with at least one lower substituent.

[0132] In embodiments, R 2w are independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In embodiments, R 2w are independently hydrogen. In embodiments, R 2w is independently substituted or unsubstituted C1-C4 alkyl. In embodiments, R 2w is independently unsubstituted C1-C4 alkyl. In embodiments, R 2w is independently unsubstituted methyl. In embodiments, R 2w is independently unsubstituted ethyl. In embodiments, R 2w is independently unsubstituted n-propyl. In embodiments, R 2w is independently unsubstituted isopropyl. In embodiments, R 2w is independently unsubstituted n-butyl. In embodiments, R 2w is independently unsubstituted tert-butyl.

[0133] In embodiments, R 3 is independently substituted or unsubstituted C1-C4 alkyl. In embodiments, R 3 is independently unsubstituted C1-C4 alkyl. In embodiments, R 3is independently unsubstituted methyl. In embodiments, R 3 is independently unsubstituted ethyl. In embodiments, R 3 is independently unsubstituted n-propyl. In embodiments, R 3 is independently unsubstituted isopropyl. In embodiments, R 3 is independently unsubstituted n-butyl. In embodiments, R 3 is independently unsubstituted tert-butyl.

[0134] In an embodiment, z3 is 0. In an embodiment, z3 is 1. In an embodiment, z3 is 2. In an embodiment, z3 is 3.

[0135] In embodiments, R 7 is independently substituted or unsubstituted C1-C4 alkyl. In embodiments, R 7 is independently unsubstituted C1-C4 alkyl. In embodiments, R 7 is independently unsubstituted methyl. In embodiments, R 7 is independently unsubstituted ethyl. In embodiments, R 7 is independently unsubstituted n-propyl. In embodiments, R 7 is independently unsubstituted isopropyl. In embodiments, R 7 is independently unsubstituted n-butyl. In embodiments, R 7 is independently unsubstituted tert-butyl.

[0136] In embodiments, R 8are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCH F2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C 10 or phenyl), or substituted or unsubstituted heteroaryl (eg, 5-10 membered, 5-9 membered, or 5-6 membered), or a bond to a binder linker.

[0137] In embodiments, R 8are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)N HNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted (e.g., at least one substituent, cyclohexyl) substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted alkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heteroalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted cycloalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heterocycloalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted aryl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heteroaryl, or a bond to a binder linker.

[0138] In embodiments, the substitution R 8 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) are substituted with at least one substituent, size-limited substituent, or lower substituent, and the substituted R 8 When R is substituted with multiple groups selected from substituents, size-limiting substituents, and lower substituents, each substituent, size-limiting substituent, and / or lower substituent may optionally be different. 8 When substituted, R is substituted with at least one substituent. 8When substituted, R is substituted with at least one size-limited substituent. 8 When substituted, it is substituted with at least one lower substituent.

[0139] In embodiments, R 8 are independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In embodiments, R 8 are independently hydrogen. In embodiments, R 8 is independently substituted or unsubstituted C1-C4 alkyl. In embodiments, R 8 is independently unsubstituted C1-C4 alkyl. In embodiments, R 8 is independently unsubstituted methyl. In embodiments, R 8 is independently unsubstituted ethyl. In embodiments, R 8 is independently unsubstituted n-propyl. In embodiments, R 8 is independently unsubstituted isopropyl. In embodiments, R 8 is independently unsubstituted n-butyl. In embodiments, R 8 is independently unsubstituted tert-butyl.

[0140] In embodiments, R 9are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCH F2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C 10 , or phenyl), or substituted or unsubstituted heteroaryl (eg, 5-10 membered, 5-9 membered, or 5-6 membered), or a bond to a binder linker.

[0141] In embodiments, R 9are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)N HNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted (e.g., at least one substituent, cyclohexyl) substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted alkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heteroalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted cycloalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heterocycloalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted aryl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heteroaryl, or a bond to a binder linker.

[0142] In embodiments, the substitution R 9 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) are substituted with at least one substituent, size-limited substituent, or lower substituent, and the substituted R 9 When R is substituted with multiple groups selected from substituents, size-limiting substituents, and lower substituents, each substituent, size-limiting substituent, and / or lower substituent may optionally be different. 9 When substituted, R is substituted with at least one substituent. 9When substituted, R is substituted with at least one size-limited substituent. 9 When substituted, it is substituted with at least one lower substituent.

[0143] In embodiments, R 9 are independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In embodiments, R 9 are independently hydrogen. In embodiments, R 9 is independently substituted or unsubstituted C1-C4 alkyl. In embodiments, R 9 is independently unsubstituted C1-C4 alkyl. In embodiments, R 9 is independently unsubstituted methyl. In embodiments, R 9 is independently unsubstituted ethyl. In embodiments, R 9 is independently unsubstituted n-propyl. In embodiments, R 9 is independently unsubstituted isopropyl. In embodiments, R 9 is independently unsubstituted n-butyl. In embodiments, R 9 is independently unsubstituted tert-butyl.

[0144] In embodiments, R 10are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCH F2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C 10 , or phenyl), or substituted or unsubstituted heteroaryl (eg, 5-10 membered, 5-9 membered, or 5-6 membered), or a bond to a binder linker.

[0145] In embodiments, R 10are independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)N HNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted (e.g., at least one substituent, cyclohexyl) substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted alkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heteroalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted cycloalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heterocycloalkyl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted aryl, substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) or unsubstituted heteroaryl, or a bond to a binder linker.

[0146] In embodiments, the substitution R 10 (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) are substituted with at least one substituent, size-limited substituent, or lower substituent, and the substituted R 10 When R is substituted with multiple groups selected from substituents, size-limiting substituents, and lower substituents, each substituent, size-limiting substituent, and / or lower substituent may optionally be different. 10 When substituted, R is substituted with at least one substituent. 10When substituted, R is substituted with at least one size-limited substituent. 10 When substituted, it is substituted with at least one lower substituent.

[0147] In embodiments, R 10 are independently hydrogen or substituted or unsubstituted C1-C4 alkyl. In embodiments, R 10 are independently hydrogen. In embodiments, R 10 is independently substituted or unsubstituted C1-C4 alkyl. In embodiments, R 10 is independently unsubstituted C1-C4 alkyl. In embodiments, R 10 is independently unsubstituted methyl. In embodiments, R 10 is independently unsubstituted ethyl. In embodiments, R 10 is independently unsubstituted n-propyl. In embodiments, R 10 is independently unsubstituted isopropyl. In embodiments, R 10 is independently unsubstituted n-butyl. In embodiments, R 10 is independently unsubstituted tert-butyl.

[0148] In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 is as described herein. In embodiments, the E3 ubiquitin ligase binding agent has the formula: [ka] In embodiments, the E3 ubiquitin ligase binding agent is a monovalent form of the...

Claims

1. A targeted protein degradation agent comprising: 1) a target protein binding agent; and 2) an E3 ubiquitin ligase binding agent, wherein the E3 ubiquitin ligase is human RNF4 or human RNF114.

2. The targeted protein degradation agent of claim 1 , wherein the E3 ubiquitin ligase binding agent is capable of forming a covalent bond with a cysteine ​​of an E3 ubiquitin ligase.

3. The targeted protein degradation agent of claim 1 , wherein the target protein binding agent and the E3 ubiquitin ligase binding agent are covalently linked by a binding agent linker.

4. The targeted protein degradation agent according to claim 1 , wherein the E3 ubiquitin ligase is human RNF4.

5. The E3 ubiquitin ligase binding agent has the formula: 【Chemistry 1】 (I) is a monovalent form of During the ceremony, R １ are independently halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to said binder linker, １ the substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R ２ are independently halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to said binder linker, ２ the substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; L ３ is a bond, -N(R ３ )-, -C(O)-, -C(O)N(R ３ ) -, -N(R ３ )C(O)-, -N(H)-, -C(O)N(H)-, -N(H)C(O)-, -C(O)O-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R ３ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R ４ are independently hydrogen, oxo, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or E; E is an electrophilic moiety; z1 is an integer from 0 to 4, z2 is an integer from 0 to 5; The only R １ or one R ２ The targeted protein degradation agent of claim 1 , wherein:

6. R １ are independently halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R １１ Substituted or unsubstituted C １ -C ８ Alkyl, R １１ substituted or unsubstituted 2-8 membered heteroalkyl, R １１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R １１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R １１ Substituted or unsubstituted C ６ -C １０ Aryl, or R １１ a substituted or unsubstituted 5-10 membered heteroaryl or a bond to the binder linker, １ The substituents are optionally joined to R １１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R １１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R １１ Substituted or unsubstituted C ６ -C １０ Aryl, or R １１ may form a substituted or unsubstituted 5-10 membered heteroaryl; R １１ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R １２ Substituted or unsubstituted C １ -C ８ Alkyl, R １２ substituted or unsubstituted 2-8 membered heteroalkyl, R １２ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R １２ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R １２ Substituted or unsubstituted C ６ -C １０ Aryl, or R １２ is a substituted or unsubstituted 5-10 membered heteroaryl; R １２ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , unsubstituted C １ -C ８ Alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C ３ -C ８ Cycloalkyl, unsubstituted 3-8 membered heterocycloalkyl, unsubstituted C ６ -C １０ aryl, or unsubstituted 5-10 membered heteroaryl; R ２ are independently halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ２１ Substituted or unsubstituted C １ -C ８ Alkyl, R ２１ substituted or unsubstituted 2-8 membered heteroalkyl, R ２１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ２１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ２１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ２１ a substituted or unsubstituted 5-10 membered heteroaryl or a bond to the binder linker, ２ The substituents are optionally joined to R ２１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ２１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ２１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ２１ may form a substituted or unsubstituted 5-10 membered heteroaryl; R ２１ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ２２ Substituted or unsubstituted C １ -C ８ Alkyl, R ２２ substituted or unsubstituted 2-8 membered heteroalkyl, R ２２ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ２２ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ２２ Substituted or unsubstituted C ６ -C １０ Aryl, or R ２２ is a substituted or unsubstituted 5-10 membered heteroaryl; R ２２ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , unsubstituted C １ -C ８ Alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C ３ -C ８ Cycloalkyl, unsubstituted 3-8 membered heterocycloalkyl, unsubstituted C ６ -C １０ aryl, or unsubstituted 5-10 membered heteroaryl; L ３ is a bond, -N(R ３ )-, -C(O)-, -C(O)N(R ３ ) -, -N(R ３ )C(O)-, -N(H)-, -C(O)N(H)-, -N(H)C(O)-, -C(O)O-, R ３ Substituted or unsubstituted C １ -C ８ Alkylene, R ３ substituted or unsubstituted 2-8 membered heteroalkylene, R ３ Substituted or unsubstituted C ３ -C ８ Cycloalkylene, R ３ Substituted or unsubstituted 3- to 8-membered heterocycloalkylene, R ３ Substituted or unsubstituted C ６ -C １０ Arylene, or R ３ is a substituted or unsubstituted 5- to 10-membered heteroarylene; R ３ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ３１ Substituted or unsubstituted C １ -C ８ Alkyl, R ３１ substituted or unsubstituted 2-8 membered heteroalkyl, R ３１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ３１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ３１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ３１ is a substituted or unsubstituted 5-10 membered heteroaryl; R ３１ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ３２ Substituted or unsubstituted C １ -C ８ Alkyl, R ３２ substituted or unsubstituted 2-8 membered heteroalkyl, R ３２ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ３２ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ３２ Substituted or unsubstituted C ６ -C １０ Aryl, or R ３２ is a substituted or unsubstituted 5-10 membered heteroaryl; R ３２ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , unsubstituted C １ -C ８ Alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C ３ -C ８ Cycloalkyl, unsubstituted 3-8 membered heterocycloalkyl, unsubstituted C ６ -C １０ aryl, or unsubstituted 5-10 membered heteroaryl; R ４ are independently hydrogen, oxo, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ４１ Substituted or unsubstituted C １ -C ８ Alkyl, R ４１ substituted or unsubstituted 2-8 membered heteroalkyl, R ４１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ４１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ４１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ４１ substituted or unsubstituted 5-10 membered heteroaryl, or E; R ４１ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ４２ Substituted or unsubstituted C １ -C ８ Alkyl, R ４２ substituted or unsubstituted 2-8 membered heteroalkyl, R ４２ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ４２ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ４２ Substituted or unsubstituted C ６ -C １０ Aryl, or R ４２ is a substituted or unsubstituted 5-10 membered heteroaryl; R ４２ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , unsubstituted C １ -C ８ Alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C ３ -C ８ Cycloalkyl, unsubstituted 3-8 membered heterocycloalkyl, unsubstituted C ６ -C １０ The targeted protein degrading agent of claim 1, which is aryl, or unsubstituted 5-10 membered heteroaryl.

7. R ２ are independently halogen, -CF ３ , -NO ２ , R ２１ Substituted or unsubstituted C １ -C ３ alkyl, unsubstituted 2-3 membered heteroalkyl, or a bond to the binder linker; ２ the substituents may optionally be combined to form an unsubstituted phenyl; R ２１ The targeted protein degrading agent of claim 6, wherein is independently -OH.

8. z2 is 1, R ２ The targeted protein degradation agent of claim 6 , wherein:

9. R １ The targeted protein degrading agent of claim 6 , wherein is independently a halogen.

10. The targeted protein degrading agent of claim 6 , wherein z1 is 0.

11. L ３ -N(R ３ ) - and R ３ is independent, R ３１ substituted methyl, unsubstituted phenyl, or unsubstituted 5-6 membered heteroaryl; R ３１ The targeted protein decomposition agent of claim 6, wherein is independently unsubstituted phenyl or unsubstituted 5- to 6-membered heteroaryl.

12. E. 【Chemistry 2】 and R １５ , R １６ and R １７ are independently hydrogen, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; X １７ The targeted protein degrading agent of claim 6 , wherein is a halogen.

13. E. 【Chemistry 3】 and R １５ , R １６ and R １７ The targeted protein degrading agent of claim 6 , wherein is independently hydrogen.

14. E. 【Chemistry 4】 And X １７ The targeted protein degrading agent of claim 6, wherein each of the groups is independently -Cl.

15. The targeted protein degradation agent of claim 1 , wherein the E3 ubiquitin ligase is human RNF114.

16. The E3 ubiquitin ligase binding agent has the formula: 【Chemistry 5】 (II) is a monovalent form of During the ceremony, R １ and R ２ are independently halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to said binder linker, １ Substituent or two R ２ the substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R ５ and R ６ are independently hydrogen, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to said binder linker; R ７ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to said binder linker, ７ the substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; L ３ is a bond, -N(R ３ )-, -C(O)-, -C(O)N(R ３ ) -, -N(R ３ )C(O)-, -N(H)-, -C(O)N(H)-, -N(H)C(O)-, -C(O)O-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R ３ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R ４ are independently hydrogen, oxo, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or E; E is an electrophilic moiety; z1 is an integer from 0 to 4, z2 is an integer from 0 to 5; z7 is an integer from 0 to 10, The only R １ , R ２ , R ５ , R ６ Or R ７ The targeted protein degradation agent of claim 1 , wherein:

17. R １ , R ２ and R ７ are independently halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ５１ Substituted or unsubstituted C １ -C ８ Alkyl, R ５１ substituted or unsubstituted 2-8 membered heteroalkyl, R ５１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ５１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ５１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ５１ a substituted or unsubstituted 5-10 membered heteroaryl or a bond to the binder linker, １ Substituent or two R ２ The substituents are optionally joined to R ５１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ５１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ５１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ５１ may form a substituted or unsubstituted 5-10 membered heteroaryl; R ５ and R ６ are independently hydrogen, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ５１ Substituted or unsubstituted C １ -C ８ Alkyl, R ５１ substituted or unsubstituted 2-8 membered heteroalkyl, R ５１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ５１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ５１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ５１ a substituted or unsubstituted 5-10 membered heteroaryl or a bond to said binder linker; R ７ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ５１ Substituted or unsubstituted C １ -C ８ Alkyl, R ５１ substituted or unsubstituted 2-8 membered heteroalkyl, R ５１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ５１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ５１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ５１ a substituted or unsubstituted 5-10 membered heteroaryl or a bond to the binder linker, ７ The substituents are optionally joined to R ５１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ５１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ５１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ５１ may form a substituted or unsubstituted 5-10 membered heteroaryl; R ５１ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , unsubstituted C １ -C ８ Alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C ３ -C ８ Cycloalkyl, unsubstituted 3-8 membered heterocycloalkyl, unsubstituted C ６ -C １０ aryl, or unsubstituted 5-10 membered heteroaryl; L ３ is a bond, -N(R ３ )-, -C(O)-, -C(O)N(R ３ ) -, -N(R ３ )C(O)-, -N(H)-, -C(O)N(H)-, -N(H)C(O)-, -C(O)O-, R ３ Substituted or unsubstituted C １ -C ８ Alkylene, R ３ substituted or unsubstituted 2-8 membered heteroalkylene, R ３ Substituted or unsubstituted C ３ -C ８ Cycloalkylene, R ３ Substituted or unsubstituted 3- to 8-membered heterocycloalkylene, R ３ Substituted or unsubstituted C ６ -C １０ Arylene, or R ３ is a substituted or unsubstituted 5- to 10-membered heteroarylene; R ３ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ３１ Substituted or unsubstituted C １ -C ８ Alkyl, R ３１ substituted or unsubstituted 2-8 membered heteroalkyl, R ３１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ３１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ３１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ３１ is a substituted or unsubstituted 5-10 membered heteroaryl; R ３１ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ３２ Substituted or unsubstituted C １ -C ８ Alkyl, R ３２ substituted or unsubstituted 2-8 membered heteroalkyl, R ３２ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ３２ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ３２ Substituted or unsubstituted C ６ -C １０ Aryl, or R ３２ is a substituted or unsubstituted 5-10 membered heteroaryl; R ３２ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , unsubstituted C １ -C ８ Alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C ３ -C ８ Cycloalkyl, unsubstituted 3-8 membered heterocycloalkyl, unsubstituted C ６ -C １０ aryl, or unsubstituted 5-10 membered heteroaryl; R ４ are independently hydrogen, oxo, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ４１ Substituted or unsubstituted C １ -C ８ Alkyl, R ４１ substituted or unsubstituted 2-8 membered heteroalkyl, R ４１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ４１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ４１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ４１ substituted or unsubstituted 5-10 membered heteroaryl, or E; R ４１ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ４２ Substituted or unsubstituted C １ -C ８ Alkyl, R ４２ substituted or unsubstituted 2-8 membered heteroalkyl, R ４２ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ４２ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ４２ Substituted or unsubstituted C ６ -C １０ Aryl, or R ４２ is a substituted or unsubstituted 5-10 membered heteroaryl; R ４２ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , unsubstituted C １ -C ８ Alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C ３ -C ８ Cycloalkyl, unsubstituted 3-8 membered heterocycloalkyl, unsubstituted C ６ -C １０ The targeted protein degrading agent of claim 16, which is aryl, or unsubstituted 5- to 10-membered heteroaryl.

18. The E3 ubiquitin ligase binding agent has the formula: 【Chemistry 6】 The targeted protein degrading agent of claim 17, which is a monovalent form of the agent.

19. L ３ Ga-CH ２ The targeted protein degrading agent of claim 17, which is NH-.

20. E. 【Chemistry 7】 and R １５ , R １６ and R １７ are independently hydrogen, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; X １７ The targeted protein degrading agent of claim 17 , wherein is a halogen.

21. E. 【Chemistry 8】 and R １５ , R １６ and R １７ The targeted protein degrading agent of claim 17 , wherein is independently hydrogen.

22. E. 【Chemistry 9】 and R １５ , R １６ and R １７ The targeted protein degrading agent of claim 17 , wherein is independently hydrogen.

23. The E3 ubiquitin ligase binding agent has the formula: 【Chemistry 10】 is a monovalent form of ( １ 、| ２ 、| ３ 、| ４ 、| ５ 、| ６ 、| ７ 、| ８ 、| ９ および| １０ The newspaper updated picture on pi. ３ 、｢r ３ 、!F ３ 、|| ３ 、!Cl ２ 、!Br ２ 、!HF ２ 、!HI ２ 、!| ２ 、、|| ２ 〒、|| ２ F、!H ２ !!||、!H、!+H ２ 、!!!!@\H ２ 、!﯁ ２ 、-SH、SO ３ 、+SO ４ 、+SO ２ . ２ 、!\H ２ 、!NH ２ 、!\\\\|| ２ 、!\\HHH ２ 、!!O ２ 、!\C\foCPH、\B\B ３ 、!C ３ 、Cr ３ 、!CI ３ 、C\l ２ 、!Cr ２ 、!CII ２ 、!CF ２ 、!CH ２ 、、!C ２ funerally, ‐flow,, ２ I、!CH ２ F、N ３ 、SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or a bond to said binder linker; The only R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ Or R １０ is the bond to the binder linker, 【Chemistry 11】 The targeted protein degrading agent of claim 1 , wherein is a single bond or a double bond.

24. R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ and R １０ are independently hydrogen, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , R ５１ Substituted or unsubstituted C １ -C ８ Alkyl, R ５１ substituted or unsubstituted 2-8 membered heteroalkyl, R ５１ Substituted or unsubstituted C ３ -C ８ Cycloalkyl, R ５１ substituted or unsubstituted 3- to 8-membered heterocycloalkyl, R ５１ Substituted or unsubstituted C ６ -C １０ Aryl, or R ５１ a substituted or unsubstituted 5-10 membered heteroaryl or a bond to said binder linker; The only R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ Or R １０ is the bond to the binder linker, R ５１ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , unsubstituted C １ -C ８ Alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C ３ -C ８ Cycloalkyl, unsubstituted 3-8 membered heterocycloalkyl, unsubstituted C ６ -C １０ The targeted protein degrading agent of claim 23, which is aryl, or unsubstituted 5-10 membered heteroaryl.

25. The E3 ubiquitin ligase binding agent has the formula: 【Chemistry 12】 The targeted protein degradation agent of claim 23, which is a monovalent form of:

26. The E3 ubiquitin ligase binding agent has the formula: 【Chemistry 13】 The targeted protein degradation agent of claim 24, which is a monovalent form of:

27. The E3 ubiquitin ligase binding agent has the formula: 【Chemistry 14】 The targeted protein degradation agent of claim 24, which is a monovalent form of:

28. The E3 ubiquitin ligase binding agent has the formula: 【Chemistry 15】 The targeted protein degradation agent of claim 24 ,

29. The E3 ubiquitin ligase binding agent has the formula: 【Chemistry 16】 The targeted protein degradation agent of claim 24 ,

30. The binder linker is L １１ -L １２ -L １３ -L １４ and L １１ directly connects to the E3 ubiquitin ligase binder; L １１ But -N(R ６１ )-, -C(O)-, -C(O)N(R ６１ ) -, -N(R ６１ )C(O)-, -N(H)-, -C(O)N(H)-, -N(H)C(O)-, -C(O)O-, -OC(O)-, -S(O) ２ -, -S(O)-, -O-, -S-, -NHC(O)NH-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, or a bioconjugate linker; L １２ , L １３ and L １４ are independently a bond, -N(R ６１ )-, -C(O)-, -C(O)N(R ６１ ) -, -N(R ６１ )C(O)-, -N(H)-, -C(O)N(H)-, -N(H)C(O)-, -C(O)O-, -OC(O)-, -S(O) ２ The targeted protein degradation agent of claim 3, which is -, -S(O)-, -O-, -S-, -NHC(O)NH-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, or a bioconjugate linker.

31. L １１ But -N(R ６１ )-, -C(O)-, -C(O)N(R ６１ ) -, -N(R ６１ )C(O)-, -N(H)-, -C(O)N(H)-, -N(H)C(O)-, -C(O)O-, -OC(O)-, -S(O) ２ -, -S(O)-, -O-, -S-, -NHC(O)NH-, R ６１ Substituted or unsubstituted C １ -C ２０ Alkylene, R ６１ substituted or unsubstituted 2- to 20-membered heteroalkylene, R ６１ Substituted or unsubstituted C ３ -C ８ Cycloalkylene, R ６１ Substituted or unsubstituted 3- to 8-membered heterocycloalkylene, R ６１ Substituted or unsubstituted C ６ -C １０ Arylene, R ６１ a substituted or unsubstituted 5-10 membered heteroarylene, or a bioconjugate linker; L １２ , L １３ and L １４ are independently a bond, -N(R ６１ )-, -C(O)-, -C(O)N(R ６１ ) -, -N(R ６１ )C(O)-, -N(H)-, -C(O)N(H)-, -N(H)C(O)-, -C(O)O-, -OC(O)-, -S(O) ２ -, -S(O)-, -O-, -S-, -NHC(O)NH-, R ６１ Substituted or unsubstituted C １ -C ２０ Alkylene, R ６１ substituted or unsubstituted 2- to 20-membered heteroalkylene, R ６１ Substituted or unsubstituted C ３ -C ８ Cycloalkylene, R ６１ Substituted or unsubstituted 3- to 8-membered heterocycloalkylene, R ６１ Substituted or unsubstituted C ６ -C １０ Arylene, R ６１ a substituted or unsubstituted 5-10 membered heteroarylene, or a bioconjugate linker; R ６１ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , unsubstituted C １ -C ８ Alkyl, unsubstituted 2-8 membered heteroalkyl, unsubstituted C ３ -C ８ Cycloalkyl, unsubstituted 3-8 membered heterocycloalkyl, unsubstituted C ６ -C １０ The targeted protein degrading agent of claim 30, which is aryl, or unsubstituted 5- to 10-membered heteroaryl.

32. The targeted protein degradation agent of claim 1 , wherein the target protein binding agent is capable of binding to a target protein associated with a disease.

33. The target protein binding agent 【Chemistry 17】 The targeted protein degradation agent of claim 1 , wherein the target protein binding agent binds to BRD4.

34. A pharmaceutical composition comprising the targeted protein degrading agent of any one of claims 1 to 33 and a pharma- ceutically acceptable excipient.

35. 2. A method for reducing the level of a cellular protein, comprising contacting the cellular protein with a targeted protein degrading agent.

36. A) ubiquitinating the cellular protein that is contacted with the targeted protein degradation agent, thereby forming a ubiquitinated cellular protein; B) contacting the ubiquitinated cellular protein with a proteasome, thereby forming a ubiquitinated cellular protein-proteasome complex; The method of claim 35, further comprising the step of: C) proteolyzing the cellular protein by the proteasome.

37. 36. The method of claim 35, wherein the E3 ubiquitin ligase binding agent contacts an E3 ubiquitin ligase cysteine ​​residue.

38. 38. The method of claim 37, wherein the E3 ubiquitin ligase binding agent covalently binds to an E3 ubiquitin ligase cysteine ​​residue.

39. 38. The method of claim 37, wherein the E3 ubiquitin ligase is human RNF4.

40. 40. The method of claim 39, wherein the cysteine ​​residue is C132 of human RNF4.

41. 40. The method of claim 39, wherein the cysteine ​​residue is C135 of human RNF4.

42. 38. The method of claim 37, wherein the E3 ubiquitin ligase is human RNF114.

43. 43. The method of claim 42, wherein the cysteine ​​residue is C8 of human RNF114.

44. 36. The method of claim 35, wherein the E3 ubiquitin ligase-binding agent contacts an amino acid of the E3 ubiquitin ligase that lacks a stable secondary or tertiary conformational structure in the absence of the E3 ubiquitin ligase-binding agent.

45. 45. The method of claim 44, wherein the amino acid that contacts the E3 ubiquitin ligase-binding agent adopts a stable secondary or tertiary conformational structure upon contact with the E3 ubiquitin ligase-binding agent.

46. 34. A method of reducing the level of a cellular protein, comprising contacting the cellular protein with a targeted protein degradation agent according to any one of claims 1 to 33.

47. A) ubiquitinating the cellular protein that is contacted with the targeted protein degradation agent, thereby forming a ubiquitinated cellular protein; B) contacting the ubiquitinated cellular protein with a proteasome, thereby forming a ubiquitinated cellular protein-proteasome complex; The method of claim 46, further comprising the step of: C) proteolyzing the cellular protein by the proteasome.

48. 47. The method of claim 46, wherein the E3 ubiquitin ligase binding agent contacts an E3 ubiquitin ligase cysteine ​​residue.

49. 49. The method of claim 48, wherein the E3 ubiquitin ligase binding agent covalently binds to an E3 ubiquitin ligase cysteine ​​residue.

50. 49. The method of claim 48, wherein the E3 ubiquitin ligase is human RNF4.

51. 51. The method of claim 50, wherein the cysteine ​​residue is C132 of human RNF4.

52. 51. The method of claim 50, wherein the cysteine ​​residue is C135 of human RNF4.

53. 49. The method of claim 48, wherein the E3 ubiquitin ligase is human RNF114.

54. 54. The method of claim 53, wherein the cysteine ​​residue is C8 of human RNF114.

55. 47. The method of claim 46, wherein the E3 ubiquitin ligase-binding agent contacts an amino acid of the E3 ubiquitin ligase that lacks a stable secondary or tertiary conformational structure in the absence of the E3 ubiquitin ligase-binding agent.

56. 56. The method of claim 55, wherein the amino acid that contacts the E3 ubiquitin ligase-binding agent adopts a stable secondary or tertiary conformational structure upon contact with the E3 ubiquitin ligase-binding agent.

57. A method of treating cancer, comprising contacting a cellular protein associated with the cancer with a targeted proteolytic agent.

58. A method for treating cancer, comprising administering a therapeutically effective amount of a targeted protein degradation agent according to any one of claims 1 to 33 to a subject in need thereof.

59. 1. A method for reducing a level of a cellular protein, comprising contacting the cellular protein with a targeted protein degrading agent, thereby forming a targeted protein degrading agent-cellular protein complex, wherein the targeted protein degrading agent: i) an E3 ubiquitin ligase binding agent; and ii) a target protein binding agent; and iii) a binder linker directly attached to said E3 ubiquitin ligase binding agent and said target protein binding agent.

60. 60. The method of claim 59, wherein the E3 ubiquitin ligase binding agent contacts an E3 ubiquitin ligase cysteine ​​residue.

61. 61. The method of claim 60, wherein the E3 ubiquitin ligase binding agent covalently binds to an E3 ubiquitin ligase cysteine ​​residue.

62. 61. The method of claim 60, wherein the E3 ubiquitin ligase is human RNF4.

63. 63. The method of claim 62, wherein the cysteine ​​residue is C132 of human RNF4.

64. 63. The method of claim 62, wherein the cysteine ​​residue is C135 of human RNF4.

65. 61. The method of claim 60, wherein the E3 ubiquitin ligase is human RNF114.

66. 66. The method of claim 65, wherein the cysteine ​​residue is C8 of human RNF114.

67. 60. The method of claim 59, wherein the E3 ubiquitin ligase-binding agent contacts an amino acid of the E3 ubiquitin ligase that lacks a stable secondary or tertiary conformational structure in the absence of the E3 ubiquitin ligase-binding agent.

68. 68. The method of claim 67, wherein the amino acid that contacts the E3 ubiquitin ligase-binding agent adopts a stable secondary or tertiary conformational structure upon contact with the E3 ubiquitin ligase-binding agent.

69. 60. The method of claim 59, wherein prior to said contacting, the targeted protein degrader is synthesized by covalently reacting an E3 ubiquitin ligase binder, a binder linker and a target protein binder to generate the targeted protein degrader.

70. 70. The method of claim 69, wherein prior to said synthesis, said E3 ubiquitin ligase binding agent is identified from candidate E3 ubiquitin ligase binding agents.

71. the E3 ubiquitin ligase binding agent i) contacting an E3 ubiquitin ligase protein with a mixture of candidate E3 ubiquitin ligase binders, thereby forming an E3 ubiquitin ligase protein-E3 ubiquitin ligase binder complex; ii) identifying the candidate E3 ubiquitin ligase binding agent as an E3 ubiquitin ligase binding agent from an E3 ubiquitin ligase protein-E3 ubiquitin ligase binding agent complex.

72. 72. The method of claim 71, wherein the candidate E3 ubiquitin ligase binding agent comprises a covalently bound cysteine ​​modifier moiety, and the candidate E3 ubiquitin ligase binding agent is identified as the E3 ubiquitin ligase binding agent by detection of covalent binding of the E3 ubiquitin ligase protein to form an E3 ubiquitin ligase protein-E3 ubiquitin ligase binding agent complex.

73. 73. The method of claim 72, wherein said detecting the E3 ubiquitin ligase protein-E3 ubiquitin ligase binding agent complex comprises the use of a detectable label or mass spectrometry.

74. 60. The method of claim 59, wherein prior to said synthesis, said target protein binding agents are identified.

75. the target protein binding agent comprising: i) contacting a cellular protein with a mixture of candidate target protein binding agents, thereby forming a cellular protein-target protein binding agent complex; ii) identifying the target protein-binding agents that form a cellular protein-target protein-binding agent complex.

76. 76. The method of claim 75, wherein the candidate target protein-binding agent that forms a cellular protein-target protein-binding agent complex is identified as a target protein-binding agent by detection of the cellular protein-target protein-binding agent complex.

77. 77. The method of claim 76, wherein said detecting the cellular protein-target protein binding agent complex comprises the use of a detectable label or mass spectrometry.

78. 60. The method of claim 59, wherein the E3 ubiquitin ligase binding agent is modified to remove a covalent cysteine ​​modifier moiety prior to said synthesis.

79. 1. A method for identifying a cellular protein contacted by a target protein-binding agent, comprising: A) contacting a first sample of a cellular proteome or cells with said target protein binding agent, thereby forming a cellular protein-target protein binding agent complex; B) both the first sample of cellular proteome or cells obtained in step A and a second sample of cellular proteome or cells not contacted with said target protein binding agent are subjected to a quantification reaction according to the formula: 【Chemistry 18】 and contacting the compound having the formula: C) contacting the obtained first sample of step B with a first detectable agent and contacting the obtained second sample of step B with a second detectable agent; D) measuring the level of the first detectable agent and the second detectable agent bound to the selected protein; E) identifying the cellular protein in the cellular protein-target protein binding agent complex by measuring the difference in the levels of the first detectable agent and the second detectable agent bound to the cellular protein capable of forming a cellular protein-target protein binding agent complex.

80. A) one of the first or second detectable agents comprises a light isotope of an atom, and the other of the first or second detectable agent comprises a heavy isotope of the atom; B) the levels of the first detectable agent and the second detectable agent of a selected protein are measured by liquid chromatography mass spectrometry; C) the cellular protein capable of binding to the target protein-binding agent is identified by a difference between the level of the cellular protein bound to the first detectable agent and the level of the cellular protein bound to the second detectable agent.

81. 80. The method of claim 79, wherein the E3 ubiquitin ligase binding agent contacts an E3 ubiquitin ligase cysteine ​​residue.

82. 82. The method of claim 81 , wherein the E3 ubiquitin ligase binding agent covalently binds to an E3 ubiquitin ligase cysteine ​​residue.

83. 82. The method of claim 81, wherein the E3 ubiquitin ligase is human RNF4.

84. 84. The method of claim 83, wherein the cysteine ​​residue is C132 of human RNF4.

85. 84. The method of claim 83, wherein the cysteine ​​residue is C135 of human RNF4.

86. 82. The method of claim 81, wherein the E3 ubiquitin ligase is human RNF114.

87. 87. The method of claim 86, wherein the cysteine ​​residue is C8 of human RNF114.

88. 80. The method of claim 79, wherein the E3 ubiquitin ligase-binding agent contacts an amino acid of the E3 ubiquitin ligase that lacks a stable secondary or tertiary conformational structure in the absence of the E3 ubiquitin ligase-binding agent.

89. 89. The method of claim 88, wherein the amino acid that contacts the E3 ubiquitin ligase-binding agent adopts a stable secondary or tertiary conformational structure upon contact with the E3 ubiquitin ligase-binding agent.

90. 1. A method of producing an E3 ubiquitin ligase-E3 ubiquitin ligase-binding agent-cellular protein complex, comprising: A) contacting the E3 ubiquitin ligase with an E3 ubiquitin ligase-binding agent, thereby forming an E3 ubiquitin ligase-E3 ubiquitin ligase-binding agent complex; B) contacting the E3 ubiquitin ligase-E3 ubiquitin ligase binding agent complex with the cellular protein, thereby forming an E3 ubiquitin ligase-E3 ubiquitin ligase binding agent-cellular protein complex.

91. 91. The method of claim 90, wherein the E3 ubiquitin ligase binding agent covalently binds a cysteine ​​of the E3 ubiquitin ligase.

92. 91. The method of claim 90, wherein the E3 ubiquitin ligase is human RNF4.

93. 1. A method for producing a cellular protein-E3 ubiquitin ligase binding agent-E3 ubiquitin ligase complex, comprising: A) contacting the cellular protein with an E3 ubiquitin ligase binding agent, thereby forming a cellular protein-E3 ubiquitin ligase binding agent complex; B) contacting the cellular protein-E3 ubiquitin ligase binding agent complex with the E3 ubiquitin ligase, thereby forming a cellular protein-E3 ubiquitin ligase binding agent-E3 ubiquitin ligase complex.

94. 94. The method of claim 93, wherein the E3 ubiquitin ligase binding agent covalently binds a cysteine ​​of the E3 ubiquitin ligase.

95. 94. The method of claim 93, wherein the E3 ubiquitin ligase is human RNF4.

96. A method for inhibiting formation of a cellular protein-E3 ubiquitin ligase complex, comprising contacting said E3 ubiquitin ligase with an E3 ubiquitin ligase-binding agent, thereby inhibiting said formation of a cellular protein-E3 ubiquitin ligase complex.

97. 97. The method of claim 96, wherein the E3 ubiquitin ligase binding agent covalently binds a cysteine ​​of the E3 ubiquitin ligase.

98. 97. The method of claim 96, wherein the E3 ubiquitin ligase-binding agent contacts the E3 ubiquitin ligase at an amino acid of the E3 ubiquitin ligase that is capable of contacting the cellular protein to form the cellular protein-E3 ubiquitin ligase complex in the absence of the E3 ubiquitin ligase-binding agent.

99. 97. The method of claim 96, wherein the E3 ubiquitin ligase is human RNF4.

100. 98. The method of claim 97, wherein the E3 ubiquitin ligase is RNF4 and the cysteine ​​is C132 of human RNF4.

101. 98. The method of claim 97, wherein the E3 ubiquitin ligase is RNF4 and the cysteine ​​is C135 of human RNF4.

102. The E3 ubiquitin ligase binding agent has the formula: 【Chemistry 19】 (I) is a monovalent form of During the ceremony, R １ are independently halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; １ the substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R ２ are independently halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; ２ the substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; L ３ is a bond, -N(R ３ )-, -C(O)-, -C(O)N(R ３ ) -, -N(R ３ )C(O)-, -N(H)-, -C(O)N(H)-, -N(H)C(O)-, -C(O)O-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R ３ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R ４ are independently hydrogen, oxo, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or E; E is an electrophilic moiety; z1 is an integer from 0 to 4, The method according to any one of claims 90 to 101, wherein z2 is an integer from 0 to 5.

103. The method of claim 102, wherein the E3 ubiquitin ligase-binding agent contacts an amino acid of the E3 ubiquitin ligase that lacks a stable secondary or tertiary conformational structure in the absence of the E3 ubiquitin ligase-binding agent.

104. The method of claim 103, wherein the amino acid that contacts the E3 ubiquitin ligase-binding agent adopts a stable secondary or tertiary conformational structure upon contact with the E3 ubiquitin ligase-binding agent.

105. The method of any one of claims 90 to 98, wherein the E3 ubiquitin ligase is human RNF114.

106. The method of claim 97 or 98, wherein the E3 ubiquitin ligase is human RNF114 and the cysteine ​​is C8 of human RNF114.

107. 106. The method of claim 105, wherein the cellular protein is p21.

108. The E3 ubiquitin ligase binding agent is a monovalent form of the formula: 【Chemistry 20】 R １ and R ２ are independently halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; １ Substituent or two R ２ the substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R ５ and R ６ are independently hydrogen, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R ７ are independently oxo, halogen, or -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; ７ the substituents may optionally be combined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; L ３ is a bond, -N(R ３ )-, -C(O)-, -C(O)N(R ３ ) -, -N(R ３ )C(O)-, -N(H)-, -C(O)N(H)-, -N(H)C(O)-, -C(O)O-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; R ３ are independently hydrogen, oxo, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R ４ are independently hydrogen, oxo, halogen, -CCl ３ , -CBr ３ , -CF ３ , -CI ３ , -CHCl ２ , -CHBr ２ , -CHF ２ , -CHI ２ , -CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OH, -NH ２ , -COOH, -CONH ２ , -NO ２ , -SH, -SO ３ H, -SO ４ H, -SO ２ N.H. ２ , -NHNH ２ , -ONH ２ , -NHC(O)NHNH ２ , -NHC(O)NH ２ , -NHSO ２ H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl ３ , -OCF ３ , -OCBr ３ , -OCI ３ , -OCHCl ２ , -OCHBr ２ , -OCHI ２ , -OCHF ２ , -OCH ２ Cl, -OCH ２ Br, -OCH ２ I, -OCH ２ F, -N ３ , -SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or E; E is an electrophilic moiety; z1 is an integer from 0 to 4, z2 is an integer from 0 to 5; The method of claim 105, wherein z7 is an integer from 0 to 10.

109. The method of claim 108, wherein the E3 ubiquitin ligase-binding agent contacts an amino acid of the E3 ubiquitin ligase that lacks a stable secondary or tertiary conformational structure in the absence of the E3 ubiquitin ligase-binding agent.

110. The method of claim 109, wherein the amino acid that contacts the E3 ubiquitin ligase-binding agent adopts a stable secondary or tertiary conformational structure upon contact with the E3 ubiquitin ligase-binding agent.

111. The E3 ubiquitin ligase binding agent is a monovalent form of the formula: 【Chemistry 21】 ( １ 、| ２ 、| ３ 、| ４ 、| ５ 、| ６ 、| ７ 、| ８ 、| ９ および| １０ The newspaper updated picture on pi. ３ 、｢r ３ 、!F ３ 、|| ３ 、!Cl ２ 、!Br ２ 、!HF ２ 、!HI ２ 、!| ２ 、、|| ２ 〒、|| ２ F、!H ２ !!||、!H、!+H ２ 、!!!!@\H ２ 、!﯁ ２ 、-SH、SO ３ 、+SO ４ 、+SO ２ . ２ 、!\H ２ 、!NH ２ 、!\\\\|| ２ 、!\\HHH ２ 、!!O ２ 、!\C\foCPH、\B\B ３ 、!C ３ 、Cr ３ 、!CI ３ 、C\l ２ 、!Cr ２ 、!CII ２ 、!CF ２ 、!CH ２ 、、!C ２ funerally, ‐flow,, ２ I、!CH ２ F、N ３ 、SF ５ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 【Chemical 22】 The method of claim 105, wherein is a single bond or a double bond.

112. The method of claim 111, wherein the E3 ubiquitin ligase-binding agent contacts an amino acid of the E3 ubiquitin ligase that lacks a stable secondary or tertiary conformational structure in the absence of the E3 ubiquitin ligase-binding agent.

113. The method of claim 112, wherein the amino acid that contacts the E3 ubiquitin ligase-binding agent adopts a stable secondary or tertiary conformational structure upon contact with the E3 ubiquitin ligase-binding agent.

Images