Polyfunctional chimeric molecules

Abstract

This invention provides a polyfunctional chemical conjugation molecule that has been found to be useful as a modifier for target substrates. [Solution] A polyfunctional chemical conjugation molecule is provided, comprising a localization portion, a chemical linker portion, an activator portion, a first orientation adapter that interconnects the chemical linker portion to the activator portion at one end, and optionally a second orientation adapter that interconnects the chemical linker molecule to the localization portion at a different end. The molecule provides a use for post-translational modification of a polymer that is not a natural substrate of the activator portion. Diseases or disorders may be treated or prevented by this molecule.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 62 / 958,696, filed on 8 January 2020, U.S. Provisional Patent Application No. 63 / 057,879, filed on 28 July 2020, and U.S. Provisional Patent Application No. 63 / 069,655, filed on 24 August 2020. All the contents of the above-specified applications are incorporated herein by reference in their entirety. Electronic sequence listing reference The contents of the electronic sequence listing ("BROD-4850WP_ST25.txt"; size 14,782 bytes, created on January 5, 2021) are incorporated herein by reference in their entirety.

[0002] The subject matter disclosed herein generally relates to polyfunctional chemical conjugation molecules used to induce modifications in target substrates. [Background technology]

[0003] Protein kinases regulate essential cellular functions such as the cell cycle, metabolism, differentiation, proliferation, and apoptosis. Kinase dysfunction is associated with various human diseases, including cancer, inflammatory conditions, autoimmune disorders, and heart disease. [Overview of the project] [Problems that the invention aims to solve]

[0004] In this technological field, there is a continuing need for effective treatments for diseases related to enzyme dysfunction and modifications such as post-translational modifications. However, nonspecific effects and other factors hinder the development of effective modifications and treatments. For example, it would be advantageous if there were no small molecule that could induce phosphorylation of any given protein, and if any small molecule could be used to phosphorylate any protein as needed. Thus, a novel small molecule that imparts new functions to enzymes through proximity-mediated effects could be useful in the research and treatment of important cellular functions and diseases. [Means for solving the problem]

[0005] In a particular exemplary embodiment, a polyfunctional chemical conjugation molecule is provided, comprising a localization portion, a chemical linker portion, an activator portion, a first orientation adapter interconnecting the chemical linker portion to the activator portion at one end, and optionally a second orientation adapter interconnecting a chemical linker molecule to the localization portion at a different end.

[0006] In a particular embodiment, the molecule is Formula IA Loc-L-(V1-Act) n (IA) (wherein Loc includes a localization portion, L is a chemical linker portion, V1 is a first orientation adapter, and Act is an activator portion, and n is at least 1); or Formula IB Loc-V2-L-(V1-Act) n (IB) (In the formula, Loc includes the localization portion, L is the chemical linker portion, V1 is the first orientation adapter, V2 is the second orientation adapter, and Act is the activator portion.) This can be represented by [formula]. The molecule may contain first and second orientation adapters independently selected from Table 1.

[0007] In one embodiment, the activator moiety binds to and activates an enzyme that modifies a target substrate associated with a localized moiety. In a particular embodiment, the target substrate is not a native substrate of the enzyme, or if the activation of the enzyme by the activator molecule is not activated by binding to the activator moiety, the enzyme modifies the target substrate at one or more novel modification sites that would otherwise remain unmodified by the enzyme.

[0008] The linker may contain PEG molecules, alkyl, heterocycloalkyl, cycloalkyl, aryl, alkylene, alkenyl, heteroaryl, amide, amine, thiol, or derivatives thereof. The linker may be a polyfunctional linker, and in some embodiments, the linker is a polyfunctional PEG linker. This polyfunctional molecule may contain about 2 to 5 activator molecules. In some embodiments, the activator portion has the ability to locate and activate an enzyme, and the enzyme may be a kinase, phosphatase, transferase, or ligase. In embodiments, the kinase is a serine / threonine kinase, a tyrosine kinase, or a bispecific protein kinase that phosphorylates protein serine / threonine and protein tyrosine.

[0009] The kinase may include AMP-activated protein kinase (AMPK), glucokinase (GK), or AGC kinase. In embodiments, the activator moiety binds to and activates protein kinase C (PKC). In embodiments, the activator moiety binds to and activates a PKC isoform selected from PKC-α, PKC-βI, PKC-βII, PKC-γ, PKC-ε, PKC-δ, PKC-η, or PKC-ζ. The activator moiety is selected from Table 1.

[0010] This molecule may contain a localization moiety that targets nucleic acids, polypeptides, or polysaccharides. In some embodiments, the localization moiety is a target polypeptide binding moiety. In some embodiments, the target polypeptide binding moiety binds to a target polypeptide containing a bromodomain and an extra-terminal motif (BET), which may be bromodomain-containing protein 4 (BRD4), BRD3, BRD2, or BRDT. The target polypeptide binding moiety may contain (+)-JQ1.

[0011] This molecule is, formula [ka] (In the formula, n=3, 5, 7, 9, 11 [ka] [1.1~1.5] It may relate to this.

[0012] This molecule is, formula [ka] (In the formula, n=0 and m=0, n=1 and m=1, n=2 and m=3, n=2 and m=1, n=2 and m=3 [PHICS2.1~2.5]) It may relate to this.

[0013] This molecule [ka] It can be selected from the following.

[0014] In one aspect, this molecule is given by the formula [ka] (Wherein, R1 is selected from JQ1, ibrutinib, dasatinib, MRX, MI-1061, gefitinib, palbociclib or foretinib, and R2 is selected from PF-06409577, benzolactam or DPH, X is CH2 or (CH2)2O, and when X = CH2, n = 1 or 5 or m = 0 or 4, and when X = (CH2)2O, n = 3 or m = 3) relates to

[0015] This molecule has the formula

Chemical formula

Chemical formula

Chemical formula

[0016] <​

[0017] A pharmaceutical composition may be provided comprising a molecule according to any one of the preceding claims and one or more pharmaceutically acceptable salts, carriers, or diluents. The compounds of the present invention may provide AMP.

[0018] A method for modifying a target substrate of a cell, comprising contacting the cell with one or more polyfunctional molecules described herein. In certain embodiments, the modification includes post-translational modifications, which may include phosphorylation, hydroxylation, acetylation, methylation, glycosylation, prenylation, amidation, eliminylation, lipidation, acylation, lipoylation, deacetylation, formylation, S-nitrosylation, S-sulfenylation, sulfonylation, sulfinylation, succinylation, sulfation, carbonylation, or alkylation.

[0019] In one embodiment, the modification includes inducing the phosphorylation of a protein in a cell. A method for phosphorylating a protein includes contacting the protein with a molecule disclosed herein, where the protein is in close proximity to a kinase specific to the activator portion of the molecule. The protein phosphorylation may include the phosphorylation of several proteins that are not substrates of the kinase. The protein may be BRD4. In a particular method, the protein is phosphorylated between BD1 and BD2 of BRD4. Methods for modifying a target substrate in a subject requiring such modification are also provided, and these methods include administering the molecule as disclosed herein. In one embodiment, the subject has a disease condition to be treated, which may be cancer.

[0020] A method for modifying a target protein is also provided, which involves contacting the target protein with a compound disclosed herein in an environment containing one or more activators. A method for treating a disease, disorder, or condition in a subject in need may include administering a molecule disclosed herein to that subject.

[0021] A method for producing a polyfunctional conjugation molecule is also provided, comprising attaching a localization moiety and an activator moiety to different ends of a linker molecule, the localization moiety and the activator moiety being optionally attached to the linker molecule via an orientation adapter, the linker molecule linking the activator molecule such that both the activator molecule and the localization moiety are active in the cell.

[0022] Those skilled in the art will be able to see the above and other aspects, purposes, features and advantages of the 91 embodiments by considering the following detailed description of the exemplary embodiments shown.

[0023] An understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings illustrating exemplary embodiments in which the principles of the present invention may be utilized. [Brief explanation of the drawing]

[0024] [Figure 1A-1C] Figure 1A illustrates the formation of PHICS-induced ternary complexes between AMPK and BRD4 (PHICS1.2) or between PKC and BRD4 (PHICS2.3), and Figure 1C illustrates the schematic explanation of how proximity-induced phosphorylation is promoted by this chimeric molecule.

[0025] [Figure 2A-2H]Biochemical characterization of PHICS1.2-induced BRD4 phosphorylation by AMPK. (Figure 2A) Ternary complex formation of BRD4, PHICS, and AMPK observed by AlphaScreen (normalized with DMSO). (R)-PHICS1.2 is an inactive analog with low affinity for BRD4. (Figure 2B) ADP-Glo® assay for AMPK-catalyzed phosphorylation of BRD4 by PHICS1.2 compared with (R)-PHICS1.2. (Figure 2C) Western blot analysis of BRD4 phosphorylation by PHICS1.2 using a phospho-AMPK substrate motif antibody. (Figure 2D) Bell-type dependence of BRD4 phosphorylation as a function of PHICS1.2 concentration, analyzed by Western blot. (Figure 2E) ADP-Glo® assay for AMPK-mediated phosphorylation of various peptide sequences from BRD4 or peptides derived from the AMPK substrate ACC (SAMS peptide). (Figure 2F) Effect of AMPK isoforms on PHICS1.2-mediated BRD4 phosphorylation. (Figure 2G) AlphaScreen showing ternary complex formation between AMPK, PHICS1.2, and various BRD proteins. (Figure 2H) Detection of PHICS1.2-catalyzed phosphorylation of various BRD proteins by Western blotting. BRD protein loading levels were determined by Kouma-Siegel.

[0026] [Figure 3A-3E] Biochemical characterization of PHICS2.3-induced BRD4 phosphorylation by PKC. (Figure 3A) Formation of BRD4, PHICS, and PKC ternary complex observed by AlphaScreen normalized with DMSO control. (Figure 3B) ADP-Glo ​​assay for PKC-catalyzed phosphorylation of BRD4 by PHICS2.3 compared with (R)-PHICS2.3. (Figure 3C) Detection of BRD4 phosphorylation by phospho-PKC substrate motif antibody in Western blot. (Figure 3D) Various BRD4 phosphorylation levels observed among PKC isoforms in the presence of PHICS2.3. (Figure 3E) Western blot analysis of PHICS2.3-mediated phosphorylation of various BRD proteins.

[0027] [Figure 4A-4B] (Figure 4A) Three-dimensional cocrystal structure of the AMPK-bound activator PF-06409577, along with the major interactions K29, K31, and D88 in the two-dimensional ligand map. (Figure 4B) Docking of the benzolactam activator and PKC, along with the major interactions T242, L251, and G253 in the two-dimensional ligand map. Solvent-exposed sites where linkers are bound are highlighted in blue.

[0028] [Figure 5] A click chemistry platform for synthesizing PHICS1 with various linkers.

[0029] [Figure 6] Biochemical validation of AMPK activators, PHICS1 intermediates, and PHICS1.2 using an ADP Glo assay with SAMS peptides as substrates.

[0030] [Figures 7A-7D] Biochemical validation of PHICS1 with various linkers to identify molecules best suited for further research. (Figure 7A) Structures of PHICS1 analogs with various linker lengths. (Figure 7B) Schematic diagram of the Alpha screen assay for BRD4-PHICS-AMPK ternary complex formation. (Figure 7C) Alpha screen assay for PHICS1 with various linkers normalized with DMSO. (Figure 7D) Western blot analysis of AMPK-catalyzed BRD4 phosphorylation by PHICS1 analogs at various concentrations.

[0031] [Figures 8A-8E]Verification of inactive analogues. (Figure 8A) Structures of PHICS1.2 and its inactive analogue (R)-PHICS1.2. (Figure 8B) ADP-Glo ​​with SAMStide peptide as a substrate for comparison of AMPK activation by PHICS1 molecule. (Figure 8C) Formation of a ternary complex of AMPK and BRD4 by induction of PHICS1.2, observed by pull-down assay. (Figure 8D) Western blot analysis of His-tagged BRD4 (49-460) phosphorylation by AMPK in the presence of PHICS1.2. (Figure 8E) Effect of AMPK concentration on PHICS1.2 or (R)-PHICS1.2-mediated BRD4 phosphorylation, observed by Western blot.

[0032] [Figures 9A-9E] Mass spectrometry identification of BRD4 phosphorylation by AMPK in the presence of PHICS1.2. (Figure 9A) Spectra of T169, (Figure 9B) T186, (Figure 9C) T221, (Figure 9D) S324 and (Figure 9E) S325 phosphorylated peptides. Fragmentation patterns are shown in each spectrum.

[0033] [Figure 10] Amino acid sequence alignments of truncated BRD4, BRD3, and BRD2 used in the experiment. The alignments were prepared using Clustal Omega. Phosphorylated residues of BRD4 identified by mass spectrometry are marked with red (AMPK-mediated) or blue (PKC-mediated) asterisks.

[0034] [Figure 11] Synthesis of PHICS2 bifunctional molecules with various linkers.

[0035] [Figure 12] An ADP-Glo ​​assay using SAMStide peptide as a substrate to determine PKC-α activation by PKC activators and PHICS2 analogs.

[0036] [Figure 13A-13C]Identification of the optimal PHICS2 for PKC-mediated BRD4 phosphorylation. (Figure 13A) Alpha screen assay for ternary complex formation with various PHICS2 analogs. (Figure 13B) Western blot analysis to compare BRD4 phosphorylation mediated by various PHICS2 molecules. (Figure 13C) Effect of PKC concentration on BRD4 phosphorylation in the presence of PHICS2.3 or (R)-PHICS2.3.

[0037] [Figure 14A-14C] Mass spectrometry identification of BRD4 phosphorylation by PKC in the presence of PHICS2.3. (Figure 14A)T229, (Figure 14B)S324, and (Figure 14C)S338 spectra of phosphorylated peptides. Fragmentation patterns are shown in each spectrum.

[0038] [Figures 15A-15B] NRM spectra: Figure 15A ¹H NMR spectrum of compound 3. Figure 15B ¹H NMR spectrum of compound 4.

[0039] [Figures 16A-16B] Figure 16A: 13C NMR of 4. Figure 16B: 1H NMR of PHICS1.1.

[0040] [Figures 17A-17B] Figure 17A: 13C NMR of PHICS1.1. Figure 17B: 1H NMR of PHICS1.2.

[0041] [Figures 18A-18B] Figure 18A: 13C NMR of PHICS1.2. Figure 18B: 1H NMR of (R)-PHICS1.2.

[0042] [Figures 19A-19B] Figure 19A-(R)-PHICS1.2 13C NMR. Figure 19B-PHICS1.3 1H NMR.

[0043] [Figures 20A-20B]Figure 20A: 13C NMR of PHICS1.3. Figure 20B: 1H NMR of PHICS1.4.

[0044] [Figures 21A-21B] Figure 21A: 13C NMR of PHICS1.3; Figure 21B: 1H NMR of PHICS1.5.

[0045] [Figures 22A-22B] Figure 22A: 13C NMR spectrum of PHICS1.5. Figure 22B: 1H NMR spectrum of 21.

[0046] [Figures 23A-23B] Figure 23A: 13C NMR spectrum of 21. Figure 23B: 1H NMR spectrum of 22.

[0047] [Figure 24] 13C NMR spectrum of 22.

[0048] [Figures 25A-25B] Figure 25A: ¹H NMR spectrum of 23. Figure 25B: ¹³C NMR spectrum of 23.

[0049] [Figures 26A-26B] Figure 26A: ¹H NMR spectrum of 24. Figure 26B: ¹³C NMR spectrum of 24.

[0050] [Figures 27A-27B] Figure 27A: ¹H NMR spectrum of 25. Figure 27B: ¹³C NMR spectrum of 25.

[0051] [Figures 28A-28B] Figure 28A: ¹H NMR spectrum of 26. Figure 28B: ¹³C NMR spectrum of 26.

[0052] [Figures 29A-29B] Figure 29A: ¹H NMR spectrum of 27. Figure 29B: ¹³C NMR spectrum of 27.

[0053] [Figures 30A-30B] Figure 30A: ¹H NMR spectrum of 28. Figure 30B: ¹³C NMR spectrum of 28.

[0054] [Figure 31A-31B] Figure 31A: ¹H NMR spectrum of 29. Figure 31B: ¹³C NMR spectrum of 29.

[0055] [Figures 32A-32B] Figure 32A: ¹H NMR spectrum of 30; Figure 32B: ¹³C NMR spectrum of 30.

[0056] [Figures 33A-33B] Figure 33A: 1H NMR spectrum of PHICS2.1. Figure 33B: 13C NMR spectrum of PHICS2.1.

[0057] [Figures 34A-34B] Figure 34A: 1H NMR spectrum of PHICS2.2. Figure 34B: 13C NMR spectrum of PHICS2.2.

[0058] [Figures 35A-35B] Figure 35A: 1H NMR spectrum of PHICS2.3. Figure 35B: 13C NMR spectrum of PHICS2.3.

[0059] [Figures 36A-36B] Figure 36A: 1H NMR spectrum of PHICS2.4. Figure 36B: 13C NMR spectrum of PHICS2.4.

[0060] [Figures 37A-37B] Figure 37A: 1H NMR spectrum of PHICS2.5. Figure 37B: 13C NMR spectrum of PHICS2.5.

[0061] [Figures 38A-38B]Figure 38A: ¹H NMR spectrum of (R)-PHICS2.3. Figure 38B: ¹³C NMR spectrum of (R)-PHICS2.3.

[0062] [Figure 39A-39B] Figure 39A - Identification of kinase binders and linker optimization for creating the PHICS molecule; Figure 39B - PHICS-kinase-BRD4 shows the "Hook effect" observed with respect to the ternary complex.

[0063] [Figure 40] Mass spectrometry indicates the neophosphorylation sites on BRD4. Identified phosphorylated residues in the kinase substrate recognition motif: T186, S324, S325; phosphorylated residues not in the kinase substrate recognition motif: T169, T221. Spectrum of the peptide phosphorylated at T169, left.

[0064] [Figure 41] A gel demonstrating that phosphatase can remove PICS-mediated BRD4 phosphorylation.

[0065] [Figure 42] Imaging studies show that PHICS cannot phosphorylate BRD4 in cells containing the kinase isoform (cytosol) and BRD4 (nucleus).

[0066] [Figure 43] Rational design of PHICS and controls for Bruton's tyrosine kinase (BTK).

[0067] [Figure 44A-44B] We investigated PHICS-mediated phosphorylation of BTK. Figure 44A: PHICS-mediated phosphorylation of BTK in vitro; Figure 44B: PHICS-mediated phosphorylation of BTK in cellulose.

[0068] [Figure 45] Immunoprecipitation of kinases and BTK mediated by PHICS.

[0069] [Figure 46] Phosphatase inhibitors increase PHICS-mediated phosphorylation levels of BTK.

[0070] [Figure 47] A drawing of the BTK pocket, showing that T474 forms a hydrogen bond; D539 and K430 form the base of the pocket (left); and a gel exhibiting phosphorylation deficiency in BTK mutants T474A, K430R, and D539N.

[0071] [Figure 48] Introducing a highly bulky group into PHICS leads to a loss of activity.

[0072] [Figure 49] PHICS for FKBP12.

[0073] [Figure 50] PHICS for c-Abl tyrosine kinase and BTK using dasatinib.

[0074] [Figures 51A-51D] (A) Detection of BRD4 phosphorylation by immunoblotting using a phospho-PKC substrate motif antibody. (B-C) ADP-Glo ​​assay comparing BRD4 phosphorylation by (B)PHICS1 or (C)PHICS2 with their respective iPHICS. (D) Western blot analysis of PHICS1-mediated BRD4 phosphorylation using a phosphor-Ser484 / 488 antibody.

[0075] [Figures 52A-52B] (A) Effects of AMPK isoforms on PHICS1-mediated BRD4 phosphorylation. (B) Effects of PKC isoforms on BRD4 phosphorylation.

[0076] [Figures 53A-53D]AMPK-mediated PHICS-mediated BTK phosphorylation in cells. (A) Structures of PHICS3 and Piv-PHICS3 inactive controls for AMPK-mediated BTK phosphorylation. (B) Detection of ternary complex formation in HEK293T cells by immunoprecipitation of AMPK and BTK-Flag in the presence of PHICS3. WCL: Whole cell lysate (C~D) Western blot analysis of PHICS3-mediated BTK phosphorylation in HEK293T cells transfected with WT BTK-Flag (C) and BTK-Flag S180A mutant (D). See SI for structures of AMPK activators and BTK inhibitors.

[0077] [Figures 54A-54D] Target association of PHICS3 in cells. (A) Competitive experiments with the covalent BTK inhibitor ibrutinib. (B) Major interaction between ibrutinib and BTK (PDB ID: 5P9I). (C) Western blot analysis of PHICS3-induced phosphorylation of WT BTK and T474 mutants. (C) Western blot analysis of BTK phosphorylation by PHICS3 and its inactive analog Piv-PHICS3.

[0078] [Figures 55A-55B] Molecular docking. (55A) Cocrystal structure of the activator PF-06409577 bound to AMPK (PDB ID: 5KQ5). The solvent-exposed site was modified for linker bonding. Highlighted in blue. (55B) Two-dimensional ligand map showing the major interactions between this molecule and Lys29, Lys31, and Asp88.

[0079] [Figures 56A-56B]Biochemical validation of modified activators and PHICS intermediates by ADP-Glo ​​assay. (56A) Biochemical validation of AMPK activation by the potent activator PF-06409577, AMPK activator, and AMPK activator with a linker using an ADP-Glo ​​assay with SAMS peptide as a substrate. (56B) ADP-Glo ​​assay using CREBtide peptide as a substrate to determine PKCα activation by modified PKC activators.

[0080] [Figure 57] Synthesis of AMPK activators, AMPK activators with linkers, bifunctional AMPK-PHICS (lead compounds obtained from PHICS1, n=5) with various linker lengths, and the inert analog iPHICS1.

[0081] [Figure 58] Synthesis of PKC activators and bifunctional PKC-PHICS (lead compounds obtained from PHICS2, n=1, m=3) with various linker lengths, as well as the inert analog iPHICS2.

[0082] [Figures 59A-59D] Identification of the optimal PHICS for BRD4 phosphorylation by AMPK. (59A) Structures of AMPK-PHICS analogs with various linker lengths. (59B) Schematic diagram of the Alpha screen assay for BRD4-PHICS-AMPK ternary complex formation. (59C) AlphaScreen assay for AMPK-PHICS with various linkers normalized with DMSO. (59D) Western blot analysis of AMPK-catalyzed BRD4 phosphorylation with AMPK-PHICS analogs at various concentrations.

[0083] [Figures 60A-60C]Identification of the optimal PHICS for PKC-mediated BRD4 phosphorylation. (60A) Structures of PKC-PHICS analogs with various linker lengths. (60B) AlphaScreen assay for ternary complex formation by various PKC-PHICS analogs normalized with DMSO. (60C) Western blot analysis to compare BRD4 phosphorylation mediated by various PKC-PHICS molecules.

[0084] [Figures 61A-61G] Verification of inactive analogs. (61A) Structures of PHICS1, PHICS2, and their inactive analogs iPHICS1 and iPHICS2. (61B) ADP-Glo ​​with SAMStide peptide as a substrate for comparing AMPK activation by PHICS1 and iPHICS1. (61C) ADP-Glo ​​with CREBtide peptide as a substrate for comparing PKC activation by PHICS2 and iPHICS2. (61D) Formation of a ternary complex of AMPK and BRD4 by induction of PHICS1, observed by pull-down assay. (61E) Western blot analysis of His-tagged BRD4 (49-460) phosphorylation by AMPK in the presence of PHICS1. (61F) Effect of AMPK concentration on PHICS1 or iPHICS1-mediated BRD4 phosphorylation, observed by Western blot. (61G) Effect of PKC concentration on BRD4 phosphorylation in the presence of PHICS2 or iPHICS2.

[0085] [Figure 62] Bell-shaped dependence of BRD4 phosphorylation as a function of PHICS1 concentration, as analyzed by Western blot.

[0086] [Figures 63A-63B] Adding AMP enhances phosphorylation. (63A) In vitro kinase assay for BRD4 phosphorylation by AMPK (1uM compounds MS231, VS804, VS806); (63B) ADP glo assay for BRD4 phosphorylation by AMPK with and without AMP.

[0087] [Figures 64A-64C] (64A) Structures of Halo-targeted and BRD4-targeted PHICS molecules based on known Abl kinase activators. L-linker. (64B~64C) Western blot analysis of PHICS-inducible phosphorylation of Halo tag (64B) and BRD4 (64C).

[0088] [Figure 65] ADP-Glo ​​assay for AMPK-mediated phosphorylation of various peptide sequences from BRD4 or peptides derived from the AMPK substrate ACC (SAMS peptide).

[0089] [Figure 66] ADP-Glo ​​assay for activation of various AMPK isoforms by PF-06409577.

[0090] [Figure 67] AlphaScreen assay for ternary complex formation between AMPK, PHICS1, and various BRD proteins.

[0091] [Figure 68] Synthesis of the non-covalent BTK inhibitor PHICS3, its analogues with various linkers, and the inactive control Piv-PHICS3.

[0092] [Figure 69] In vitro biochemical verification of BTK phosphorylation by AMPK in the presence of PHICS. Western blot analysis of BTK phosphorylation using 10 μM BTK-targeted AMPK-PHICS with various linkers to identify the optimal molecule for further study. The molecule with an 8-carbon alkyl linker (PHICS3) was identified as the optimal molecule for phosphorylation.

[0093] [Figures 70A-70D]Cell-based studies on PHICS-mediated BTK phosphorylation by AMPK. (70A) Western blot analysis of phosphorylation of overexpressed Flag-BTK in HEK293 cells using PHICS with various linkers. Compounds were treated for 4 hours, and phosphorylation was detected by phospho-BTK(Ser180) antibody. The molecule with an 8-carbon alkyl linker (PHICS3) was identified as the optimal molecule for phosphorylation. (70B) Western blot analysis to compare BTK Ser180 phosphorylation in the presence of 5 μM PHICS3, 5 μM activator, or incrementally increasing concentrations of the potent AMPK activator PF-06409577. (70C) Time-dependent and (70D) PHICS3 dose-dependent BTK phosphorylation observed by Western blotting. 5 μM PHICS3 was used for the time-dependent study.

[0094] [Figure 71A-71C] Verification of PHICS3 target association with BTK in cells. (71A) Structure of ibrutinib and covalent binding of ibrutinib to BTK. Pretreatment of cells with ibrutinib leads to irreversible covalent modification of Cys481, blocking PHICS3 binding to BTK. (71B) Crystal structure of ibrutinib bound to BTK, along with key interactions (PDB ID: 5P9I). Thr474 forms hydrogen bonds with the four amino groups of ibrutinib. Asp539 (cyan) forms hydrogen bonding interactions (magenta) with ibrutinib and Lys430 (green). (71C) Detection of S180 phosphorylation in T474A, K430R and D539N mutants of BTK compared to WT.

[0095] [Figure 72] 1H NMR spectrum of 37.

[0096] [Figure 73] 37 13C NMR spectra.

[0097] [Figure 74]1H NMR spectrum of PHICS3 recorded in DMSO-d6.

[0098] [Figure 75] 13C NMR spectrum of PHICS3 recorded in DMSO-d6.

[0099] [Figure 76] 1H NMR spectrum of Piv-PHICS3 recorded in a CDCl3:CD3OD(1:1) solvent mixture.

[0100] [Figure 77] 13C NMR spectrum of Piv-PHICS3 recorded in a CDCl3:CD3OD(1:1) solvent mixture.

[0101] [Figure 78] Two concepts are presented for using N-acyl-N-alkylsulfonamide (NASA) for PKC activation. The upper panel shows Concept 1: The activator (PKC) is labeled with a localization site, e.g., a binder for the target protein, using a NASA warhead. The lower panel shows Concept 2: PKC is labeled with trans-cyclooctene (TCO) using a NASA warhead. This can then be attached to a localization site, e.g., a binder for any target protein, using tetrazine click chemistry.

[0102] [Figures 79A-79B] Phosphorylation of transcription factors disrupts their (79A) protein-DNA and (79B) protein-protein bonds.

[0103] [Figure 80] Modular synthesis of exemplary PHICS molecules for kinase evaluation, including exemplary activator moieties such as ABL activators, IR activators, MEK inhibitors, and AKT inhibitors, as well as localization moieties identified as AR and BRD4 binders.

[0104] [Figure 81A-81D] A binding agent for transcription factors targeted by PHICS.

[0105] [Figures 82A-82B] (82A) Cellular localization of PHICS targets. (82B) Timeline and workflow.

[0106] [Figure 83A-83C] (83A) Structures of DPH and (83B) DPH-L6-azide. (83C) ADP-Glo ​​assay for DPH and DPH-L6-azide.

[0107] [Figure 84A-84D] (84A) Binding agents for selected protein targets (localization moieties) or (84B) kinases (activator moieties) functionalized with reactive handles (red and green for A and B, respectively). (84C-84D) Synthetic schemes for constructing PHICS molecules. Note: The names below the structures represent the parent binding agents, with the corresponding proteins provided in parentheses.

[0108] [Figure 85] Cooperative action in three-body equilibrium.

[0109] [Figure 86] A schematic diagram illustrating the kinetic process of pharmacokinetics from drug administration to disease progression. Definitions: Plasma (Cp); near target (C); compound concentration in the complex with the target (CT); free target concentration (T); kin is the generation rate; kout is the dissipation rate; kon is the on-rate constant; koff is the off-rate constant.

[0110] [Figure 87] The chemical structures of the CDK8 inhibitors initially considered in this study and their experimental residence times. The common 1-(3-tert-butyl-1-p-tolyl-1H-pyrazole-5-yl)urea scaffold is shown in blue.

[0111] [Figure 88]The active site of the human CDK8 crystal structure in complexes with compounds 1-7 shown in Figure 87. The interactions between the urea group of the crystallization inhibitor and Glu66 and Asp173 are indicated by dashed lines. Protein carbon atoms are shown in white. The protein backbone is shown as a white sketch, excluding the hinge region (gray sketch).

[0112] [Figure 89] Two-dimensional representations of eight p38α inhibitors: SB5, SB6, SB7, B12, B96, BR5, BR8, and BMU. Common substructures are highlighted.

[0113] [Figure 90] Inhibition response rates of various Abl1 inhibitors, including dasatinib, imatinib, ponatinib, and nilotinib.

[0114] [Figure 91] Evaluation of tyrosine phosphorylated Abl-BRD4(pTyr Millipore) compounds according to exemplary embodiments.

[0115] [Figure 92] Evaluation of a tyrosine phosphorylated Abl-BRD4 (DPH activator) compound according to an exemplary embodiment. [Modes for carrying out the invention]

[0116] The figures in this specification are for illustrative purposes only and are not necessarily drawn to a specific scale.

[0117] general definition Unless otherwise defined, scientific and technical terms used herein have the same meanings as those generally understood by those skilled in the art in the field relating to this disclosure. For definitions of general terms and techniques in molecular biology, see Molecular Cloning: A Laboratory Manual, 2 ndedition(1989)(Sambrook,Fritsch,and Maniatis);Molecular Cloning:A Laboratory Manual,4 th edition(2012)(Green and Sambrook);Current Protocols in Molecular Biology(1987)(F.M.Ausubel et al.eds.);the series Methods in Enzymology(Academic Press,Inc.):PCR 2:A Practical Approach(1995)(M.J.MacPherson,B.D.Hames,and G.R.Taylor eds.):Antibodies,A Laboratory Manual(1988)(Harlow and Lane,eds.):Antibodies A Laboratory Manual,2 ndedition 2013(EAGreenfield ed.);Animal Cell Culture(1987)(RIFreshney,ed.);Benjamin Lewin,Genes IX,published by Jones and Bartlet,2008(ISBN 0763752223);Kendrew et al.(eds.),The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd.,1994(ISBN 0632021829); Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley&Sons(New York, NY1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2 nd See edition (2011).

[0118] As used herein, the singular forms "a," "an," and "it" refer to both singular and plural objects unless otherwise explicitly indicated by the context.

[0119] The terms "optional" or "optional" mean that the event, situation, or substituent described thereafter may or may not occur, and that the description includes both cases in which the event or situation occurs and cases in which it does not.

[0120] Numerical ranges indicated by endpoints include all numbers and fractions within each range, as well as the endpoints themselves.

[0121] When the terms “about” or “approximately” are used herein to refer to measurable values ​​such as parameters, quantities, or temporal durations, they are intended to encompass the specified value and its variations, to the extent that variations relating to the disclosed invention, such as variations of ±10%, ±5%, ±1%, and ±0.1%, are appropriate. It should be understood that the values ​​referred to by the modifying phrases “about” or “approximately” are themselves also specifically and preferably disclosed.

[0122] As used herein, “biological sample” may include whole cells and / or living cells and / or cell debris. A biological sample may include (or be derived from) “body fluids.” The present invention encompasses embodiments in which body fluids are selected from amniotic fluid, aqueous humor, vitreous fluid, bile, serum, breast milk, cerebrospinal fluid, earwax, chyle, erosion, endolymph, perilymph, exudate, feces, female semen, gastric acid, gastric juice, lymph, mucus (including rhinorrhea and sputum), pericardial fluid, ascites, pleural fluid, pus, mucosal secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more of these. Biological samples include cell culture media, body fluids, and cell culture media derived from body fluids. Body fluids may be obtained from mammalian organisms, for example, by puncture or other collection or sampling procedures.

[0123] The terms “subject,” “individual,” and “patient” are used herein synonymously to refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, mice, monkeys, humans, farm animals, sports animals, and pets. Tissues, cells, and their offspring of biological entities obtained in vivo or cultured in vitro are also included.

[0124] A “diastereoisomer” is a stereoisomer having at least two chiral atoms but not being mirror images of each other. Absolute stereochemistry is specified according to the Kahn-Ingold-Prelogue RS scheme. When a compound is an enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Divided compounds with an unknown absolute configuration can be indicated as (+) or (-) depending on the direction of rotation of plane-polarized light at the wavelength of the sodium D line (dextrorotatory or levorotatory). Some of the compounds described herein contain one or more chiral centers and thus may result in enantiomers, diastereomers, and other stereoisomeric forms that can be defined as (R)- or (S)- in terms of absolute stereochemistry at each chiral atom. The chemical entities, pharmaceutical compositions, and methods described herein are intended to include all such possible isomers, including racemic mixtures, optically substantially pure forms, and intermediate mixtures. In some chemical structures, stereocenters may be identified by "wavy" bonds, indicating that the stereocenter can be in an R or S configuration unless otherwise specified. However, stereocenters without wavy bonds (i.e., "linear" bonds) can also be in an (R) or (S) configuration unless otherwise specified. Compositions containing compounds may contain stereocenters, each of which can independently be in an (R) configuration, an (S) configuration, or a racemic mixture.

[0125] Optically active (R) and (S) isomers can be prepared, for example, using chiral synthons or chiral reagents, or they can be separated using prior art. Enantiomers can be separated from racemic mixtures by any method known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC), chiral salt formation and crystallization, or they can be prepared by asymmetric synthesis.

[0126] Optical isomers can be obtained by conventional methods, such as the separation of a racemic mixture by treatment with an optically active acid or base, or by the formation of diastereoisomer salts. Examples of suitable acids are tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, ditoluyltartaric acid, and camphorsulfonic acid. Separation of the diastereoisomer mixture is obtained by crystallization and subsequent liberation of optically active bases from their salts. Another method involves the synthesis of covalent diastereoisomer molecules by reacting the disclosed compound with an activated form of an optically pure acid or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization, or sublimation, and then hydrolysis can yield the enantioconcentrated compound.

[0127] Optically active compounds can also be obtained by using active starting materials. In some embodiments, these isomers may be in the form of free acids, free bases, esters, or salts.

[0128] In certain embodiments, the disclosed compounds may be tautomers. As used herein, the term “tautomer” refers to a type of isomer comprising two or more interconvertible compounds resulting from at least one formal transfer of a hydrogen atom and at least one change in valence (e.g., from a single bond to a double bond, from a triple bond to a single bond, or vice versa). Tautomerization includes prototropic or proton transfer tautomerization, which is considered part of acid-base chemistry. Prototropic or proton transfer tautomerization involves the rearrangement of protons with a change in bond order. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. If tautomerization is possible (e.g., in solution), tautomers can reach chemical equilibrium. Tautomerization (i.e., the reaction that provides a pair of tautomers) can be catalyzed by an acid or a base, or it can occur without the action or presence of an external substance. Exemplary tautomerizations include, but are not limited to, keto-enol; amide-imide; lactam-lactim; enamine-imine; and enamine-(another) enamine tautomerizations. A specific example of keto-enol tautomerization is the interconversion of pentan-2,4-dione and 4-hydroxypenta-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridine-4-ol and pyridine-4(1H)-one tautomers.

[0129] Unless otherwise specified, all chiral, diastereomer, racemic, and geometric isomeric forms of the structure are intended. All processes used in the preparation of the compounds and the intermediates produced therein are included in this disclosure. All tautomers of the illustrated or described compounds are also included in this disclosure.

[0130] When used herein, bond substitutions resulting from a ring, for example, [ka] This means that this substitution can occur at any of the available positions on the ring.

[0131] Various embodiments are described below. It should be noted that these specific embodiments are not intended to be exhaustive or to limit the scope to a broader range of embodiments considered herein. An embodiment described in conjunction with a detailed embodiment is not necessarily limited to that embodiment and can be implemented in one or more other embodiments. Whenever the terms “one embodiment,” “a certain embodiment,” or “a certain exemplary embodiment” are used throughout this specification, it means that a particular feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases “in one embodiment,” “in a certain embodiment,” or “a certain exemplary embodiment” in various places throughout this specification does not necessarily refer to the same embodiment, although it may. Furthermore, a particular feature, structure, or characteristic can be combined in one or more embodiments in any preferred manner, as will be apparent to those skilled in the art from this disclosure. Furthermore, some embodiments described herein include some features included in other embodiments, and do not include others, but combinations of features from different embodiments are intended to be within the scope of the present invention. For example, any of the claimed embodiments in the appended claims can be used in any combination.

[0132] All publications, published documents, and patent applications referenced herein are referred to by reference to the same extent as each individual publication, published document, or patent application is specifically and individually indicated to be referred to by reference.

[0133] overview Embodiments disclosed herein provide compounds for inducing, achieving, or promoting the modification of a target substrate. These compounds are polyfunctional conjugation molecules comprising a localization moiety, a chemical linker moiety, an activator moiety, and a first orientation adapter interconnecting the chemical linker moiety at one end to the activator moiety. Optionally, a second orientation adapter exists, interconnecting the chemical linker moiety at a different end to the localization moiety. The terms “polyfunctional conjugation compound,” “polyfunctional molecule,” “chimeric molecule,” and “chimeric conjugation molecule” are used synonymously herein.

[0134] Polyfunctional chemical conjugation molecules can be used to modify target substrates. In one embodiment, the modification is post-translational modification. Modification, as used herein, may include the addition or removal of functional groups. Exemplary embodiments disclosed herein provide small molecule compounds that induce phosphorylation and / or have protein phosphorylation ability.

[0135] The compounds of the present invention preferably modify polymer substrates, such as polypeptides, oligosaccharides, and polysaccharides, as well as nucleic acids such as DNA or RNA. Association with polymers may include covalent bonding, non-covalent bonding, electrophilic association, or other associations. Bonding may include reversible or irreversible bonding.

[0136] Polyfunctional chimeric molecules can be modified and designed to allow for time-dependent and dose-dependent control, as detailed herein.

[0137] Modifications achieved by activators, either directly or indirectly, with the activator moiety of a polyfunctional compound, may be reversible or irreversible. In embodiments, modifications may include the addition of chemical groups, such as phosphorylation, hydroxylation, acetylation, or methylation. In one embodiment, modifications are the addition of complex molecules, such as prenylation, glycosylation, ADP-ribosylation, and AMPation. In another embodiment, modifications may include cleavage, such as proteolysis. Amino acid modifications, such as amide degradation and elimination, are achieved.

[0138] In one exemplary embodiment, a polyfunctional chimeric molecule or polyfunctional chemical conjugation molecule comprises a localization moiety, a chemical linker moiety, an activator moiety, a first orientation adapter interconnecting the chemical linker moiety at one end to the activator moiety, and optionally a second orientation adapter interconnecting the chemical linker molecule at the other end to the localization moiety. One or more activator moieties may be used in the polyfunctional chimeric molecule.

[0139] In certain exemplary embodiments, the molecule is of formula IA Loc-L-(V1-Act) n (IA) (In the formula, Loc includes the localization portion, L is the chemical linker portion, V1 is the first orientation adapter, and Act is the activator portion, and n is 1 to 5.) It can be represented by:

[0140] In certain other exemplary embodiments, the molecule is of formula IB Loc-V2-L-(V1-Act) n (IB) (In the formula, Loc includes the localization portion, L is the chemical linker portion, V1 is the first orientation adapter, V2 is the second orientation adapter, and Act is the activator portion, and n is 1 to 5.) It is represented by [this].

[0141] The use of the polyfunctional compounds disclosed herein includes use in proximity-inducible modification of polymeric substrates. When phosphorylation is used as an exemplary modification, the compounds can advantageously induce phosphorylation of a target protein of interest. For example, a protein that is a non-substrate of the activator moiety of a polyfunctional compound can be phosphorylated by exemplary embodiments of the polyfunctional compounds disclosed herein. The polyfunctional compounds disclosed herein result in a novel class of molecules capable of modifying target substrates without the need to use them in conjunction with the native substrate of the activator moiety, e.g., inducing phosphorylation of target proteins (e.g., bromodomain family proteins). Using the principle of proximity-inducible reactivity, kinases such as AMPK or PKC can phosphorylate non-substrate proteins (e.g., BRD4).

[0142] These multifunctional chimeric molecules can be designed and modified according to the localization moiety, activator moiety, and the proximity required to enable the desired modification of the target substrate, as described in further detail herein.

[0143] localized part The localization moiety represented as Loc in formulas IA and IB provides targeting, binding to, or association with a polymer. In embodiments, the polymer is a polypeptide, a nucleic acid such as DNA or RNA, or a sugar molecule. In embodiments, the polypeptide is a protein, such as an enzyme.

[0144] In one embodiment, the localization moiety binds to the target substrate to be modified. The function of the localization moiety is to bind the substrate to be modified by the activator bound to the activator moiety, thereby bringing the target substrate into close proximity to the activator moiety. This reaction may allow the activator to modify more substrates, including non-natural target substrates of the activator, and increase the reaction rate / efficiency of such substrate modification. To this end, the localization moiety must be capable of binding to a specific substrate and may include a number of molecules suitable for this purpose and capable of binding to the activator moiety, as further described herein. Various exemplary classes of localization moieties are provided herein, based on the substrate to be modified.

[0145] In embodiments, the localization region is selected based on the desired association and modification to be achieved. Therefore, the desired modification can be adjusted based on specific conditions, diseases, treatments, or other desired effects, and may be a design consideration when selecting the localization region. For example, a positively charged DNA-binding domain interacts with a negatively charged DNA backbone. Phosphorylation converts a neutral residue to a negatively charged residue, where charge neutralization results in lower DNA-binding affinity. See, for example, Gallagher, et al., Nature Methods, “Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity”; and Slaymaker et al., Science, “Rationally engineered Cas9 nucleases with improved specificity.”

[0146] Localization site for binding to polypeptide substrates In certain exemplary embodiments, the localization moiety is a target polypeptide binding moiety used for binding a polypeptide substrate. The target polypeptide binding moiety can be selected with respect to a specific protein of interest that may be located at various localization sites in the cell, e.g., the nucleus, cytoplasm, mitochondria, or cell surface. Exemplary target polypeptide binding moieties are disclosed, for example, in Sun et al., Signal Transduction and Targeted Therapy, 4:64 (2019), which provides exemplary proteins and corresponding ligands (i.e., target polypeptide binding moieties), see in particular Figures 5 to 48 (which are incorporated herein by reference). The target polypeptide binding moiety may bind to proteins that undergo conformational changes upon binding, such as androgen receptors (ARs).

[0147] The target polypeptide binding moiety can bind to, for example, bromodomains and extraterminal domain (BRD) family proteins (e.g., BRD2, BRD3, BRD4). Bromodomains are a family of structurally and evolutionarily conserved protein interaction modules (approximately 110 amino acids) that specifically recognize substrate proteins, particularly acetylated lysine present in histones. Bromodomains exist as components of large, multi-domain nucleoproteins involved in chromatin remodeling, cell signaling, and transcriptional regulation. Examples of bromodomain-containing proteins with known functions include (i) histone acetyltransferases (HATs), including CREBBP, GCN5, PCAF, and TAFII250; (ii) methyltransferases such as ASH1L and MLL; (iii) components of chromatin remodeling complexes such as Swi2 / Snf2; and (iv) several transcriptional regulators (Florence et al. Front. Biosci. 2001, 6, D1008-1018, which is incorporated herein by reference as a whole).

[0148] In other exemplary embodiments, the target polypeptide binding moiety is a small molecule target polypeptide binding moiety. In one exemplary embodiment, the target polypeptide binding moiety is a JQ1-derived moiety. For example, the target polypeptide binding moiety may be as follows: [ka]

[0149] In some embodiments, the portion derived from JQ1 is either derived from (+)-JQ1 or from (-)-JQ1. [ka]

[0150] Further target polypeptide binding molecules. For example, the target polypeptide binding molecule may be selected from p53 binders (e.g., 2,5-bis(5-hydroxymethyl-2-thienyl)furan or RITA), Max binders KI-MS2-008 (Figure 81A), ER inhibitors raloxifene or β-catenin inhibitors (UU-T02).

[0151] The localized region may also include molecules such as ibrutinib (BTK), dasatinib (BCR-ABL), MRTX (KRAS), MI-1061 (MDM2), gefitinib (EGFR), palbociclib (CDK4 / 6), and foretinib (C-MET), as shown in Figure 84A.

[0152] In one embodiment, the localization portion is an antibody or its binding portion. The localization portion may be a nanobody containing a single-domain antibody fragment that possesses the structural and functional properties of naturally occurring heavy-chain-only antibodies; see, for example, Bannas et al, Front.Immunol., doi:10.3389 / fimmu.2017.01603. The term “binding portion” (or “antibody portion”) of an antibody includes one or more complete domains, e.g., a pair of complete domains, and antibody fragments that retain the ability to specifically bind to a target molecule. It has been shown that the antibody binding function can be performed by full-length antibody fragments. Binding fragments are prepared by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, Fv, single-chain, VHH, single-chain antibodies, e.g., scFv, and single-domain antibodies.

[0153] In certain embodiments, a "humanized" non-human antibody contains amino acid residues in its frame region that are similar to those in the frame region of a human antibody. In certain embodiments, the frame region of a camelid antibody or heavy chain antibody is modified. In certain embodiments, a "humanized" non-human antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin (e.g., a camelid). In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues in the hypervariable region of the recipient are replaced with residues in the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capabilities. In some examples, FR residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, a humanized antibody may contain residues not found in the recipient antibody or donor antibody. Such modifications are made to further refine the performance of the antibody. Generally, humanized antibodies will contain substantially all of at least one, typically two, variable domains, where all or substantially all of the hypervariable region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region corresponds to the human immunoglobulin sequence. Optionally, humanized antibodies will also contain at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0154] Examples of antibody or epitope-binding protein segments included in this definition include: (i) Fab fragments having VL, CL, VH, and CH1 domains; (ii) Fab' fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragments having VH and CH1 domains; (iv) Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CHI domain; (v) Fv fragments having VL and VH domains of a single arm of the antibody; (vi) dAb fragments consisting of an antigen-binding VH domain or VL domain (Ward et al., 341 Nature 544 (1989)); (vii) isolated CDR regions or isolated CDR regions provided to a functional framework; (viii) F(ab')2 fragments, which are bivalent fragments containing two Fab' fragments linked by disulfide crosslinking in a hinge region; (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., 242 Science Examples include (x) a "diabody" having two antigen-binding sites, including a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (see, for example, European Patent No. 404,097; International Publication No. 93 / 11161; Hollinger et al., PNAS 6444 (1993)); and (xi) a "linear antibody" containing a tandem Fd segment pair (VH-Ch1-VH-Ch1) that, together with a complementary light chain polypeptide, forms a pair of antigen-binding regions (Zapata et al., Protein Eng. 8(10):1057-62 (1995); and U.S. Patent No. 5,641,870).

[0155] "Specific binding" of an antibody means that the antibody exhibits a reasonable degree of affinity to a specific antigen or epitope, and generally does not exhibit significant cross-reactivity. "A reasonable degree of" binding refers to binding with an affinity of at least 25 μM. (1 × 10⁻⁶) 7 M -1Antibodies with greater affinity (or a dissociation coefficient of 1 μM or less or a dissociation coefficient of 1 nM or less) typically bind with correspondingly higher specificity. Intermediate values among those shown herein are also intended to be within the scope of the invention, and the antibodies of the invention can bind, for example, within an affinity range of 100 nM or less, 75 nM or less, 50 nM or less, 25 nM or less, for example 10 nM or less, 5 nM or less, 1 nM or less or in embodiments 500 pM or less, 100 pM or less, 50 pM or less or 25 pM or less. An antibody that "does not exhibit significant cross-reactivity" will not show appreciable binding to entities other than its target (e.g., different epitopes or different molecules). For example, an antibody that specifically binds to a target molecule will bind to that target molecule to an appreciable extent but will not significantly react with non-target molecules or peptides. An antibody specific for a particular epitope will not, for example, significantly cross-react with a remote epitope on the same protein or peptide. Specific binding can be determined by any means recognized in the art for determining such binding. Preferably, specific binding is determined by Scatchard analysis and / or competitive binding assays.

[0156] As used herein, the term "affinity" refers to the strength with which a single antigen-binding site binds to an antigenic determinant. Affinity depends on the tightness of the stereochemical fit between the antibody binding site and the antigenic determinant, the size of the contact area between them, the distribution of charged and hydrophobic groups, etc. Antibody affinity can be measured by equilibrium dialysis or by the kinetic BIAcore™ method. The dissociation constant Kd and the association constant Ka are quantitative measures of affinity.

[0157] Localization moiety for binding to a polynucleotide substrate Polynucleotide-binding proteins have been identified as factors that exacerbate inflammation. These proteins can be identified from nucleotide-binding folds within proteins, such as the Rossmann fold (see, e.g., Kleiger et al., J. of Mol. Biol. 323:69-76) and the P-loop-containing nucleotide hydrolase fold (see, e.g., Saraste et al., Trends in Bio Sci, 15:430-434). Chauhan et al. have developed methods for identifying ATP and GTP-binding residues, and Ansari et al. have designed a method specifically for NAD. Parca et al. (2012) identified nucleotide-binding sites in protein structures, listing the nucleotides to which proteins bind, the protein names, and the organism names in Table S1 of DOI:10.1371 / journal.pone.0050240 (incorporated herein by reference). Therefore, the nucleotide-binding localization moieties are known in the art and can be identified by those skilled in the art for use as localization moieties in the present composition.

[0158] Localization site for binding to oligosaccharide substrates The oligosaccharide-binding moiety includes carbohydrate-binding proteins and is an important target when considering antiviral and anticancer drugs. The localization moiety can be, for example, a lectin that promotes carbohydrate interaction sites. Exemplary molecules include small molecule boronolectins, nucleic acid-based boronolectins, and peptide boronolectins. For example, (Jin et al., Med. Res. Rev. 2010 March; 30(2):171-257; doi:10.1002 / med.20155) (incorporated herein by reference), specifically, see Figures 1-50 for binding molecules and the complexes formed. Publicly available computational methods for selecting small molecules with the ability to bind carbohydrates using developed bioinformatics are available, for example, see Zhao et al., Current Protocols in Protein Science 94:1 10.1002 / cpps.75; Shionyu-Mitsuyama C, Shirai T, Ishida H, Yamane T (2003) Protein Eng 16:467-478; and Kulharia M, Bridgett SJ, Goody RS, Jackson RM (2009) “InCa-SiteFinder: a method for structure-based prediction of inositol and carbohydrate binding sites on proteins)”. J Mol Graph Model 28:297-303.

[0159] By analyzing the binding site residues in a protein-carbohydrate complex together with the stabilizing residues, it becomes possible to identify the folding and binding of the complex and understand the interactions in addition to non-covalent interactions such as hydrogen bonds and non-polar interactions. For example, see Shanmugam et al., doi.:10.2174 / 0929866525666180221122529. Using publicly available tools, the carbohydrate-binding moiety, including the binding site and the predicted folding, can be used in the design of multifunctional molecules containing such carbohydrate-binding localization moieties.

[0160] Localization site for binding to lipid substrates Lipid-binding moieties can be used as localization sites in the polyfunctional molecules disclosed herein. Modifications of their composition, distribution, or transport may be useful in the treatment, regulation, and / or modification of pathways, processes, and conditions as regulators of cell stability and signal transduction. Lipids include charged lipids, such as phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI) and PI phosphate, PI diphosphate and PI triphosphate (PIP - a family of seven anionic charged lipids), and gangliosides (GM). Zwitterionic lipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) lipids, ceramide (CER), diacylglycerol (DAG) and lysophosphatidylcholine (LPC) lipids, sphingolipids, glycerophospholipids, cholesterol, and phosphatidylglycerol.

[0161] Lipid-binding moieties of proteins and other proteins can either bind specifically to lipids (in which case a clear binding site on a given lipid can be identified) or bind nonspecifically (in which case the lipid acts as a medium), and physical properties such as thickness, fluidity, or curvature regulate protein function. Phosphoinositide-binding domains, such as the FRRG motif in the β-propeller of FYVE, PX, or PROPPIN, are more common domains and can be used to identify lipid-binding proteins. The FYVE domain, named after the first four proteins containing the motif (Fab1, YOTB, Vac1, EEA1), contains several conserved regions, which can also be used to identify related domains. See, for example, AHLystad, A. Simonsen, “Phosphoinositide-binding proteins in autophagy”, FEBS Lett., 590 (2016), pp. 2454-2468, 10.1002 / 1873-3468.12286. Further FYVE domain-containing proteins include SARA, FRABIN, DFCP1 FGD1, ANKFY1, EEA1 FGD1, FGD2, FGD3, FGD4, FGD5, FGD6, FYCO1, HGS MTMR3, MTMR4, PIKFYVE, PLEKHF1, PLEKHF2, RUFY1, RUFY2, WDF3, WDFY1, WDFY2, WDFY3, ZFYVE1, ZFYVE16, ZFYVE19, ZFYVE20, ZFYVE21, ZFYVE26, ZFYVE27, ZFYVE28, and ZFYVE9.

[0162] Eukaryotic cells can degrade intracellular components through a lysosomal degradation pathway called macroautophagy, and dysfunction of this pathway is associated with several diseases. Dikic et al., “Mechanism and medical implications of mammalian autophagy”. Nat. Rev. Mol. Cell Biol., 19(2018), pp.349-364, doi:10.1038 / s41580-018-0003-4. Therefore, autophagy-related (ATG) proteins, including LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2, can be used as lipid-binding moieties in this invention. De la Ballina (2019), doi.org / 10.1016 / j.jmb.2019.05.051. Lipid-binding proteins include HCLS1-binding protein 3 (HS1BP3), which can negatively regulate the activity of phospholipase D1 (PLD1).

[0163] Linker section A linker or linking portion is a bifunctional or polyfunctional portion that can be used to link one or more activator portions to a localization portion. In some embodiments, the linker has a functional group capable of reacting with such portions for covalent attachment. The linker portion is preferably a chemical linker portion, represented as L in formulas IA and IB. The linker may be cleavable or incleavable. In embodiments, the linker may be cleavable and can be selected in relation to in vivo efficacy, safety and degradation rate. The linker may be immobile, mobile or cleavable in vivo and can be reasonably designed based on the properties of the polyfunctional molecular portion and further design considerations detailed herein. For considerations of the rational design of linkers in pharmaceutical formulations, see, for example, Chen et al., Adv Drug Deliv Rev. 2013 Oct 15;65(10):1357-1369. Linker lengths can vary, and the degree of ternary complex formation and various linker lengths can be studied to determine the level of modification by the activator moiety. Linkers can be inherently bifunctional or polyfunctional, and multiple activator moieties can be attached to one or more functional groups of the linker. In a detailed example, polyfunctional linkers allow for the attachment of vectors and activator molecules to multiple sites on the linker. Branched linkers with length and functionality tailored for each activator molecule are within the scope of the present invention.

[0164] The linkers that can be used include PEG molecules, alkyls, heterocycloalkyls, cycloalkyls, aryls, alkylenes, alkenyls, heteroaryls, amides, amines, thiols, or derivatives thereof. The linker may be a polyfunctional linker, or in embodiments, a polyfunctional PEG linker.

[0165] In certain embodiments, the linker is the product of an azide / alkyne[3+2] cycloaddition or selected from amides, carbamates, esters, ureas, thioureas, and PEG molecules. In preferred embodiments, the linker is a PEG molecule, an alkyl or click chemistry linker, such as trans-cyclooctene, cyclooctin, or terminal alkynes, for example, the reagent N-(1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-ylmethyloxycarbonyl-1,8-diamino-3,6-dioxaoctane ("BCNamine").

[0166] In one aspect, the connecting portion relates to the following formula. [ka]

[0167] In the formula, n is between 1 and 15, preferably n = 3, 5, 7, 9, or 11.

[0168] In the embodiment, the connecting portion is formula [ka] (In the formula, n is 0 to 10, 0 to 5, or 0 to 2, and m is 0 to 10, 0 to 5, or 0 to 3) This relates to the matter.

[0169] In this embodiment, the linker portion is given by formula: [ka] (In the formula, n is between 1 and 50) This relates to the matter.

[0170] Linker, [ka] (In the formula, X is either O or CH2, and n is between 0 and 10.) It may relate to. In a detailed embodiment, when X is O, n = 1 or 2, and when X = CH2, n = 1.

[0171] In an embodiment, the linker moiety

Chemical formula

Chemical formula

[0172] Activator moiety The activator moiety can be selected based on the type of modification desired. As used herein, the activator moiety can effect a modification on a substrate that inhibits or activates that substrate. In one embodiment, the activator moiety has the ability to find and modulate an enzyme, e.g., activate or inhibit it. In certain embodiments, the activator moiety is an inhibitor molecule such as an allosteric inhibitor. In an embodiment, the enzyme is a kinase, phosphatase, transferase, ligase, histone acetylase (HAT) or histone deacetylase (HDAC), hydroxylase, glutamine synthetase adenyl transferase (GSATase), an enzyme that catalyzes the hydroxylation of protein residues, oxygenase or sulfotransferase.

[0173] The activator moiety may be selected based on the type of modification, such as post-translational modification. In a detailed example, the activator moiety has the ability to locate and activate an enzyme, and therefore the type of enzyme to be activated may be selected in relation to the desired modification of the target substrate. One type of modification performed is post-translational modification (PTM). This may include peptide bond cleavage, disulfide bond formation, acylation, prenylation, lipoylation, acetylation, deacetylation, formylation, alkylation, carbonylation, phosphorylation, glycosylation or lipidation, hydroxylation, S-nitrosylation, S-sulfenylation, sulfinylation, sulfonylation, succinylation, sulfation, and malonylation (Taherzadeh et al. 2018). Thus, post-translational modifying enzymes constitute a set of activators intended for use in the present invention.

[0174] The activator may be selected based on the desired substrate modification. In embodiments, the activator provides modification to the amino acid; see, for example, Table 1 in Karve et al., Journal of Amino Acids Volume 2011, Article ID 207691, 13 pages, DOI: 10.4061 / 2011 / 207691 (incorporated herein by reference).

[0175] In one embodiment, the activator is a kinase activator moiety. The kinase activator moiety may be a small molecule or compound that activates the kinase. As used herein, a kinase is an enzyme that adds a phosphate group to an amino acid of another molecule, typically a protein substrate. The kinase activator enhances phosphorylation. In detailed embodiments, the kinase activator moiety promotes the activity conformation of the enzyme, in one aspect through binding interactions with a regulatory subunit. See, for example, Zorn et al., Nat. Chem. Biol. (2010), doi:10.1038 / NCHEMBIO.318. In embodiments, the kinase acts on the amino acids serine, threonine, tyrosine, or a combination thereof.

[0176] In some embodiments, the activator is an activator of protein kinase C isozyme. In some embodiments, the activator is an activator of AMPK. In some embodiments, the activator is an activator of Src kinase or shares sequence homology with the Src kinase family. In some embodiments, the kinase is c-Abl, a non-receptor tyrosine kinase. In some embodiments, the kinase is Bruton's tyrosine kinase (BTK). The activator may be an activator of insulin receptor tyrosine kinase. In some embodiments, the activator moiety is an inhibitor of RAC-α-serine / threonine-protein kinase (AKT) or mitogen-activated protein kinase (MEK).

[0177] The activator portion can be identified from activators known in the art. The activator may be a derivative of a activator known in the art and may contain fewer or additional functional groups, while still being usable as an activator, it may enhance or promote the desired formation, conformation, or binding site for the polyfunctional molecules described herein. Exemplary modifications may include derivatives that increase solubility, charge, or functionality for use with orientation adapters or linkers detailed in other parts of this specification.

[0178] FKBP activator part In a detailed embodiment, the activator can be designed as an activator of FK506-binding protein (FKBP). In one embodiment, the FKBP is FKBP12, which binds to intracellular calcium release channels and TGF-β type I receptors. In one embodiment, the FKBP activator moiety is [ka] That is the case.

[0179] PKC activator part In detailed embodiments, the activator can be designed as an activator for diacylglycerol (DAG)-reactive C1 domain-containing proteins, such as protein kinase C. Protein kinase C (PKC) comprises multiple isozymes and exhibits tissue-specific expression and various biological roles by playing a role in signaling pathways. The activator of PKC can be used in the small chimeric molecules disclosed herein, and this activating moiety is selective with respect to PKC isoforms.

[0180] Activators of DAG-reactive proteins may include DAG-indolactones as described in LCGarcia et al., Bioorg. Med. Chem., 22(2014) 3123-3140. An exemplary DAG-indolactone is given by formula [ka] (In the formula, R is indole.) This may relate to the following: R may be, for example, 1-methyl,1H-indole5-yl, 1-methyl,1H-indole6-yl, 1-methyl,1H-indole4-yl, or 1-methyl,1H-indole7-yl. In embodiments, this compound is selective with respect to PKCα or PKCε.

[0181] The DAG lactone is a DAG lactone such as AJH-836, as described in Cooke, et al., J. Biol. Chem. (2018) 293(22) 8330-8341. In embodiments, the DAG lactone is a DAG lactone of formula [ka] It may relate to the following. As provided to Cooke, the formula for AJH-836 is, [ka] It is selective with respect to PKCδ and PKC.

[0182] Teleocidines such as (-)-indolactam-V (ILV) and benzolactam-V8, for example, 7-substituted benzolactam-V8, can be used as PKC activators. PKC activators may be as described in Ma, et al., Org. Lett. 4:14 (2002) DOI: 10.1021 / ol026125l.

[0183] In the embodiment, the PKC activator is, [ka] (In the formula, R1, R3, and R4 are each independently alkyl, alkenyl, and alkynyl, and R2 is saturated or unsaturated alkylene (e.g., branched alkylene, linear alkylene, cycloalkylene, C1-C) 22 Branched alkylene, C1~C 22 Linear alkylenes, C3~C 22 Cycloalkylene, C1~C 10 Branched alkylene, C1~C 10 Linear alkylenes, C3~C 10 Cycloalkylene, C1-C8 branched alkylene, C1-C8 linear alkylene, C3-C8 cycloalkylene), C1-C 22 Saturated or unsaturated heteroalkylenes (e.g., branched heteroalkylenes, linear heteroalkylenes, heterocycloalkylenes, C1-C) 22 Branched heteroalkylene, C1~C 22 Linear heteroalkylene, C3~C 22 Heterocycloalkylene, C1~C 10 Branched heteroalkylene, C1~C 10 Linear heteroalkylene, C3~C 10 Heterocycloalkylenes, C1-C8 branched heteroalkylenes, C1-C8 linear heteroalkylenes, C3-C8 heterocycloalkylenes), arylenes (e.g., C5-C) 22 Arylenes), heteroarylenes (e.g., C5~C) 22 (Can be selected from heteroarylenes or divalent hydrocarbons selected from combinations thereof.) This relates to; where each of the foregoing may have one or more (e.g., two, three, four, or five) substitution sites, substitution amides, including those selected from those listed in Table 1 on page 366 of Kozikowski et al. J. Med. Chem, 2003, 46:3, 364-373 (as specifically incorporated herein by reference). R2 is -(C(R a )(R a )) 1~8 -,-(OC(R a )(R a )) 1~8 -,-(OC(R a )(R a )-C(R a )(R a )) 1~8 -, -N(R a )-, -O-, -C(O)-, C6 arrine substituted by choice, C substituted by choice 5~12 Heteroarylene, hydroxyl-substituted C 3~6 It may be selected from one or more cycloalkylenes or hydroxylated C4 heterocycloalkylenes; where each of the foregoing may have one or more (e.g., 2, 3, 4, 5) substitution sites; and R a Each instance is independently selected from hydrogen or alkyl (e.g., C1-C7 alkyl, C1-C3 alkyl).

[0184] In a detailed embodiment, this formula is: [ka] (In the formula, R1, R3, and R4 are each independently alkyl, alkenyl, and alkynyl, and R2 is saturated or unsaturated alkylene (e.g., branched alkylene, linear alkylene, cycloalkylene, C1-C) 22 Branched alkylene, C1~C 22 Linear alkylenes, C3~C 22 Cycloalkylene, C1~C 10 Branched alkylene, C1~C 10 Linear alkylenes, C3~C 10Cycloalkylene, C1-C8 branched alkylene, C1-C8 linear alkylene, C3-C8 cycloalkylene), C1-C 22 Saturated or unsaturated heteroalkylenes (e.g., branched heteroalkylenes, linear heteroalkylenes, heterocycloalkylenes, C1-C) 22 Branched heteroalkylene, C1~C 22 Linear heteroalkylene, C3~C 22 Heterocycloalkylene, C1~C 10 Branched heteroalkylene, C1~C 10 Linear heteroalkylene, C3~C 10 Heterocycloalkylenes, C1-C8 branched heteroalkylenes, C1-C8 linear heteroalkylenes, C3-C8 heterocycloalkylenes), arylenes (e.g., C5-C) 22 Arylenes), heteroarylenes (e.g., C5~C) 22 (Can be selected from heteroarylenes or divalent hydrocarbons selected from combinations thereof.) This relates to; where each of the foregoing may have one or more (e.g., two, three, four, or five) substitution sites, substitution amides, including those selected from those listed in Table 1 on page 366 of Kozikowski et al. J. Med. Chem, 2003, 46:3, 364-373 (specifically incorporated herein by reference).

[0185] R2 is -(C(R a )(R a )) 1~8 -,-(OC(R a )(R a )) 1~8 -,-(OC(R a )(R a )-C(R a )(R a )) 1~8 -, -N(R a )-, -O-, -C(O)-, C6 arrine substituted by choice, C substituted by choice 5~12 Heteroarylene, hydroxyl-substituted C 3~6It may be selected from one or more cycloalkylenes or hydroxylated C4 heterocycloalkylenes; where each of the foregoing may have one or more (e.g., 2, 3, 4, 5) substitution sites; and R a Each instance is independently selected from hydrogen or alkyl (e.g., C1-C7 alkyl, C1-C3 alkyl).

[0186] In a detailed embodiment, this formula is: [ka] (In the formula, R1, R3, and R4 are independently alkyl, alkenyl, and alkynyl, and R2 may be selected from the following.) This relates to the above. In embodiments, the PKC activator is a benzolactam analog of ILV, as described in Kozikowski et al., J. Med. Chem., 1997, 40:9 1316-1326, where R may be CC(CH2)7CH3 or (CH2)9CH3.

[0187] In some embodiments, R1, R3, and R4 are alkyl groups, and in some embodiments, R1, R3, and R4 are methyl groups. In certain embodiments, this formula is [ka] This relates to the matter.

[0188] In embodiments, the PKC activator is a natural product activator, such as DPP, prostratin, mezelein, octahydromezelein, thimereatoxin, (-)-octylindolactam V, OAG, or resiniferatoxin, as described in Kazanietz et al., Mol. Pharma. 44:296-307 (1993).

[0189] In embodiments, the PKC activator is selective with respect to PKCδ. In detailed embodiments, the PKC activator is 7α-acetoxy-6β-benzoyloxy-12-O benzoylleureanone (Roy-Bz) as described in Bessa et al., Cell Death and Disease (2018) 9:23.

[0190] The PKC activator may be an ILV derivative, such as n-hexyl ILV or 10-membered ring 1-hexyl indolactam V10 or derivatives thereof, as described in Yanagita, et al., J. Med. Chem., 2008, 51:1, 46-56 (incorporated herein by reference). The PKC activator is [ka] (In the formula, R1 and R2 = H, R1 = H and R2 = Cl, or R1 = Br and R2 = H) It can be PKCδ, PKCε, or PKCη in some cases, and in some cases it can be PKCδ, PKCε, or PKCη.

[0191] In embodiments, the activator portion is 6-chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic acid (PF-06409577), benzolactam, DPP, prostratin, mezelein, octahydromezelein, thimeleatoxin, (-)-indolactam V, (-)-octylindolactam V, OAG, or derivatives thereof.

[0192] In embodiments, the activating agent portion is thieno[2,3-b]pyridine, thienopyridone, quinoxalindione, imidazo[4,5-b]pyridine, [2,3-d]pyridine, benzimidazole, pyrrolo[2,3-d]pyrimidine, spiro-ring indolinone, tetrahydroquinoline, thieno[2,3-b]pyridinedione, and derivatives thereof. See Expert Opin Ther. Patents (2012) 22(12) (incorporated herein by reference). [Table 1] JPEG2026076184000028.jpg250170JPEG2026076184000029.jpg255169JPEG2026076184000030.jpg255170 JPEG2026076184000031.jpg238170JPEG2026076184000032.jpg255169JPEG2026076184000033.jpg255169 JPEG2026076184000034.jpg242170JPEG2026076184000035.jpg255169JPEG2026076184000036.jpg253170 JPEG2026076184000037.jpg255169JPEG2026076184000038.jpg253170JPEG2026076184000039.jpg251170

[0193] Using NASA chemistry, the activator moiety can be functionalized, and a localization moiety can be further added thereto. NASA chemistry is generally described in Nat Commun 9, 1870 (2018) (incorporated herein by reference). In certain embodiments, the PKC activator moiety can be attached to the localization moiety using an N-acyl N-alkylsulfonamide (NASA) warhead. In one embodiment, the NASA warhead is [ka] (In the formula, R includes a fluorescent dye, a BRD4 binder, an FKBP binder, an MDM2 binder, an ER binder, or any other binder for the desired protein.) Includes.

[0194] In another method, the PKC activator moiety is labeled with tetracyclooctene (TCO) using NASA chemistry, which allows it to be clicked to any target protein binder using tetrazine click chemistry. Thus, in one embodiment, the PKC activator is given by formula: [ka] Prepared according to, then this, [ka] (In the formula, R is a binder for any target protein, such as a localization site.) It can be reacted with functionalized tetrazine according to the following. Accordingly, one embodiment includes a method for preparing the compositions disclosed herein using NASA chemistry and as further described in the examples.

[0195] AMPK activator part AMPK is a serine / threonine kinase that organizes into a heterotrimeric complex composed of a catalytic α-subunit and two regulatory β and γ-subunits. See, for example, Wells et al. (2012). Small molecules that mimic AMP binding to the γ-subunit are thought to be able to directly activate AMPK. AMPK activators can be selected from the AMPK activators disclosed herein.

[0196] In this embodiment, the AMPK activator is [ka] The following are selected. Other AMPK activators include A769662 (Cool et al., Cell Metab. 3, 403-416 (2006)) and PT1 (Pang et al., J. Biol. Chem. 283, 16051-16060 (2008)).

[0197] AMPK activators may be, for example, as described in U.S. Patent Application Publication No. 20050038068 (incorporated herein by reference), [ka] This may relate to the following. AMPK activators may be as described in International Publication Nos. 2007019914, 2009124636, 2009135580, 2008006432, or 2009152909 (incorporated herein by reference). In embodiments, the activator is [ka] It may relate to this.

[0198] AMPK activators may be as described in International Publication No. 2009100130 (incorporated herein by reference). In one embodiment, the AMPK activator is [ka] This relates to the matter.

[0199] AMPK activators may be as described in International Publication Nos. 2010036613, 2010047982, 2010051176, 2010051206, 2011106273, or 2012116145. In embodiments, the AMPK activator is [ka] This relates to the matter.

[0200] In certain exemplary embodiments, the AMPK activator may be as described in International Publication Nos. 2011029855, 2011138307, 2012119979, and 2012119978 (incorporated herein by reference). In one embodiment, the AMPK activator is [ka] It can be selected from the following.

[0201] In certain exemplary embodiments, the AMPK activator may be as described in International Publication Nos. 2011032320, 2011033099, 2011069298, 2011070039, 2011128251, and 2012001020 (incorporated herein by reference). In one embodiment, the AMPK activator is [ka] It can be selected from the following.

[0202] In certain exemplary embodiments, the AMPK activator may be as described in International Publication No. 2011080277 (incorporated herein by reference). In one embodiment, the AMPK activator is [ka] It is possible.

[0203] In certain exemplary embodiments, the AMPK activator may be as described in International Publication No. 2012033149 (incorporated herein by reference). In one embodiment, the AMPK activator is [ka] It can be selected from the following.

[0204] Bruton's tyrosine kinase activator portion Bruton's tyrosine kinase (Btk) is involved in multiple signaling cascades and plays a role in B cell development and oncogenic signaling. See, for example, Singh et al., 2018; Pal et al., 2018. In embodiments, the Btk activator is ibrutinib or a derivative thereof. [ka]

[0205] In a particular exemplary embodiment, the Btk activator is [ka] Selected from.

[0206] In a particular exemplary embodiment, the Btk activator portion includes: [ka] The targeting portion is provided.

[0207] The linker, if present, may include an alkyl chain containing 5 to 10, more preferably 6 to 8 carbon atoms. In certain exemplary embodiments, the molecule containing the Btk activator moiety is: [ka] That is the case.

[0208] ABL kinase activator portion Abelson kinase (c-Abl) is a ubiquitously expressed non-receptor tyrosine kinase that plays a crucial role in cell differentiation and survival. (Simpson, et al., J.Med.Chem. 2019 62, 2154-2171). ABL tyrosine kinase can be found in the nucleus, cytoplasm, and mitochondria. In embodiments, the c-Abl kinase activator is (5-[3-(4-fluorophenyl)-1-phenyl-1H-pyrazole-4-yl]-2,4-imidazolidinedione or 5-(1,3-diaryl-1H-pyrazole-4-yl)hydantoin) as described in Yang et al., “Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site”, Chem.&Biol., 18, 177-186, Feb. 25, 2011; DOI:10.1016 / j.chembiol.2010.12.013): [ka] (DPH). In certain embodiments, the c-Abl kinase activator is [ka] These may be selected from those that demonstrated in vivo activation of c-Abl in Simpson et al. 2019. The novel aminopyrazoline small molecule activators listed in Table 6 of Simpson et al. are specifically incorporated herein by reference.

[0209] In a particular exemplary embodiment, the c-Abl kinase activator moiety is [ka] That is the case.

[0210] In a particular exemplary embodiment, the compound is of the formula [ka] (In the formula, R is, [ka] (is) This relates to the matter.

[0211] In certain exemplary embodiments, DPH is functionalized. [ka]

[0212] In a particular exemplary embodiment, the ABL kinase activator is [ka] (In the formula, the dashed circles indicate the parts that attach to the orientation adapter and / or linker.) The functional groups depicted within the dashed circles of the ABL kinase activator can be used in a method of attaching the linker and orientation adapter before attaching them to the localization site.

[0213] The activator moiety can be functionalized for the attachment of the orientation adapter and linker. The ABL kinase activator parent molecule DPH can be functionalized for the attachment of the orientation adapter and linker. Exemplary molecules are, [ka] It is possible.

[0214] After functionalization is complete, orientation adapters and linkers can be added sequentially or all at once, where the orientation adapter and linker are added as a single molecule. Exemplary molecules are provided below, where the R group represents the localization moiety. [ka] Optionally, two or more activator moieties may be attached to the localization moiety. In any example, the identified activator moieties can be functionalized as described herein in the method for attaching the linker and orientation adapter, and then attached to the localization moiety, for example, using the functional groups shown in the dashed circles.

[0215] In one embodiment, the Abl kinase activator is DPH or a dihydropyrazole activator. Exemplary molecules are: [ka] (wherein X is (CH2)n, which may be substituted with one or more of the amide, acetal, aminal, amine, alkyl, ether, hydrocarbyl and derivatives thereof, or other groups as described in other parts of this specification.) This may include: In a particular embodiment, n is 0 to 20, more preferably n is 1 to 10 or 2 to 7, and R is [ka] In one embodiment, attachment to the ABL kinase activator dihydropyrazole is via various linkers, see, for example (PHICS 10.1-10.5, Figure 64A). In one embodiment, when the localization portion is for BRD4, [ka] Exemplary molecules are, [ka] It may include.

[0216] Insulin receptor (IRTK) activators In certain exemplary embodiments, the kinase activator moiety is a membrane-bound insulin receptor kinase. In one embodiment, the activator for ITRK is kojic acid or a derivative thereof.

[0217] In some embodiments, the target is AR. In some embodiments, the localization moiety may contain enzalutamide. In some embodiments, the enzalutamide is attached to a linker containing an azide terminus via an ether bond. Thus, in certain embodiments, it may be possible to link the activator moiety by bioorthogonal click chemistry by adding an alkyne functional group. See, for example, Figure 80. In certain embodiments, the insulin receptor is of formula [ka] (In the formula, X is C, N, O, S, or P) This relates to the matter.

[0218] CARM-1 activator In certain exemplary embodiments, the kinase activator portion is a CARM1 activator.

[0219] In a particular exemplary embodiment, the CARM1 activator is [ka] or (In the formula, the dashed circles indicate the parts that attach to the orientation adapter and linker.) Selected from the above. The functional groups depicted within the dashed circles of the CARM1 activator can be used in a method of attaching the linker and orientation adapter before attaching them to the localization portion.

[0220] Abl1 inhibitors Abl1 is a tyrosine protein kinase that is thought to be involved in the processes of cell differentiation, cell division, cell adhesion, and stress response. The structures of exemplary inhibitors dasatinib, imatinib, ponatinib, and nilotinib are provided in Example 7. In some embodiments, these inhibitors are selected to adjust the pharmacological effect on a dose basis in terms of their reaction rate and residence time, reduce off-target effects, and optimize residence time. The effects of the polyfunctional chimeric molecules of the present invention can be optimized using the methods described in Example 7.

[0221] MEK inhibitors In one embodiment, the moiety is a mitogen-activated protein kinase inhibitor. In one embodiment, the MEK inhibitor is trametinib functionalized with an alkyne used in a bioorthogonal click chemistry reaction with an azide-functionalized localization moiety. The MEK moiety can be synthesized according to the guidance and designs provided herein, taking into account the MEK binding moiety as disclosed, for example, in Sweeney et al. Ann Rheum Dis. 65(3):iii83-iii88 (2006); Wu et al. Pharm Ther 156:59-68 (2015); Suplatov et al. J Biomol Struct Dyn 37(8):2049-2060 (2018); Heald et al. J Medicin Chem 55(10):4594-4604 (2012); Force et al. Circulation 109:1196-1205 (2004). [ka]

[0222] As the activating portion of the polyfunctional chimeric molecule of the present invention, exemplary p38α mitogen-activated protein kinase inhibitors SB5, SB6, SB7, B12, B96, BR5, BR8, and BMU, and their derivatives, as shown in Figure 89, can be used.

[0223] AKT inhibitors In one embodiment, the portion is an RAC-α-serine / threonine-protein kinase (AKT) inhibitor. In one embodiment, the ATK inhibitor is borussertib functionalized with an alkyne used in a bioorthogonal click chemistry reaction with an azide-functionalized localization portion as described in other parts of this specification. The AKT portion can be synthesized according to the guidance and designs provided herein, taking into account the AKT binding portion, as disclosed, for example, in Panicker et al. Adv Exp Med Biol 1163:253-278 (2019); Botello-Smith et al. PLoS Comp Biol 13(8):e1005711 (2017); Mou et al. Chem Biol Drug Des 89(5):723-731 (2017); Ruiz-Carillo et al. Sci Rep 8:7365 (2018) and Budas et al. Biochem Soc Trans 35:1021-1026 (2007). [ka]

[0224] Orientation adapter A polyfunctional molecule may include one or more orientation adapters. In embodiments, the orientation adapter can be an example of either a localization moiety or an activator moiety. In one embodiment, the orientation adapters are attached to both ends of a linker molecule, where the orientation adapters are attached to each activator moiety of the polyfunctional molecule and optionally provide orientation adapters that interconnect a chemical linker molecule to another end of a localization moiety.

[0225] In embodiments, the orientation adapter is a small molecule group that assists in the orientation of the localization moiety and the activator. In embodiments, the orientation adapter is selected so that the small molecule compound binds in one of its preferred low-energy conformations. For example, when a protein is the substrate, the protein dissipates strain energy more easily by changing little over its degrees of freedom compared to when the small molecule adopts an undesirable conformation by distorting its few rotatable bonds. Therefore, “soft” energy or low-energy twist barriers are useful in designing small molecule compounds. When designing orientation molecules between aryl rings, priority can be considered. Anisoles (ArOCH2R) and anilines (ArNHR) preferentially adopt coplanar conformations, while alkylaryls (ArCH2R), arylsulfonamides, and arylsulfones preferentially adopt orthogonal conformations. The orientation adapter atom controls both distance and direction. For example, see Brameld et al. J. Chem. Inf. Model. 2008, 48, 1-24.

[0226] Orientation adapters are sometimes referred to as exit vectors in embodiments. Exit vector parameters can be identified, in part, based on the average orientation of substituents attached to the variable group site, which can be generated using cheminformatics software. Exit vectors may include bonds emanating from a chemical moiety. In certain embodiments, this bond is selected to act energetically favorably, preferably increasing bond affinity. Orientation adapters may be represented together with a linker in certain embodiments and may be adjusted depending on the linker used for a polyfunctional molecule. In embodiments, the orientation adapter is a chemical moiety or bond that promotes stereochemical protrusion, which may further promote subsequent coupling, bonding, and / or ease of contact. In embodiments, first and second orientation adapters are provided as bonds on a linker, providing conformation that connects the linker with the activator moiety and / or localization moiety.

[0227] In the embodiment, the first and second orientation adapters, if present, are independently selected from Table 2 (Orientation Adapter Table). [Table 2]

[0228] Methods for modifying target substrates The polyfunctional molecules disclosed herein can be used in methods for modifying target substrates. Methods for modifying target substrates may include contacting the target substrate with the polyfunctional molecules of the present invention. Contact may enable binding or association with the target substrate or molecules adjacent to the target substrate. In some embodiments, if the target substrate is not a native substrate of the enzyme, or if the activation of the enzyme by an activator molecule is not activated by binding to the activator moiety, then enzymatic modification of the target substrate occurs at one or more novel modification sites that would otherwise remain unmodified by the enzyme. Modifications may include, for example, post-translational modifications as disclosed herein, including phosphorylation, hydroxylation, acetylation, methylation, glycosylation, prenylation, amidation, elimination, lipidation, acylation, lipoylation, deacetylation, formylation, S-nitrosylation, S-sulfenylation, sulfonylation, sulfinylation, succinylation, sulfation, carbonylation, or alkylation. In some embodiments, the method includes inducing the phosphorylation of a protein in a cell. In one embodiment, the insulin phosphorylation cascade can be replicated by the use of small molecule compounds. This method may involve contacting a target substrate with a polyfunctional molecule. In a detailed embodiment, the target substrate is in close proximity to a kinase specific to the activator portion of the molecule. The polyfunctional molecule that induces phosphorylation may optionally be adenosine monophosphate (AMP) or another molecule that provides an additional phosphate group. Although not constrained by theory, the addition of a molecule that provides AMP or another phosphate may enhance phosphorylation.

[0229] target substrate The activator portion of the present invention binds to, associates with, and / or activates an enzyme that modifies a target substrate associated with a localization portion. In some embodiments, the target substrate is not the enzyme's native substrate, i.e., not the substrate to which the activator portion associates. In one embodiment, when the enzyme is activated by the activator molecule, it results in enzyme modification of the target substrate at one or more new modification sites that would otherwise remain unmodified by the enzyme if not activated by binding to the activator portion. The target substrate may be a substrate to which the localization portion can bind, or to which the localization portion can associate in other ways, or a substrate known to be in close proximity to the localization portion, and therefore the activator portion is within a distance range that modifies the target substrate, for example, within a close range. The target substrate does not need to be the enzyme's native substrate. The target substrate may be a protein, and the considerations herein regarding genes include products of gene expression. Further applications may include modification of protein-DNA interactions, e.g., Myc, or protein-protein interactions. Applications may include, for example, phosphorylation to alter the charge of nucleic acids or protein molecules.

[0230] As a non-limiting example, proteins associated with secretase disorders include PSENEN (presenilin enhancer 2 homolog (C. elegans)), CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid β (A4) precursor protein), APH1B (prepharyngeal deficiency 1 homolog B (C. elegans)), PSEN2 (presenilin 2 (Alzheimer's disease 4)), BACE1 (β-site APP cleavage enzyme 1), ITM2B (membrane-intrinsic protein 2B), and CTSD (cathepsin D). NOTCH1 (Notch homolog 1, translocation-related (Drosophila)), TNF (tumor necrosis factor (TNF superfamily, member 2)), INS (insulin), DYT10 (dystonia 10), ADAM17 (ADAM metallopeptidase domain 17), APOE (apolipoprotein E), ACE (angiotensin I-converting enzyme (peptidyl dipeptidase A) 1), STN (statin), TP53 (tumor protein p53), IL6 (interleukin 6 (interferon, beta 2)), NGFR (neuronal Growth factor receptor (TNFR superfamily, member 16), IL1B (interleukin 1, beta), ACHE (acetylcholinesterase (Yt blood type)), CTNNB1 (catenin (cadherin-related protein), beta 1, 88kDa), IGF1 (insulin-like growth factor 1 (somatomedin C)), IFNG (interferon, gamma), NRG1 (neuregulin 1), CASP3 (caspase 3, apoptosis-related cysteine ​​peptidase), MAPK1 (mitogen-activated protein kinase 1), CDH1 (cadherin 1, Type 1, E-cadherin (epithelial), APBB1 (amyloid β (A4) precursor protein binding, Family B, Member 1 (Fe65)), HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase), CREB1 (cAMP response element binding protein 1), PTGS2 (prostaglandin endoperoxide synthase 2 (prostaglandin G / H synthase and cyclooxygenase)), HES1 (hairy and split enhancer 1, (Drosophila)), CAT (catalase),TGFB1 (transforming growth factor, beta-1), ENO2 (enolase 2 (gamma, neuron)), ERBB4 (v-erb-a erythroblastic leukemia virus oncogene homolog 4 (tori)), TRAPPC10 (transport protein particle complex 10), MAOB (monoamine oxidase B), NGF (nerve growth factor (beta polypeptide)), MMP12 (matrix metallopeptidase 12 (macrophage elastase)), JAG1 (jagged 1 (Alagille syndrome)), CD40LG (CD40 ligand), PPARG (peroxy Somal growth factor-activated receptor gamma), FGF2 (fibroblast growth factor 2 (basic)), IL3 (interleukin 3 (colony-stimulating factor, multiple)), LRP1 (low-density lipoprotein receptor-related protein 1), NOTCH4 (Notch homolog 4 (Drosophila)), MAPK8 (mitogen-activated protein kinase 8), PREP (prolyl endopeptidase), NOTCH3 (Notch homolog 3 (Drosophila)), PRNP (prion protein), CT SG (Cathepsin G), EGF (Epidermal Growth Factor (Betaurogastron)), REN (Renin), CD44 (CD44 molecule (Indian blood type)), SELP (Selectin P (Granulosa membrane protein 140kDa, Antigen CD62)), GHR (Growth hormone receptor), ADCYAP1 (Adenylyl cyclase-activated polypeptide 1 (Pituitary gland)), INSR (Insulin receptor), GFAP (Glial fibrillary acidic protein), MMP3 (Matrix metallopeptidase 3 (Stromelysin 1, Progelatinase)), MAPK10 (Mitojing 10) , SP1 (Sp1 transcription factor), MYC (v-myc myelocytoma virus oncogene homolog (Tri)), CTSE (Cathepsin E), PPARA (Peroxisome Proliferator-Activated Receptor Alpha), JUN (Jun Oncogene), TIMP1 (TIMP Metallopeptidase Inhibitor 1), IL5 (Interleukin 5 (Colony Stimulating Factor, Eosinophil)), IL1A (Interleukin 1, Alpha), MMP9 (Matrix Metallopeptidase 9 (Gelatinase B, 92kDa Gelatinase, 92kDa Type IV Collagenase)),HTR4 (5-hydroxytryptamine (serotonin) receptor 4), HSPG2 (heparan sulfate proteoglycan 2), KRAS (v-Ki-ras2 Carsten rat sarcoma virus oncogene homolog), CYCS (cytochrome c, somatic), SMG1 (SMG1 homolog, phosphatidylinositol 3-kinase-related kinase (C. elegans)), IL1R1 (interleukin 1 receptor, type I), PROK1 (prokinethicin 1), MAPK3 (mitogen-activated protein kinase 3), NTRK1 (neurotrophic cytotoxicin 1 (Prostaglandin kinase, receptor, type 1), IL13 (interleukin 13), MME (membrane-type metalloendopeptidase), TKT (transketolase), CXCR2 (chemokine (CXC motif) receptor 2), IGF1R (insulin-like growth factor 1 receptor), RARA (retinoic acid receptor, alpha), CREBBP (CREB-binding protein), PTGS1 (prostaglandin endoperoxide synthase 1 (prostaglandin G / H synthase and cyclooxygenase)), GALT (galactose-1-phosphate uridilyltransferase) (Lase), CHRM1 (Cholinergic receptor, muscarinergic 1), ATXN1 (Ataxin 1), PAWR (PRKC, apoptosis, WT1, regulator), NOTCH2 (Notch homolog 2 (Drosophila)), M6PR (Mannose-6-phosphate receptor (cation-dependent)), CYP46A1 (Cytochrome P450, Family 46, Subfamily A, Polypeptide 1), CSNK1D (Casein kinase 1, Delta), MAPK14 (Mitogen-activated protein kinase 14, also known as p38-α), PR G2 (Proteoglycan 2, bone marrow (natural killer cell activator, major basic protein of eosinophil granules)), PRKCA (protein kinase C, alpha), L1CAM (L1 cell adhesion molecule), CD40 (CD40 molecule, TNF receptor superfamily member 5), NR1I2 (nuclear receptor subfamily 1, group I, member 2), JAG2 (jagged 2), CTNND1 (catenin (cadherin-related protein), delta 1), CDH2 (cadherin 2, type 1, N-cadherin (neuron)), CMA1 (chymase 1, mast cell),SORT1 (Sortilin 1), DLK1 (Delta-like 1 homolog (Drosophila)), THEM4 (Thioesterase superfamily member 4), JUP (Junction placoglobin), CD46 (CD46 molecule, complement regulatory protein), CCL11 (Chemokine (CC motif) ligand 11), CAV3 (Caveolin 3), RNASE3 (Ribonuclease, RNase A family, 3 (eosinophil cationic protein)), HSPA8 (Heat shock 70kDa protein 8), CASP9 (Caspase 9, apoptosis-related cysteine ​​peptidase), CYP3A4 (Cytochrome P450) Family 3, subfamily A, polypeptide 4), CCR3 (chemokine (CC motif) receptor 3), TFAP2A (transcription factor AP-2 alpha (activating enhancer-binding protein 2 alpha)), SCP2 (sterol carrier protein 2), CDK4 (cyclin-dependent kinase 4), HIF1A (hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)), TCF7L2 (transcription factor 7-like 2 (T cell specific, HMG box)), IL1R2 (interleukin 1 receptor, type II), B3GALTL (beta-1,3-galactosyltransferase-like), MDM2 (Mdm2 p53 binding protein homolog (mouse), RELA (v-rel reticuloendotheliopathy virus oncogene homolog A (bird)), CASP7 (caspase 7, apoptosis-related cysteine ​​peptidase), IDE (insulin-degrading enzyme), FABP4 (fatty acid binding protein 4, adipocyte), CASK (calcium / calmodulin-dependent serine protein kinase (MAGUK family)), ADCYAP1R1 (adenylate cyclase-activated polypeptide 1 (pituitary) receptor type I), ATF4 (activating transcription factor 4 (tax-reactive enhancer element B67)), PDGFA (platelet-derived growth factor alpha polypeptide), C21orf33 (chromosome 21 open reading frame 33), SCG5 (secretogranin V (7B2 protein)), RNF123 (RING finger protein 123), NFKB1 (nuclear factor 1 of kappa light chain polypeptide gene enhancer in B cells),ERBB2 (v-erb-b2 erythroblastic leukemia virus oncogene homolog 2, neuronal / glioblastoma-derived oncogene homolog (bird)), CAV1 (caveolin 1, caveolae protein, 22kDa), MMP7 (matrix metallopeptidase 7 (matrilysin, uterus)), TGFA (transforming growth factor, alpha), RXRA (retinoid X receptor, alpha), STX1A (syntaxin 1A (brain)), PSMC4 (proteasome (prosome, macropain) 26S subunit, ATPase, 4), P2RY2 (purine receptor P2Y, G protein-coupled, 2), TNFRSF21 (tumor necrosis factor receptor superfamily, member 21), DLG1 (discs, large homolog 1 (Drosophila genus (D) Examples include rosophila, NUMBL (numb homolog (Drosophila)-like), SPN (sialophorin), PLSCR1 (phospholipid scramblase 1), UBQLN2 (ubiquilin 2), UBQLN1 (ubiquilin 1), PCSK7 (proprotein convertase subtilisin / kexin type 7), SPON1 (spongin 1, extracellular matrix protein), SILV (silver homolog (mouse)), QPCT (glutaminyl peptide cyclotransferase), HESS (hairy and split enhancer 5 (Drosophila)), GCC1 (GRIP and coiled-coil domain-containing 1), and any combination thereof.

[0231] Further targets include those thought to be related to fatty acid disorders. In certain embodiments, the target is one or more of ACADM, HADHA, and ACADVL. In embodiments, the targeted editing is the activity of an intracellular gene selected from the medium-chain fatty acid acyl coenzyme A dehydrogenase (ACADM) gene, the long-chain fatty acid long-chain 3-hydroxyl coenzyme A dehydrogenase (HADHA) gene, and the very long-chain fatty acid acyl coenzyme A dehydrogenase (ACADVL) gene. In one embodiment, the disease is medium-chain acyl coenzyme A dehydrogenase deficiency (MCADD), long-chain 3-hydroxyl coenzyme A dehydrogenase deficiency (LCHADD), and / or very long-chain acyl coenzyme A dehydrogenase deficiency (VLCADD). A further target may be angiopoietin-like 4 (ANGPTL4). Treatable ANGPTL4-related diseases or disorders include ANGPTL4, which is associated with dyslipidemia, low plasma triglyceride levels, angiogenesis regulation, tumorigenesis regulation, and severe diabetic retinopathy.

[0232] In certain embodiments, the disease or disorder is related to apolipoprotein C3 (APOCIII), which can be targeted for modification. In some embodiments, targets include recombinant activator gene 1 (RAG1), BCL11 A, PCSK9, laminin, alpha 2 (lama2), ATXN3, alanine-glyoxylate aminotransferase (AGXT), type vii collagen alpha 1 chain (COL7a1), spinocerebellar ataxia type 1 protein (ATXN1), angiopoietin-like 3 (ANGPTL3), frataxin (FXN), superoxide dismutase 1, soluble (SOD1), synuclein, alpha (SNCA), sodium channel, voltage-opening, type X alpha subunit (SCN10A), spinocerebellar ataxia type 2 protein (ATXN2), myotonic dystrophy protein kinase (DMPK), and the beta-globin gene on chromosome 11. It may contain, medium-chain fatty acid acyl coenzyme A dehydrogenase (ACADM), long-chain fatty acid long-chain 3-hydroxyl coenzyme A dehydrogenase (HADHA), very long-chain fatty acid acyl coenzyme A dehydrogenase (ACADVL), apolipoprotein C3 (APOCIII), transthyretin (TTR), angiopoietin-like 4 (ANGPTL4), voltage-opening sodium channel alpha subunit 9 (SCN9A), interleukin-7 receptor (IL7R), glucose-6-phosphatase, catalytic (G6PC), hemochromatosis (HFE), SERPINA1, C9ORF72, β-globin, dystrophin, and γ-globin.

[0233] In certain embodiments, the target is associated with a specific gene. The target may be AAVS1 (PPPIR12C), ALB gene, Angptl3 gene, ApoC3 gene, ASGR2 gene, CCR5 gene, FIX (F9) gene, G6PC gene, Gys2 gene, HGD gene, Lp(a) gene, Pcsk9 gene, serpine al gene, TF gene, and TTR gene. To evaluate the efficiency of cDNA knock-in to the first exon mediated by HDR / NHEJ, the following regions may be used, e.g.: ApoC3 (chr11:116829908~116833071), Angptl3 (chr1:62,597,487~62,606,305), serpine al (chr14:94376747~94390692), Lp(a) (chr6:160531483~160664259), Pcsk9(chr1:55,039,475~55,064,852), FIX(chrX:139,530, 736~139,563,458), ALB(chr4:73,404,254~73,421,411), TTR(chr18:31,591,766~31,599,0 23), TF(chr3:133,661,997~133,779,005), G6PC(chr17:42,900,796~42,914,432), Gys2(ch r12:21,536,188~21,604,857), AAVS1(PPP1R12C)(chr19:55,090,912~55,117,599), HGD(ch cDNA knock-in to a "safe harbor" site can be utilized, such as single-stranded or double-stranded DNA having homology arms with one of the following: r3 (120,628,167~120,682,570), CCR5 (chr3:46,370,854~46,376,206), or ASGR2 (chr17:7,101,322~7,114,310). In one embodiment, the target is superoxide dismutase 1, soluble (SOD1).

[0234] In some embodiments, the disease is cancer-related. In some embodiments, neophosphorylation of oncogenic targets can elicit an immune response, or phosphorylation is used as an autoantigen. In some embodiments, phosphorylation is used in multiple sclerosis, e.g., SLE with αB-crystallin or multiple targets (see, e.g., Doyle and Mamula, Curr Opin Immunol. 2012). In some embodiments, the disease is related to the expression of tumor antigens, such as proliferative disorders, precancerous conditions, cancer, or non-cancer-related indications related to the expression of tumor antigens, and the tumor antigens are, in some embodiments, B2M, CD247, CD3D, CD3E, CD3G, TRAC, TRBC1, TRBC2, HLA-A, HLA-B, HLA-C, DCK, CD52, FKBP1A, CIITA, NLRC5, RFXANK, RFX5, RFXAP, or NR3C1, HAVCR 2, LAG3, PDCD1, PD-L2, CTLA4, CEACAM (CEACAM-1, CEACAM-3 and / or CEACAM-5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD113), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD107), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGF beta or PTPN11 DCK, CD52, NR3C1, LILRB1, CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; Epidermal growth factor receptor variant III (EGFRvIII); Ganglioside G2 (GD2); Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA);Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin-11 receptor alpha (IL-11Ra); Prostate stem cell antigen (PSCA); Protease serine 21 (Testicin or PRSS21); Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific fetal antigen-4 (SSEA-4); CD 20; Folate receptor alpha; Receptor tyrosine protein kinase ERBB2 (Her2 / neu); n kinase ERBB2 (Her2 / neu); Mucin 1, cell surface-related (MUC1); Epidermal growth factor receptor (EGFR); Neuronal cell adhesion molecule (NCAM); Prostase; Prostatic acid phosphatase (PAP); Elongation factor 2 mutant (ELF2M); Ephrin B2; Fibroblast-activating protein α (FAP); Insulin-like growth factor 1 receptor (IGF-I receptor), Carbonic anhydrase IX (CAIX); Proteasome (prosome, macropain) sub Unit, beta type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein (bcr-abl) consisting of cleavage cluster region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-Acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7 related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globo-H glycoceramide (globo-H);Mammary gland differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenoceptor beta 3 (ADRB3); Panexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, gene locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR gamma surrogate leading frame protein (TARP); Wilms tumor protein (WT1); Cancer / testicular antigen 1 (NY-ESO-1); Cancer / testicular antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); Sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie 2); Melanoma cancer testicular antigen-1 (MAD-CT-1); Melanoma cancer testicular antigen-2 (MAD-CT-2); Fos-related antigen 1; Tumor protein p53 (p53); p53 mutant; Prostain; Surviving; Telomerase; Prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), Melanoma antigen 1 recognized by T cells (Melan A or MART1); Rat sarcoma (Ras) mutant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Melanoma apo Ptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc trimyelocytosis virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (brother of the Regulator of Imprinted Sites) (BORIS or imprinting site regulator), squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired-box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1);Lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-section 2 (SSX2); late glycation end product receptor (RAGE-1); renal ubiquitous receptor 1 (RU1); renal ubiquitous receptor 2 (RU2); regmine; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); enteric carboxylesterase; heat shock protein 70-2 mutant (mut) Targets may include hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRLS); and immunoglobulin lambda-like polypeptide 1 (IGLL1), CD19, BCMA, CD70, G6PC, dystrophin including modification of exon 51 by deletion or excision, DMPK, CFTR (cystic fibrosis membrane conductance regulator). In embodiments, the targets include knock-in of CD70 or CD33 and knock-out of B2M. In embodiments, the targets include knock-out of TRAC and B2M or TRAC, B2M and PD1, and may or may not include additional target genes. In a particular embodiment, the disease is cystic fibrosis in which the SCNN1A gene is targeted. In one application, the small molecules disclosed herein are utilized in the presentation of human leukocyte antigens (HLA) and immune responses.

[0235] In detailed embodiments, it is envisioned that genes involved in modifying the quantity and / or quality of lipids produced by algal cells will be specifically modified. Examples of genes encoding enzymes involved in the fatty acid synthesis pathway may encode proteins having fatty acid thioesterase or malate enzyme activity, such as acetyl-CoA carboxylase, fatty acid synthase, 3-ketoacyl acyl carrier protein synthase III, glycerol-3-phosphate dehydrogenase (G3PDH), enoyl acyl carrier protein reductase (enoyl-ACP-reductase), glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase or diacylglycerol acyltransferase, phospholipid: diacylglycerol acyltransferase, phosphatidic acid phosphatase, palmitoyl protein thioesterase, etc. In further embodiments, it is envisioned that diatoms with increased lipid accumulation will be created. This can be achieved by targeting genes that reduce lipid catabolism. Genes involved in the activation of both triacylglycerols and free fatty acids, as well as genes directly involved in fatty acid β-oxidation, such as acyl-CoA synthetase, 3-ketoacyl-CoA thiolase, acyl-CoA oxidase activity, and phosphoglucumutase, are particularly useful for use in the methods of the present invention. Using the systems and methods described herein, such genes in diatoms can be specifically activated to increase their lipid content.

[0236] In some embodiments, the disease is metachromatic leukodystrophy and the target is arylsulfatase A; the disease is Viscott-Aldrich syndrome and the target is Viscott-Aldrich syndrome protein; the disease is adrenoleukodystrophy and the target is ATP-binding cassette DI; the disease is human immunodeficiency virus and the target is receptor type 5 CC chemokine or CXCR4 gene; the disease is β-thalassemia and the target is hemoglobin β subunit; the disease is X-linked severe combined immunodeficiency receptor subunit It is nit gamma, and the target is interleukin-2 receptor subunit gamma, the disease is multisystem lysosomal storage disorder, cystine storage disorder, and the target is cystinosine, the disease is Diamond-Blackfan anemia, and the target is ribosomal protein S19, the disease is Fanconi anemia, and the target is Fanconi anemia complements (e.g., FNACA, FNACB, FANCC, FANCD1, FANCD2, FANCE, FANCF, RAD51C), the disease is Schwakman-Bodian-Diamond syndrome, and The target is the Schubakmann syndrome gene, the disease is Gaucher disease, and the target is glucocerebrosidase; the disease is hemophilia A, and the targets are antihemophilic factor or factor VIII, Christmas factor, serine protease, hemophilia B factor IX; the disease is adenosine deaminase deficiency (ADA-SCID), and the target is adenosine deaminase; the disease is GM1 gangliosidosis, and the target is β-galactosidase; the disease is type II glycogen storage disease, Pompe disease; the disease is acid maltase deficiency, and the target is α- Glucosidase, disease is Niemann-Pick disease, SMPDl-related (sphingomyelin phosphodiesterase type 1 or type A and B) acid, and target is sphingomyelinase, disease is Krabbe disease, globoid cell type leukodystrophy, and target is galactosylceramidase or galactosylceramide lipidosis, target is galactocerebrosidase, human leukocyte antigen DR-15, DQ-6, disease is multiple sclerosis (MS), DRB1, disease is herpes simplex virus type 1 or 2, and target is RS1,This involves knockdown of one, two, or three copies of the RL2 and / or LAT genes. In embodiments, the disease is an HPV-associated cancer accompanied by a therapy comprising edited cells containing binding molecules such as the TCR or its antigen-binding fragment, and antibodies and their antigen-binding fragments, such as those that recognize or bind to human papillomavirus. The disease may be hepatitis B targeting one or more of the PreC, C, X, PreS1, PreS2, S, P, and / or SP genes.

[0237] Proteins encoded by chromosome sequence: ALAS2 δ-aminolevulinic acid synthase 2 (ALAS2), ABCA1 ATP-binding cassette transporter (ABCA1), ACE angiotensin I-converting enzyme (ACE), APOE apolipoprotein E precursor (APOE), APP amyloid precursor protein (APP), AQP1 aquaporin 1 protein (AQP1), BIN1 Myc-box-dependent interaction protein 1 or cross-linking integrator 1 protein (BIN1), BDNF brain-derived neurotrophic factor (BDNF), BTNL8 butyrophyllin-like protein 8 (BTNL8), C1ORF49 chromosome 1 open reading frame 49, CDH4 cadherin 4, CHRNB2 neuronal acetylcholine receptor subunit β-2, CKLFSF2 CKLF-like MARVEL transmembrane domain-containing protein 2 (CKLFSF2), CLEC4E C-type lectin domain family 4, member e (CLEC4E), CLU clusterin protein (also known as apolipoprotein J) CR1 Erythrocyte complement receptor 1 (CR1, also known as CD35, C3b / C4b receptor and immunoadhesive receptor) CR1L Erythrocyte complement receptor 1 (CR1L) CSF3R Granulocyte colony-stimulating factor 3 receptor (CSF3R) CST3 Cystatin C or cystatin 3 CYP2C Cytochrome P450 2C DAPK1 Cell death-related protein kinase 1 (DAPK1) ESR1 Estrogen receptor 1 FCAR Fc fragment of IgA receptor (FCAR, also known as CD89) FCGR3B Fc fragment of IgG, low affinity IIIb, receptor (FCGR3B or CD16b) FFA2 Free fatty acid receptor 2 (FFA2) FGA Fibrinogen (factor I) GAB2 GRB2-associated binding protein 2 (GAB2) GAB2 GRB2-associated binding protein 2 (GAB2) GALP Galanine-like peptide GAPDHS Glyceraldehyde-3-phosphate dehydrogenase, spermatogenesis (GAPDHS) GMPB GMBP HP haptoglobin (HP) HTR7 5-hydroxytryptamine (serotonin) receptor 7 (adenylate cyclase-coupled) IDE insulinase IF127 IF127 IFI6 interferon, α-inducible protein 6 (IFI6) IFIT2Interferon-inducible protein 2 (IFIT2) with tetratricopeptide repeats; IL1RN; Interleukin-1 receptor antagonist (IL-1RA); IL8RA; Interleukin-8 receptor, α (IL8RA or CD181); IL8RB; Interleukin-8 receptor, β (IL8RB); JAG1; KCNJ15; Potassium inward rectifying channel, subfamily J, member 15 (KCNJ15); LRP6; Low-density lipoprotein receptor-associated protein 6 (LRP6); MAPT; Microtubule-binding protein τ (MAPT); MARK4; MAP / microtubule affinity-regulated kinase 4 (MARK4); MPHOSPH1; MTHFR; 5,10-methylenetetrahydrofolate reductase; MX2; Interferon-inducible GTP-binding protein; Mx2; NBN; Nibrin, also known as NBN; NCSTN; Nicastrin; NIACR2 Niacin receptor 2 (NIACR2, also known as GPR109B) NMNAT3 Nicotinamide nucleotide adenylyltransferase 3 NTM Neurotrimin (or HNT) ORM1 Orosmucoid 1 (ORM1) or α-1-acid glycoprotein 1 P2RY13 P2Y Purine receptor 13 (P2RY13) PBEF1 Nicotinamide phosphoribosyltransferase (NAmPRTase or Nampt) also known as pre-B cell colony enhancing factor 1 (PBEF1) or bisfatin PCK1 Phosphoenolpyruvate carboxykinase PICALM Phosphatidylinositol-binding clathrin aggregate protein (PICALM) PLAU Urokinase-type plasminogen activator (PLAU) PLXNC1 Plexin C1 (PLXNC1) PRNP Prion protein PSEN1 Presenilin 1 protein (PSEN1) PSEN2 Presenilin 2 protein (PSEN2) PTPRA protein tyrosine phosphatase receptor A protein (PTPRA) RALGPS2 Ral GEF2 (RALGPS2) with PH domain and SH3 binding motif RGSL2 G protein signaling-like regulator 2 (RGSL2) SELENBP1 selenium-binding protein 1 (SELNBP1) SLC25A37 Mitoferrin 1 SORL1Sortirin-related receptor L (DLR class) A repeat-containing protein (SORL1)TF Transferrin TFAM Mitochondrial transcription factor A TNF Tumor necrosis factor TNFRSF10C Tumor necrosis factor receptor superfamily member 10C (TNFRSF10C) TNFSF10 Tumor necrosis factor receptor superfamily, (TRAIL) member 10a (TNFSF10) UBA1 Ubiquitin-like modifier activating enzyme 1 (UBA1) UBA3 NEDD8 activating enzyme E1 catalytic subunit protein (UBE1C) UBB Ubiquitin B protein (UBB) UBQLN1 Ubiquitin 1 UCHL1 Ubiquitin carboxyl-terminal esterase L1 protein (UCHL1) UCHL3 Ubiquitin carboxyl-terminal hydrolase isozyme L3 protein (UCHL3) VLDLR Very low density lipoprotein receptor protein (VLDLR).

[0238] The targets include very low-density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, ubiquitin-like modifier activator 1 (UBA1) encoded by the UBA1 gene, NEDD8 activator E1 catalytic subunit protein (UBE1C) encoded by the UBA3 gene, aquaporin 1 protein (AQP1) encoded by the AQP1 gene, ubiquitin carboxyl-terminal esterase L1 protein (UCHL1) encoded by the UCHL1 gene, ubiquitin carboxyl-terminal hydrolase isozyme L3 protein (UCHL3) encoded by the UCHL3 gene, ubiquitin B protein (UBB) encoded by the UBB gene, microtubule-associated protein τ (MAPT) encoded by the MAPT gene, and PTPRA gene. Examples of proteins encoded include tyrosine phosphatase receptor type A protein (PTPRA), phosphatidylinositol-binding clathrin aggregate protein (PICALM) encoded by the PICALM gene, clusterin protein (also known as apolipoprotein J) encoded by the CLU gene, presenilin 1 protein encoded by the PSEN1 gene, presenilin 2 protein encoded by the PSEN2 gene, sorotirin-related receptor L (DLR class) A repeat-containing protein (SORL1) protein encoded by the SORL1 gene, amyloid precursor protein (APP) encoded by the APP gene, apolipoprotein E precursor (APOE) encoded by the APOE gene, or brain-derived neurotrophic factor (BDNF) encoded by the BDNF gene.In an exemplary embodiment, the animal undergoing genetic modification is a rat, and the edited chromosome sequence encoding the AD-related proteins is as follows: APP Amyloid Precursor Protein (APP) NM_019288 AQP1 Aquaporin 1 Protein (AQP1) NM_012778 BDNF Brain-Derived Neurotrophic Factor NM_012513 CLU Clusterin Protein (also known as NM_053021 Apolipoprotein J) MAPT Microtubule-Associated Protein NM_017212 τ (MAPT) PICALM Phosphatidylinositol-Binding NM_053554 Clathrin Assembly Protein (PICALM) PSEN1 Presenilin 1 Protein (PSEN1) NM_019163 PSEN2 Presenilin 2 Protein (PSEN2) NM_031087 PTPRA Protein Tyrosine Phosphatase NM_012763 Receptor A Protein (PTPRA) SORL1 Sortillin-related receptor L (DLR NM_053519, class) A repeat-containing XM_001065506, protein (SORL1) XM_217115 UBA1 ubiquitin-like modifier activation NM_001014080 enzyme 1 (UBA1) UBA3 NEDD8 activating enzyme E1 NM_057205 catalytic subunit protein (UBE1C) UBB ubiquitin B protein (UBB) NM_138895 UCHL1 ubiquitin carboxyl terminus NM_017237 esterase L1 protein (UCHL1) UCHL3 ubiquitin carboxyl terminus NM_001110165 hydrolase isozyme L3 protein (UCHL3) VLDLR very low density lipoprotein NM_013155 receptor protein (VLDLR).

[0239] Delivery of compounds Methods for modifying the target of interest include, without limitation, administering or delivering cells or otherwise contacting them by one or more methods known in the art, including microinjection, electroporation, sonoporation, microparticle guns, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, phototransfection, enhancement of nucleic acid uptake by proprietary drugs, and delivery by liposomes, immunoliposomes, virosomes, or artificial virions. In some methods, the composition is introduced into the embryo by microinjection. The composition may be injected into the nucleus or cytoplasm of the embryo by microinjection.

[0240] Actively targeting lipid particles or nanoparticles or liposomes or lipid bilayer delivery systems (generally, with respect to embodiments of the present invention, “lipid entities of the present invention” delivery systems) are prepared by conjugating targeting moieties, including small molecule ligands, peptides, and monoclonal antibodies, onto the surface of lipids or liposomes; for example, certain receptors, such as folate receptors and transferrin (Tf) receptors (TfRs), are overexpressed in many cancer cells and are used to make liposomes tumor cell-specific. Liposomes accumulating in the tumor microenvironment can then be taken up into cells via endocytosis by subsequently interacting with specific cell surface receptors. To efficiently target cells such as cancer cells with liposomes, it is useful that the targeting moieties have affinity for cell surface receptors and that a sufficient amount of targeting moieties are linked to have optimal affinity for the cell surface receptors; and determining these aspects is within the scope of the art. In the field of active targeting, there are several cell-specific, e.g., tumor-specific targeting ligands.

[0241] Furthermore, regarding active targeting, in relation to targeting cell surface receptors such as cancer cell surface receptors, targeting ligands on liposomes can provide attachment from liposomes to cells, such as vascular cells, via non-internalized epitopes; and this can increase the extracellular concentration of what is being delivered, and therefore the amount delivered to the target cells. Strategies for targeting cell surface receptors, such as cell surface receptors on cancer cells, including those overexpressed on cancer cells, involve the use of receptor-specific ligands or antibodies. Many cancer cell types exhibit upregulation of tumor-specific receptors. For example, TfR and folate receptor (FR) are significantly overexpressed by many tumor cell types in response to their increased metabolic requirements. Folate can be used as a targeting ligand for specialized delivery due to its ease of conjugation to nanocarriers, its high affinity for FR, and its relatively low frequency of FR in normal tissues compared to its overexpression in activated macrophages and cancer cells, such as certain ovarian, breast, lung, colon, kidney, and brain tumors. Overexpression of FR on macrophages is an indicator of inflammatory diseases such as psoriasis, Crohn's disease, rheumatoid arthritis, and atherosclerosis; therefore, folate-mediated targeting of the present invention can also be used in the research, treatment, or therapy of inflammatory disorders and cancer. The folate-bound lipid particles, nanoparticles, liposomes, or lipid bilayers of the present invention ("lipid entities of the present invention") deliver their cargo into cells via receptor-mediated endocytosis. Intracellular transport can be directed towards acidic compartments that facilitate cargo release, and most importantly, cargo release can be altered or delayed until it reaches the cytoplasm or the vicinity of a target organelle. Cargo delivery using lipid entities of the present invention having a targeting moiety, such as the folate-bound lipid entities of the present invention, may be superior to that of untargeted lipid entities of the present invention. Directly attaching folic acid to the lipid head group may be undesirable for intracellular delivery of the folic acid conjugate-type lipid entity of the present invention, because such folic acid may not bind to cells as efficiently as folic acid attached to the surface of the lipid entity of the present invention by a spacer that allows for more efficient entry into cancer cells.The lipid entities of the present invention coupled to folic acid can be used for the delivery of lipid complexes, such as liposomes, such as anionic liposomes, and viruses, such as adenoviruses or AAVs, as considered herein, or capsids, envelopes, or viral coat proteins. Tf is a monomeric serum glycoprotein of approximately 80 kDa involved in the transport of iron throughout the body. Tf binds to TfR and translocates into cells via receptor-mediated endocytosis. TfR expression can be elevated in certain cells, such as tumor cells (compared to normal cells), and in rapidly proliferating cancer cells, it is accompanied by increased iron requirements. Therefore, the present invention includes TfR-targeted lipid entities of the present invention for, for example, hepatocytes, liver cancer cells, mammary cells such as breast cancer cells, colon cancer cells such as colon cancer cells, ovarian cancer cells such as ovarian cancer cells, head, neck and lung cells such as head, neck and non-small cell lung cancer cells, and oral tumor cells such as oral tumor cells.

[0242] Regarding active targeting, the lipid entities of the present invention may also be polyfunctional, meaning that two or more targeting moieties such as CPP can be used together with Tf; a bifunctional system; for example, a combination of Tf and poly-L-arginine which can provide transport across the blood-brain barrier endothelium. EGFR, which is a tyrosine kinase receptor belonging to the ErbB receptor family that mediates cell growth, differentiation and repair in cells, especially non-cancerous cells, is overexpressed in certain cells, including many solid tumors such as colorectal cancer, non-small cell lung cancer, ovarian squamous cell carcinoma, renal cancer, head cancer, pancreatic cancer, cervical cancer and prostate cancer and especially breast cancer. The present invention comprises one or more EGFR-targeted monoclonal antibodies linked to the lipid entities of the present invention. HER-2 is often overexpressed in breast cancer patients and is also associated with lung cancer, bladder cancer, prostate cancer, brain cancer and gastric cancer. HER-2 is encoded by the ERBB2 gene. The present invention encompasses lipid entities of the present invention that target HER-2, e.g., anti-HER-2 antibody (or its conjugated fragment)-lipid entity of the present invention, PEGylated lipid entities of the present invention that target HER-2 (e.g., having an anti-HER-2 antibody or its conjugated fragment), and maleimide-PEG polymer-lipid entity of the present invention that target HER-2 (e.g., having an anti-HER-2 antibody or its conjugated fragment). Upon cell association, this receptor-antibody complex can be internalized by the formation of endosomes for delivery to the cytoplasm. With respect to receptor-mediated targeting, those skilled in the art will take into consideration that ligand / target affinity, the amount of receptor on the cell surface, and PEGylation can act as barriers to interaction with the receptor. The use of antibody-targeting lipid entities of the present invention may be advantageous. The targeting portion in a polyvalent form can also increase the uptake and signaling properties of the antibody fragment. In carrying out the present invention, those skilled in the art will take ligand density into consideration (e.g., a high ligand density on the lipid entity of the present invention may be advantageous for increased binding to target cells).Early prevention by macrophages can be addressed by linking ligands to the ends of molecules such as PEG that are anchored in the sterically stable lipid entities of the present invention (e.g., lipid particles or nanoparticles or liposomes or lipid bilayers). This allows for targeting of the cell aggregate microenvironment, such as the tumor microenvironment; for example, targeting cell aggregate vascular structures, such as the tumor vascular microenvironment, may be advantageous. Accordingly, the present invention encompasses targeting VEGF. VEGF and its receptor are well-known pro-angiogenic molecules and well-characterized targets for anti-angiogenic therapies. Many small molecule inhibitors of receptor tyrosine kinases, such as VEGFR or basic FGFR, have been developed as anticancer agents, and the present invention encompasses coupling one or more of these peptides, for example, one or more phage IVO peptides (e.g., via or with the PEG terminus), or tumor-homing peptides such as APRPG-PEG modified, to the lipid entities of the present invention. VCAM, vascular endothelium, plays a crucial role in the pathogenesis of inflammation, thrombosis, and atherosclerosis. CAM is involved in inflammatory disorders, including cancer, and is a logical target. E- and P-selectins, VCAM-1, and ICAM can be used to target the lipid entities of the present invention, for example, by PEGylation. Matrix metalloproteinases (MMPs) belong to the family of zinc-dependent endopeptidases. They are involved in tissue remodeling, tumor invasiveness, apoptosis resistance, and metastasis. There are four MMP inhibitors called TIMP1-4, which determine the balance between tumor growth inhibition and metastasis; the protein involved in angiogenesis of tumor blood vessels is MT1-MMP, which is expressed in newly formed blood vessels and tumor tissue. The proteolytic activity of MT1-MMP cleaves proteins such as fibronectin, elastin, collagen, and laminin at the cell membrane, activating soluble MMPs such as MMP-2, which degrade the matrix. In the implementation of the present invention, antibodies such as anti-human MT1-MMP monoclonal antibodies or fragments thereof such as Fab' fragments can be used, such as anti-human MT1-MMP monoclonal antibodies linked to the lipid entities of the present invention via spacers such as PEG spacers.αβ-integrins, or integrins, are a group of transmembrane glycoprotein receptors that mediate the adhesion of cells to their surrounding tissues or the extracellular matrix. Integrins contain two distinct chains (heterodimers) called α- and β-subunits. Tumor tissue-specific expression of integrin receptors can be utilized for targeted delivery in the present invention, and the targeting moiety can therefore be, for example, an RGD peptide such as cyclic RGD. Aptamers are ssDNA or RNA oligonucleotides that confer high affinity and specific recognition of target molecules through electrostatic, hydrogen bonding, and hydrophobic interactions, in contrast to the Watson-Crick base pairing typical of oligonucleotide binding interactions. Aptamers as targeting moieties may have advantages over antibodies: aptamers may demonstrate higher target antigen recognition compared to antibodies; aptamers may be more stable and smaller in size compared to antibodies; aptamers may be easily synthesized and chemically modified for molecular conjugation; and aptamers may be sequence-modified to improve selectivity and developed to recognize targets with low immunogenicity. Such portion as an sgc8 aptamer can be used as a targeting portion (for example, by covalently linking to the lipid entity of the present invention via a spacer such as a PEG spacer). The targeting portion may be stimulus-sensitive, such as to external stimuli such as magnetic fields, ultrasound, or light; and pH-triggered may also be used, and an easily dissociable linkage can be used between a hydrophilic portion such as PEG and a hydrophobic portion such as the lipid entity of the present invention, which is cleaved only when exposed to relatively acidic conditions specific to a particular environment or microenvironment, such as an endocytic vacuole or an acidic tumor mass.pH-sensitive copolymers can also be incorporated into embodiments of the present invention, which may provide shielding; dioltoesters, vinyl esters, cysteine-cleavable lipopolymers, biesters, and hydrazones are a few examples of pH-sensitive bonds that are extremely stable at pH 7.5 but hydrolyzed relatively quickly below pH 6, for example, a copolymer with alkylated ends of N-isopropylacrylamide and methacrylic acid, which promotes the destabilization and release of the lipid entities of the present invention within its compartment as the pH value decreases; or the present invention may encompass ionic polymers for generating the pH-responsive lipid entities of the present invention (e.g., poly(methacrylic acid), poly(diethylaminoethyl methacrylate), poly(acrylamide), and poly(acrylic acid)). Temperature-induced delivery is also within the scope of the present invention. Many pathological ranges, such as inflamed tissues and tumors, exhibit characteristic abnormally high temperatures compared to normal tissues. Since abnormally high temperatures are associated with increased tumor permeability and enhanced uptake, utilizing these abnormally high temperatures is an attractive strategy in cancer therapy. This technique involves locally heating the site to increase the pore size and blood flow of microvessels, which may result in increased extravasation in embodiments of the present invention. The temperature-sensitive lipid entities of the present invention can be prepared from heat-sensitive lipids or polymers with low critical eutectic temperatures. Above the lower critical eutectic temperature (e.g., in a site such as a tumor or inflamed tissue), the polymer precipitates, and the liposomes are broken and released. These lipid entities of the present invention are prepared using lipids having specific gel-liquid phase transition temperatures; and the lipid for the heat-sensitive embodiments may be dipalmitoylphosphatidylcholine. Heat-sensitive polymers can also be destabilized and subsequently released, and a useful heat-sensitive polymer is poly(N-isopropylacrylamide). Another temperature-induced system may utilize lysolipid temperature-sensitive liposomes. The present invention also encompasses redox-induced delivery: the difference in redox potential between normal tissue and inflammatory or tumor tissue, and between the intracellular and extracellular environments, is utilized for delivery; for example, GSH is a reducing agent that is abundant in cells, particularly in the cytosol, mitochondria, and nucleus.The GSH concentrations in blood and the extracellular matrix are only 1 / 100 to 1 / 1000 of the intracellular concentration, respectively. This large difference in redox potential caused by GSH, cysteine, and other reducing agents breaks reducing bonds, destabilizing the lipid entities of the present invention and resulting in the release of the payload. Disulfide bonds can be used as cleavable / reversible linkers in the lipid entities of the present invention because they give rise to redox sensitivity for disulfide-thiol reduction reactions; the lipid entities of the present invention can be made reduction-sensitive by using two forms (e.g., two forms of disulfide-conjugate polyfunctional lipids), where cleavage of the disulfide bond (e.g., by tris(2-carboxyethyl)phosphine, dithiothreitol, L-cysteine, or GSH) can cause removal of the hydrophilic head group of the conjugate, altering the membrane organization and potentially leading to payload release. Calcein release from the reduction-sensitive lipid entities of the present invention containing disulfide conjugates may be more useful than in reduction-insensitive embodiments. Enzymes can also be used as factors that induce payload release, such as MMPs (e.g., MMP2), phospholipase A2, and alkaline enzymes. Enzymes including riphosphatase, transglutaminase, or phosphatidylinositol-specific phospholipase C have been shown to be overexpressed in certain tissues, such as tumor tissue. In the presence of these enzymes, specially modified enzyme-sensitive lipid entities of the present invention can be disrupted and release a payload. An octapeptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln) (SEQ ID NO: 1) cleavable by MMP2 can be incorporated into the linker, which may have antibody targeting capabilities, such as antibody 2C5. The present invention also encompasses light or energy-induced delivery; for example, the lipid entities of the present invention may be photosensitive, and therefore light or energy may promote structural and conformational changes, leading to direct interactions between the lipid entities of the present invention and target cells by membrane fusion, photoisomerization, photocleavage, or photopolymerization; therefore, such a portion may be a benzoporphyrin photosensitizer.Ultrasound can be one form of energy that induces delivery; the lipid entities of the present invention can be induced and released with ultrasound, such as low-frequency ultrasound (LFUS), along with small amounts of specific gases, including air, or perfluorinated hydrocarbons. Magnetic delivery: The lipid entities of the present invention can be magnetized by the incorporation of magnetite such as Fe3O4 or γ-Fe2O3, for example, those with a size of less than 10 nm. Therefore, targeted delivery can be achieved by exposure to a magnetic field.

[0243] Regarding active targeting, the present invention also encompasses intracellular delivery. Liposomes follow the endocytosis pathway, becoming trapped within endosomes (pH 6.5-6), subsequently fusing with lysosomes (pH < 5), where they undergo degradation, thus reducing their potential therapeutic capacity. The low pH of endosomes can be utilized to avoid degradation. Membrane-fusion lipids or peptides destabilize the endosomal membrane after conformational transition / activation at lower pH. At acidic pH, amines are protonated, causing endosome swelling and rupture due to buffering effects. Unsaturated dioleoylphosphatidylethanolamine (DOPE) readily adopts an inverted hexagonal shape at low pH, which causes fusion of liposomes and endosomal membranes. This process destabilizes the DOPE-containing lipid entity, releasing cargo into the cytoplasm; membrane-fusion lipids GALA, cholesteryl-GALA, and PEG-GALA may exhibit highly efficient endosomal release; the pore-forming protein Listeriolysin O may provide an endosomal escape mechanism; and histidine-rich peptides, having the ability to fuse with the endosomal membrane, can lead to pore formation, buffer the proton pump, and induce membrane lysis.

[0244] Regarding active targeting, cell-permeable peptides (CPPs) also promote the uptake of macromolecules across the cell membrane, thus enhancing the delivery of CPP-modified molecules within the cell. CPPs can be divided into two classes: amphiphilic helical peptides such as transportans and MAPs (where lysine residues are the primary contributors to the positive charge); and Arg-rich peptides such as TATp, Antennapedia, or penetratin. TATp is an 86-amino acid transcription activator containing a strongly basic (two Lys and six Arg) protein transduction domain, which leads to nuclear localization and RNA binding. Other CPPs used for liposome modification include: the minimal protein transduction domain of Antennapedia, a Drosophila homeoprotein called penetratin, which is a 16-mer peptide (residues 43-58) located in the third helix of the homeodomain; a 27-amino acid chimeric CPP containing a peptide sequence from the amino terminus of the neuropeptide galanin, which is linked to the wasp venom peptide mastoparan via a Lys residue; VP22, a major structural component of HSV-1 that promotes intracellular transport; and a transportan (18-mer) amphiphilic model peptide that migrates across the cell membranes of mast cells and endothelial cells by both energy-dependent and energy-independent mechanisms. The present invention encompasses lipid entities of the present invention modified with one or more CPPs for intracellular delivery that may proceed via energy-dependent micropinocytosis and subsequent endosomal escape. The present invention further encompasses organelle-specific targeting. Lipid entities of the present invention whose surface is functionalized with a triphenylphosphonium (TPP) moiety, or lipid entities of the present invention having the lipophilic cation rhodamine 123, may be effective in the delivery of cargo to mitochondria. DOPE / sphingomyelin / stearyloctaarginine can deliver cargo into the mitochondria via membrane fusion. Lipid entities of the present invention whose surface is modified with the lysosomal-tropic ligand octadecylrhodamine B can deliver cargo to lysosomes.Ceramides are useful for inducing lysosomal membrane translocation; the present invention encompasses intracellular delivery of the lipid entities of the present invention having ceramides. The present invention further encompasses the lipid entities of the present invention that target the nucleus, for example, via DNA intercalation moieties. The present invention also encompasses polyfunctional liposomes for targeting, i.e., attaching, two or more functional groups to the surface of the lipid entities of the present invention, such as enhancing accumulation at a desired site and / or promoting organelle-specific delivery and / or targeting specific types of cells and / or responding to local stimuli such as temperature (e.g., an increase therein), pH (e.g., a decrease therein), responding to externally applied stimuli such as magnetic fields, light, energy, heat or ultrasound, and / or promoting intracellular delivery of cargo. All of these are considered active targeting moieties.

[0245] Embodiments of the system may include lipid particles, nanoparticles, liposomes, or lipid bilayer delivery systems, or targeting portions that thereby have active targeting or the targeting portion is an active targeting portion. The targeting portion may consist of one or more targeting portions, and the targeting portion may be any desired type of targeting, such as targeting cells as listed herein; or targeting organelles as listed herein; or targeting responses to chemical or physical conditions such as heat, energy, ultrasound, light, pH, or enzymatic stimulation; or targeting achieving a specific outcome, such as delivery of a payload to a specific location, such as by cell permeation.

[0246] With respect to each possible targeting or active targeting portion discussed herein, it should be understood that there are embodiments of the present invention in which the delivery system includes such targeting or active targeting portion.

[0247] Pharmaceutical composition The methods described herein include the manufacture and use of pharmaceutical compositions comprising one or more of the agents described herein as active ingredients. The pharmaceutical compositions themselves are also included.

[0248] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, and combinations of two or more of these, which are compatible with the drug administration. Supplementary active compounds may also be incorporated into the composition.

[0249] Pharmaceutical compositions are typically formulated to be compatible with the intended route of administration. Examples of particularly useful routes of administration in this method include parenteral (e.g., intravenous), intrathecal, oral, and nasal or intranasal administration (e.g., administration as drops or by inhalation). In some embodiments, for compounds that do not cross the blood-brain barrier, direct delivery to the CNS or CSF can be used, for example, using an implantable intrathecal pump (see, e.g., Borrini et al., Archives of Physical Medicine and Rehabilitation 2014;95:1032-8; Penn et al., N.Eng.J.Med.320:1517-21(1989); and Rezai et al., Pain Physician 2013;16:415-417) or nanoparticles, such as gold nanoparticles (e.g., glucose-coated gold nanoparticles, see, e.g., Gromnicova et al.(2013) PLoS ONE 8(12):e81043). Methods for formulating and delivering suitable pharmaceutical compositions are known in the art; see, for example, the books in the series Drugs and the Pharmaceutical Sciences: A Series of Textbooks and Monographs (Dekker, NY) and Allen et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Lippincott Williams & Wilkins; 8th edition (2004).

[0250] Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to pass easily through an injection needle. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols like mannitol and sorbitol, and sodium chloride in the composition. By including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition, sustained absorption of the injectable composition can be achieved.

[0251] Sterile injectable solutions can be prepared by compounding the required amount of the active compound with one or a combination of the components listed above, as needed, in a suitable solvent, followed by sterilization by filtration. Generally, dispersions are prepared by compounding the active compound in a sterile medium containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, thereby yielding powders of the active component + any additional desired components from those solutions that have been previously sterile filtered.

[0252] For oral administration, the composition may be formulated with an inert diluent or food carrier. For oral therapeutic administration, the active compound may be formulated with excipients and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or auxiliary materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any or similar compounds of the following components: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; fluidizers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; and flavorings such as peppermint, methyl salicylate, or orange flavoring.

[0253] For administration by inhalation, the compound can be delivered in the form of an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant, such as a gas including carbon dioxide. Such methods include those described in U.S. Patent No. 6,468,798.

[0254] Therapeutic compounds that are nucleic acids or contain nucleic acids can be administered by any method suitable for the administration of nucleic acid agents, such as DNA vaccines. These methods include needle-free methods such as gene guns, bioinjectors and skin patches, and microparticle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, as well as percutaneous needle-free mammalian vaccination with vaccines in powder form as disclosed in U.S. Patent No. 6,168,587. In addition, intranasal delivery is possible, in particular, as described in Hamajima et al., Clin.Immunol.Immunopathol., 88(2), 205-10 (1998).

[0255] Liposomes (e.g., as described in U.S. Patent No. 6,472,375) and microencapsulation can also be used for the delivery of the compounds described herein. Biodegradable microparticle delivery systems can also be used (e.g., as described in U.S. Patent No. 6,471,996).

[0256] In one embodiment, the therapeutic compound is prepared with a carrier that protects the therapeutic compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques or are commercially available from, for example, Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeted to selected cells by monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared by methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0257] The pharmaceutical composition may be contained in a container, pack, or dispenser, such as a single-dose dispenser, along with instructions for administration. The container, pack, or dispenser may also be included as part of a kit that may contain, for example, enough single-dose dispensers for a day, a week, or a month of treatment.

[0258] Dosage The dosage, toxicity, and therapeutic efficacy of a compound can be determined, for example, by standard pharmaceutical procedures for determining the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population) in cell cultures or experimental animals. The dose-to-therapeutic ratio is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds exhibiting a high therapeutic index are preferred. Although compounds exhibiting toxic side effects may be used, care must be taken to design a delivery system that targets such compounds to the site of the affected tissue in order to minimize the possibility of damage to uninfected cells and thus reduce side effects.

[0259] Using data obtained from cell culture assays and animal studies, a dosage range for use in humans can be formulated. The dosage of such compounds is preferably within the range of circulating concentrations that include an ED50 with little to no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration used. For any compound used in the method of the present invention, the therapeutically effective dose can initially be estimated from cell culture assays. The dose can be formulated to achieve a circulating plasma concentration range in animal models that includes the IC50 (i.e., the concentration of the test compound that achieves half of the maximum symptom inhibition) as determined under cell culture conditions. Using this information, a more accurate determination of a useful dose in humans can be made. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0260] An "effective dose" is an amount sufficient to achieve a beneficial or desired outcome. For example, a therapeutic dose is the amount that achieves the desired therapeutic effect, which may depend on the degree of modification. This amount may be the same as or different from the preventive effective dose, which is the amount required to prevent the onset of a disease or disease symptoms. An effective dose may be administered in one or more doses, applications, or prescriptions. The therapeutic effective dose of a composition depends on the composition selected. Compositions may be administered at doses ranging from once a day to once a week; or every other day. Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, past treatments, the subject's overall health and / or age and other pre-existing conditions, may influence the dose and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutic effective dose of a composition described herein may include a single treatment or a series of treatments.

[0261] Treatment method The present invention also intends to utilize the systems described herein for the treatment of various diseases and disorders. Because the localization portion can bind to or localize to a target of interest, the activation portion can modify the target. The present invention also intends to utilize the polyfunctional molecules described herein for the treatment of various diseases and disorders. Exemplary applications include use as a small molecule analog of insulin and rewiring of cellular signaling pathways. See Lim et al., Nat Rev Mol Cell Biol 2010, 11(6), 393-403. The need for cellular signaling can be addressed by adding phosphoryl groups to specific signaling proteins of interest, while enabling rewiring of kinase signaling pathways in disease or health with controlled dose and timing. The polyfunctional systems described herein may enable targeted degradation of proteins, where the phosphorylation site is a target that recruits ubiquitin ligases and signals degradation. See Toure et al., Angewandte Chemie (Inter'l ed. In English) 2016, 55(6), 1966-73. Similarly, preventing protein aggregation may be beneficial in cancer treatment. As described herein, adding negatively charged phosphoryl groups using polyfunctional molecules to proteins prone to aggregation can increase solubility and reduce self-aggregation. Guo et al., FEBS Letters, 2005, 579(17), 3574-3578; Zhang et al., Protein Expression and Purification 2004 36(2) 207-216. Finally, neophosphorylation to elicit an immune response may find applications in cancer immunotherapy. Treatment for kinasopathy is also being considered; for an overview, please refer to Lahiry et al., Nature Reviews Genetics, 2011 (the disclosure in Table 1 regarding hereditary kinasopathy is incorporated herein by reference).A method is provided for modifying a target substrate in a subject that requires it, the method comprising administering the molecule to the subject as disclosed herein. Delivery may be as described in other parts thereof. In embodiments, the invention described herein relates to a method for therapy in which cells are ex vivo modified with a polyfunctional molecule to modify at least one target substrate, and the edited cells are subsequently administered to a patient that requires it.

[0262] In embodiments, the treatment may be the treatment of a disease / disorder of a single organ, including liver disease, eye disease, muscle disease, heart disease, blood disease, brain disease, and kidney disease, or it may include the treatment of autoimmune diseases, central nervous system diseases, cancer and other proliferative disorders, neurodegenerative disorders, inflammatory diseases, metabolic disorders, musculoskeletal disorders, etc.

[0263] Specific diseases / disorders include chondrodysplasia, color blindness, acid maltase deficiency, adrenoleukodystrophy, Aicardi syndrome, α-1 antitrypsin deficiency, α-thalassemia, androgen insensitivity syndrome, Apert syndrome, arrhythmogenic right ventricle, dysplasia, ataxia telangiectasia, Barth syndrome, β-thalassemia, blue rubber ball nevus syndrome, Canavan disease, chronic granulomatous disease (CGD), cat-cry syndrome, cystic fibrosis, Darkham disease, ectodermal dysplasia, Fanconi anemia, fibrodysplasia ossificans progressive, fragile X syndrome, galactosemia, Gaucher disease, systemic gangliosidosis (e.g., GM1), hemochromatosis, hemoglobin C mutation at codon 6 of β-globin (HbC), hemophilia, Huntington's disease, Harler syndrome, hypophosphatasia, Klinefelter syndrome, Krabbe disease, and Langer-Giddy syndrome. Onn syndrome, leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome, mucopolysaccharidosis (MPS), onychopatellar syndrome, nephrogenic diabetes insipidus, neurofibromatosis, Niemann-Pick disease, osteogenesis imperfecta, porphyria, Prader-Willi syndrome, progeria, Proteus syndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybe syndrome, Sanfilippo syndrome, severe combined immunodeficiency (SCID), Schubakmann syndrome, sickle cell anemia, Smith-Majenis syndrome, Stickler syndrome, Tay-Sachs disease, thrombocytopenic radial aplasia (TAR) syndrome, Treacher-Collins syndrome, trisomy, tuberous sclerosis, Turner syndrome, urea cycle disorder, von Hippel-Lindau disease, Waardenburg syndrome, Williams syndrome, Wilson's disease, and Wiscott-Aldrich syndrome.

[0264] In embodiments, the disease is related to the expression of tumor antigens, such as proliferative disorders, precancerous conditions, cancer, or non-cancer-related indications related to the expression of tumor antigens, and the tumor antigens are, in some embodiments, B2M, CD247, CD3D, CD3E, CD3G, TRAC, TRBC1, TRBC2, HLA-A, HLA-B, HLA-C, DCK, CD52, FKBP1A, CIITA, NLRC5, RFXANK, RFX5, RFXAP, or NR3C1, HAVCR2, LAG3, PDCD1, PD-L2, CTLA4, CEACAM (CEACAM-1, CEACAM-3 and / or CEACAM-5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD113), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD107), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGF beta or PTPN11 DCK, CD52, NR3C1, LILRB1, CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; Epidermal growth factor receptor variant III (EGFRvIII); Ganglioside G2 (GD2); Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); proteaseserine 21 (testicin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen;CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific fetal antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine protein kinase ERBB2 (Her2 / neu); n kinase ERBB2 (Her2 / neu); Mucin 1, cell surface-related (MUC1); Epidermal growth factor receptor (EGFR); Neuronal adhesion molecule (NCAM); Prostase; Prostatic acid phosphatase (PAP); Elongation factor 2 mutant (ELF2M); Ephrin B2; Fibroblast-activating protein α (FAP); Insulin-like growth Factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein (bcr-abl) consisting of cleavage cluster region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGl cp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma quina -ase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globo-H glycoceramide (globo-H); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); panexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, gene locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma surrogate leading frame protein (TARP); Wilms tumor protein (WT1);Cancer / testicular antigen 1 (NY-ESO-1); Cancer / testicular antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation mutant gene 6, located on chromosome 12p (ETV6-AML); Sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie 2); Melanoma cancer testicular antigen-1 (MAD-CT-1); Melanoma cancer testicular antigen-2 (MAD-CT-2); Fos-associated antigen 1; Tumor protein p53 (p53); p53 mutant; Prostain; Surviving; Telomerase; Prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), Melanoma antigen 1 recognized by T cells (Melan A or MART1); Rat sarcoma (Ras) mutant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Melanoma apo Ptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc trimyelocytosis virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (Brother of the Regulator of Imprinted Sites) (BORIS or imprinting site regulator); squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-cleavage 2 (SSX2); late glycation end product receptor (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); regmine; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); enteric carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a;This may include targets selected from CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of the IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); type C lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRLS); and immunoglobulin lambda-like polypeptide 1 (IGLL1), CD19, BCMA, CD70, G6PC, dystrophin, DMPK, and CFTR (cystic fibrosis membrane conductance regulator).

[0265] In each embodiment, the disease is metachromatic leukodystrophy and the target is arylsulfatase A; the disease is Viscott-Aldrich syndrome and the target is Viscott-Aldrich syndrome protein; the disease is adrenoleukodystrophy and the target is ATP-binding cassette DI; the disease is human immunodeficiency virus and the target is receptor type 5 CC chemokine or CXCR4 gene; the disease is β-thalassemia and the target is hemoglobin β subunit; the disease is X-linked severe combined immunodeficiency receptor subunit It is unit gamma, and the target is interleukin-2 receptor subunit gamma, the disease is multisystem lysosomal storage disorder, cystine storage disorder, and the target is cystinosine, the disease is Diamond-Blackfan anemia, and the target is ribosomal protein S19, the disease is Fanconi anemia, and the target is Fanconi anemia complements (e.g., FNACA, FNACB, FANCC, FANCD1, FANCD2, FANCE, FANCF, RAD51C), the disease is Schwakman-Bodian-Diamond syndrome Yes, and the target is the Schbaeckmann syndrome gene, the disease is Gaucher disease, and the target is glucocerebrosidase, the disease is hemophilia A, and the targets are antihemophilic factor or factor VIII, Christmas factor, serine protease, hemophilia B factor IX, the disease is adenosine deaminase deficiency (ADA-SCID), and the target is adenosine deaminase, the disease is GM1 gangliosidosis, and the target is β-galactosidase, the disease is type II glycogen storage disease, Pompe disease, the disease is acid maltase deficiency. Furthermore, the target is α-glucosidase, the disease is Niemann-Pick disease; SMPDl-related (sphingomyelin phosphodiesterase type 1 or type A and B) acid, and the target is sphingomyelinase, the disease is Krabbe disease, globoid cell type leukodystrophy, and the target is galactosylceramidase or galactosylceramide lipidosis, the target is galactocerebrosidase, human leukocyte antigen DR-15, DQ-6, the disease is multiple sclerosis (MS), DRB1, the disease is herpes simplex virus type 1 or 2.The disease may be hepatitis B targeting one or more of the PreC, C, X, PreS1, PreS2, S, P, and / or SP genes.

[0266] In some embodiments, the immune disease is severe combined immunodeficiency (SCID) or Omen syndrome, and in one embodiment, the target is recombinant activator gene 1 (RAG1) or interleukin-7 receptor (IL7R). In a more detailed embodiment, the disease is transthyretin amyloidosis (ATTR) or familial amyloid cardiomyopathy, and in one embodiment, the target is the TTR gene, including one or more mutations in the TTR gene. In some embodiments, the disease is alpha-1 antitrypsin deficiency (AATD) or another disease in which alpha-1 antitrypsin is thought to be involved, such as GvHD, transplant organ rejection, diabetes mellitus, liver disease, COPD, emphysema, and cystic fibrosis, and in a more detailed embodiment, the target is SERPINA1.

[0267] In embodiments, the disease is primary hyperoxaluria, where, in certain embodiments, the target comprises one or more lactate dehydrogenase A (LDHA) and hydroxy acid oxidase 1 (HAO 1). In embodiments, the disease is adenocarcinoma, chronic alcoholism, Alzheimer's disease, Cooley's anemia, aneurysm, anxiety disorder, asthma, breast malignancy, cutaneous malignancy, renal cell carcinoma, cardiovascular disease, cervical malignancy, coronary artery sclerosis, coronary heart disease, diabetes mellitus, diabetes mellitus vera, insulin-independent diabetes mellitus vera, diabetic nephropathy, eclampsia, eczema, subacute bacterial endocarditis, glioblastoma, type II glycogen storage disease, sensorineural hearing loss (impairment), hepatitis, hepatitis A, hepatitis B, homocystinuria, hereditary sensory autonomic neuropathy type 1, hyperoxaluria Dosteronism, hypercholesterolemia, hyperoxaluria, primary hyperoxaluria, hypertensive diseases, inflammatory bowel disease, kidney stones, kidney disease, chronic renal failure, leiomyosarcoma, metabolic diseases, congenital metabolic disorders, mitral valve prolapse syndrome, myocardial infarction, neoplasm metastasis, nephrotic syndrome, obesity, ovarian diseases, periodontitis, polycystic ovary syndrome, renal failure, adult respiratory distress syndrome, retinal diseases, cerebrovascular attacks, Turner syndrome, viral hepatitis, tooth loss, early ovarian dysfunction, essential hypertension, left ventricular hypertrophy, migraine disorders, cutaneous melanoma, hypertensive heart Diseases, chronic glomerulonephritis, migraine with aura, secondary hypertension, acute myocardial infarction, aortic atherosclerosis, allergic asthma, pineoblastoma, malignant neoplasm of the lung, primary hyperoxaluria type 1, primary hyperoxaluria type 2, inflammatory breast cancer, cervical cancer, restenosis, hemorrhagic ulcer, systemic glycogen storage disease in children, nephrolithiasis, chronic kidney transplant rejection, urinary tract stones, tingling pain in the skin, metabolic syndrome X, maternal hypertension, atherosclerosis of the carotid artery, carcinogenesis, breast cancer, lung cancer, nephronoplasia, microalbuminuria, home These include retinoblastoma rheumatoid arthritis, systolic heart failure, ischemic stroke, left ventricular systolic dysfunction, cauda equina ganglia, hepatocyte carcinogenesis, chronic kidney disease, glioblastoma multiforme, non-neoplastic disorders, calcium oxalate nephrolithiasis, apallidosis-macrostomia syndrome, coronary artery disease, liver cancer, chronic kidney disease stage 5, allergic rhinitis (disorder), Crigler-Nadjar syndrome type 2, and ischemic cerebrovascular accidents, as well as primary hyperoxaluria type 1 (ph1) and other alanine-glyoxylate aminotransferase (AGXT) gene-related conditions or disorders. In certain embodiments, the treatment is targeted to the liver.In this embodiment, the gene is AGXT with a cytogenetic location of 2q37.3, and its genomic coordinates are 240,868,479–240,880,502 on the forward strand of chromosome 2.

[0268] Treatment includes treatment for malignant neoplasms of the skin, squamous cell carcinoma, colorectal neoplasms, Crohn's disease, epidermolysis bullosa, inguinal hernia, pruritus, schizophrenia, dermatological disorders, hereditary skin diseases, teratomas, Cockayne-Turaine disease, acquired epidermolysis bullosa, dystrophic epidermolysis bullosa, junctional epidermolysis bullosa, Allopor-Siemens disease, bullous skin diseases, corpus callosum agenesis, onychodystrophy, bullous stomatitis, epidermolysis bullosa with congenital focal skin defects and nail deformities, juvenile myoclonic epilepsy, esophageal squamous cell carcinoma, and Kindler's polymorphism. Conditions or disorders related to the type VII collagen α1 chain (col7a1) gene can also be targeted, including atrophy, pretibial epidermolysis bullosa, dominant dystrophic epidermolysis bullosa white papular type (disorder), focal recessive dystrophic epidermolysis bullosa, systemic dystrophic epidermolysis bullosa, cutaneous squamous cell carcinoma, prurigopathic epidermolysis bullosa, mammary neoplasm, superficial simple epidermolysis bullosa, solitary onychomycosis, transient bullous dermatolysis of the newborn, autosomal recessive dystrophic epidermolysis bullosa focal variant, and autosomal recessive dystrophic epidermolysis bullosa opposite type.

[0269] In embodiments, the disease is Wilms tumor I (WTI) and acute myeloid leukemia (AML) targeting HLA-expressing cells. In embodiments, the therapy is a T-cell therapy comprising modified T cells having a WTI-specific TCR, as described in other parts of this specification. In certain embodiments, the target is CD157 in AML.

[0270] In some embodiments, the disease is a blood disorder. In certain embodiments, the disease is hemophilia, and in one embodiment, the target is factor XI. In other embodiments, the disease is an abnormal hemoglobinosis, such as sickle cell disease, sickle cell phenotype, hemoglobin C disease, hemoglobin C phenotype, hemoglobin S / C disease, hemoglobin D disease, hemoglobin E disease, thalassemia, conditions related to hemoglobin with increased oxygen affinity, conditions related to hemoglobin with decreased oxygen affinity, unstable hemoglobinosis, and methemoglobinemia. Deficiencies in hemostatic factors as well as factors X and XII can also be treated. In the embodiment, the target is the BCL11A gene (e.g., the human BCL11a gene), the BCL11a enhancer (e.g., the human BCL11a enhancer) or the HFPH region (e.g., the human HPFH region), β-globulin, fetal hemoglobin, the γ-globin gene (e.g., HBG1, HBG2, or HBG1 and HBG2), the red blood cell-specific enhancer of the BCL11A gene (BCL11Ae), or a combination thereof.

[0271] In embodiments, the target loci are RAC, TRBCl, TRBC2, CD3E, CD3G, CD3D, B2M, CIITA, CD247, HLA-A, HLA-B, HLA-C, DCK, CD52, FKBP1A, NLRC5, RFXANK, RFX5, RFXAP, NR3C1, CD274, HAVCR2, LAG3, PD CD1, PD-L2, HCF2, PAI, TFPI, PLAT, PLAU, PLG, RPOZ, F7, F8, F9, F2, F5, F7, F10, F11, F12, F13A1, F13B, STAT1, FOXP3, IL2RG, DCLRE1C, ICOS, MHC2TA, GALNS, HGSNAT, ARSB, RFXAP, CD 20, CD81, TNFRSF13B, SEC23B, PKLR, IFNG, SPTB, SPTA, SLC4A1, EPO, EPB42, CSF2, CSF3, VFW, SERPINCA1, CTLA4, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD113), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD107), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFβ, PTPN11, and one or more combinations thereof. In embodiments, Chrl 1: 5,250,094~5,250,237, -chain, hg38; Chrl l: 5,255,022~5,255,164, -chain, hg38; undefected HFPH region; Chrl 1: 5,249,833~Chrl 1: 5,250,237, -chain, hg38; Chrl 1: 5,254,738~Chrl 1: 5,255,164, -chain, hg38; Chrl 1: 5,249,833~5,249,927, -chain, hg3; Chrl 1: 5,254,738~5,254,851, -chain, hg38; Chrl 1:5,250,139~5,250,237, -chain, is a target sequence in the genomic nucleic acid sequence in hg38.

[0272] In some embodiments, the disease is related to high cholesterol, and cholesterol regulation is provided, and in some embodiments, the regulation is achieved by modification of the target PCSK9. Other diseases in which PCSK9 may be involved, and therefore may be targeted by the systems and methods described herein, include abetalipoproteinemia, adenoma, arteriosclerosis, atherosclerosis, cardiovascular disease, cholelithiasis, coronary artery disease, coronary heart disease, insulin-independent diabetes mellitus, hypercholesterolemia, familial hypercholesterolemia, hyperinsulinemia, hyperlipidemia, familial combined hyperlipidemia, hypobetalipoproteinemia, chronic renal failure, liver disease, hepatic neoplasms, melanoma, myocardial infarction, narcolepsy, neoplasm metastasis, nephroblastoma, obesity, peritonitis, pseudoxanthoma elasticum, cerebrovascular accident, vascular disease, and xanthomatous disease. These include peripheral vascular disease, myocardial ischemia, dyslipidemia, impaired glucose tolerance, xanthomas, polygenic hypercholesterolemia, secondary malignant neoplasms of the liver, dementia, overweight, hepatitis C, chronic conditions, atherosclerosis, hyperlipoproteinemia type Ha, intracranial atherosclerosis, ischemic stroke, acute coronary syndrome, aortic calcification, cardiovascular pathological conditions, hyperlipoproteinemia type lib, peripheral artery disease, familial hyperaldosteronism type II, familial hypobetalipoproteinemia, autosomal recessive hypercholesterolemia, autosomal dominant hypercholesterolemia type III, coronary artery disease, liver cancer, ischemic cerebrovascular attack, and atherosclerotic cardiovascular disease NOS. In embodiments, the treatment may be targeted to the liver, which is the primary active site of PCSK9.

[0273] In embodiments, the disease or disorder is characterized by hyper-IGM syndrome or a CD40 signaling deficiency. In certain embodiments, insertion of the CD40L exon restores proper CD40 signaling and B-cell class switch recombination. In detailed embodiments, the target is the CD40 ligand (CD40L) - one or more exons 2-5 of the CD40L gene are edited in cells, e.g., T cells or hematopoietic stem cells (HSCs).

[0274] In embodiments, the disease is merosine-deficient congenital muscular dystrophy (mdcmd) and other laminin α2 (lama2) gene-related conditions or disorders. Therapies can target muscles, e.g., skeletal muscle, smooth muscle, and / or cardiac muscle. In certain embodiments, the target is laminin α2 (LAMA2), which may also be referred to as laminin-12 subunit α, laminin-2 subunit α, laminin-4 subunit α3, merosine heavy chain, laminin M chain, LAMM, congenital muscular dystrophy, and merosine. LAMA2 has a cytogenetic location at 6q22.33 and genomic coordinates at 128,883,141-129,516,563 on the forward strand of chromosome 6. In embodiments, the diseases treated include merosine-deficient congenital muscular dystrophy (MDCMD), amyotrophic lateral sclerosis, bladder neoplasms, Charcot-Marie-Tooth disease, colorectal cancer, contractures, cysts, Duchenne muscular dystrophy, fatigue, hyperopia, renovascular hypertension, melanoma, intellectual disability, myopathy, muscular dystrophy, myopia, myositis, neuromuscular diseases, peripheral neuropathy, refractive errors, schizophrenia, severe intellectual disability (IQ 20-34), thyroid neoplasms, tobacco use disorder, severe combined immunodeficiency, synovial cysts, lung adenocarcinoma (disorder), tumor progression, cutaneous strawberry nevi, muscle degeneration, microdentistry (disorder), and Walker's disease. It may be Warburg congenital muscular dystrophy, chronic periodontitis, leukoencephalopathy, cognitive impairment, Fukuyama type congenital muscular dystrophy, scleroatonic muscular dystrophy, Eichsfeld type congenital muscular dystrophy, neuropathy, myo-ophthalmological-brain disease, limb-girdle muscular dystrophy, congenital muscular dystrophy (disorder), muscle fibrosis, cancer recurrence, drug-resistant epilepsy, respiratory failure, mucinous cysts, abnormal respiration, merosin-deficient congenital muscular dystrophy, colorectal cancer, congenital muscular dystrophy due to partial LAMA2 deficiency, and autosomal dominant craniometrial dysplasia.

[0275] In one embodiment, the target is superoxide dismutase 1, soluble (SOD1), which may be useful in treating diseases or disorders associated with this gene. In a detailed embodiment, the disease or disorder is associated with SOD1 and includes, for example, adenocarcinoma, albuminuria, chronic alcoholism, Alzheimer's disease, amnesia, amyloidosis, amyotrophic lateral sclerosis, anemia, autoimmune hemolytic anemia, sickle cell anemia, anoxia, anxiety disorders, aortic disease, arteriosclerosis, rheumatoid arthritis, neonatal asphyxia, asthma, atherosclerosis, autism spectrum disorder, autoimmune diseases, Barrett's esophagus, Behçet's syndrome, bladder neoplasms, brain neoplasms, breast neoplasms, oral candidiasis, colon malignancies, bronchogenic carcinoma, and non-small cell carcinoma. Lung cancer, squamous cell carcinoma, transitional cell carcinoma, cardiovascular disease, carotid thrombosis, neoplastic cell transformation, cerebral infarction, cerebral ischemia, transient ischemic attack, Charcot-Marie-Tooth disease, cholera, colitis, colorectal cancer, coronary artery sclerosis, coronary heart disease, Cryptococcus neoformans infection, hearing loss, cessation of vital functions, dysphagia, presenile dementia, depressive disorder, contact dermatitis, diabetes mellitus, diabetes mellitus vera, experimental diabetes mellitus vera, insulin-dependent diabetes mellitus vera, insulin-independent diabetes mellitus vera, diabetic vascular disease, diabetic nephropathy, diabetic retinopathy, Down syndrome Syndrome, dwarfism, edema, Japanese encephalitis, toxic epidermal necrolysis, temporal lobe epilepsy, rash, fasciculations, alcoholic fatty liver, fetal growth restriction, fibromyalgia, fibrosarcoma, fragile X syndrome, giardiasis, glioblastoma, glioma, headache, partial hearing loss, cardiac arrest, heart failure, atrial septal defect, helminthiasis, hemochromatosis, hemolysis (disorder), chronic hepatitis, HIV infection, Huntington's disease, hypercholesterolemia, hyperglycemia, hyperplasia, hypertensive disorders, hyperthyroidism, hypothyroidism, hypothyroidism, hypoproteinemia, hypotension, spontaneous hypothermia Immunodeficiency syndrome, immune system disorders, inflammation, inflammatory bowel disease, influenza, intestinal disease, ischemia, Kearns-Sayre syndrome, keratoconus, kidney stones, kidney disease, acute renal failure, chronic renal failure, polycystic kidney disease, leukemia, myeloid leukemia, acute promyelocytic leukemia, cirrhosis, liver disease, hepatic neoplasms, locked-in syndrome, chronic obstructive airway disease, pulmonary neoplasms, systemic lupus erythematosus, non-Hodgkin lymphoma, Machado-Joseph disease, malaria, gastric malignant neoplasms, animal mammary neoplasms, Marfan syndrome, meningomyelocele, intellectual disability, mitral stenosis, acquired dental fluorosis.Movement disorders, multiple sclerosis, muscle rigidity, muscle spasms, muscular atrophy, spinal muscular atrophy, myopathy, fungal infections, myocardial infarction, myocardial reperfusion injury, necrosis, nephrotic syndrome, neurodegeneration, nervous system disorders, neuralgia, neuroblastoma, neuroma, neuromuscular diseases, obesity, occupational diseases, ocular hypertension, oligospermia, degenerative polyarthritis, osteoporosis, ovarian cancer, pain, pancreatitis, Papillon-Lefevre disease, paresis, Parkinson's disease, phenylketonuria, pituitary disorders, pre-eclampsia, prostate neoplasms, protein deficiency, proteinuria, psoriasis, pulmonary fibrosis, renal artery occlusion, reperfusion injury, retinal degeneration, retinal diseases Retinoblastoma, schistosomiasis, schistosomiasis mansoni, schizophrenia, scrapie, seizures, age-related cataracts, spinal cord compression, cerebrovascular accident, subarachnoid hemorrhage, progressive supranuclear palsy, tetanus, trisomy, Turner syndrome, unipolar depression, urticaria, vitiligo, vocal cord paralysis, intestinal volvulus, weight gain, HMN (Hereditary Motor Neuropathy) proximal type I, holoprosencephalopathy, motor neuron disease, neurofibrillary degeneration (morphological abnormalities), burning sensation, apathy, mood swings, synovial cyst, cataract, migraine disorder, sciatic neuropathy, sensory neuropathy, atrophic skin conditions, muscle weakness, esophageal cancer, lingual, facial, and buccal dyskinesia Nesia, idiopathic pulmonary hypertension, lateral sclerosis, migraine with aura, mixed hearing loss, iron deficiency anemia, malnutrition, prion disease, mitochondrial myopathy, MELAS syndrome, chronic progressive extraocular palsy, generalized paralysis, progeria syndrome, fibrillation, psychiatric symptoms, memory impairment, muscle degeneration, neurological symptoms, gastric hemorrhage, pancreatic cancer, cerebral pick's disease, hepatic fibrosis, malignant neoplasms of the lung, age-related macular degeneration, Parkinson's disease-like disorders, disease progression, hypocorcemia, cytochrome c oxidase deficiency, essential tremor, familial motor neuron disease, lower motor neuron disease, degenerative myelopathy, diabetic polyneuropathy, hepatic and intrahepatic cholangiocarcinoma, Persian Gulf syndrome, senile plaques, atrophic frontotemporal dementia, semantic dementia, common migraine, cognitive impairment, hepatic neoplasm, pancreatic neoplasm, prostate neoplasm, pure autonomic dysfunction, motor symptoms, spastic dementia, neurodegenerative disorders, chronic hepatitis C, Guam amyotrophic lateral sclerosis, rigid limbs, multiple system disorders, hair loss, prostate cancer, hepatopulmonary syndrome, Hashimoto's disease, progressive neoplasms, breast cancer, terminal disease, lung cancer, tardive dyskinesia, lymph node-associated malignant neoplasms, colon cancer, gastric cancer, central nervous system blastoma, dissecting thoracic aortic aneurysm, diabetic macular edema, microalbuminuria, middle cerebral artery occlusion, middle cerebral artery infarction,Upper motor neuron sign, frontotemporal lobar degeneration, memory loss, classical phenylketonuria, CADASIL syndrome, neurogenic gait disorder, spinocerebellar ataxia type 2, spinal cord ischemia, Lewy body disease, muscular atrophy, bulbar spinal disease, monosomy of chromosome 21, thrombocytosis, skin spots, drug-induced liver injury, Leber's hereditary optic nerve atrophy, cerebral ischemia, ovarian neoplasm, tauopathy, macrovascular disease, persistent pulmonary hypertension, malignant ovarian neoplasm, mucinous cyst, drusen, sarcoma, weight loss, major depressive disorder, mild cognitive impairment, degenerative disorders, partial trisomy, cardiovascular pathology, hearing impairment, cognitive changes, ureteral stones, breast neoplasm, colorectal cancer, chronic kidney disease, minimal change nephropathy Possible adverse events include: Rose syndrome, non-neoplastic disorders, X-linked spinal atrophy, normal-tension glaucoma susceptibility (based on mammography density and findings), vitiligo-associated multiple autoimmune disease susceptibility (based on findings), amyotrophic lateral sclerosis and / or frontotemporal dementia (1), amyotrophic lateral sclerosis (1), sporadic amyotrophic lateral sclerosis, monoliary muscular atrophy, coronary artery disease, degenerative migraine, reflux, urothelial carcinoma, motor disorders, liver cancer, protein folding abnormalities, TDP-43 proteinosis, promyelocytic leukemia, weight gain adverse events, mitochondrial cell death, idiopathic pulmonary hypertension, progressive cGVHD, infection, GRN-associated frontotemporal dementia, mitochondrial lesions, and hearing loss.

[0276] In detailed embodiments, the disease is associated with the genes ATXN1, ATXN2, or ATXN3, which may be therapeutic targets. In some embodiments, the CAG repeat region located in exon 8 of ATXN1, exon 1 of ATXN2, or exon 10 of ATXN3 is targeted. In embodiments, the disease is spinocerebellar ataxia type 3 (SCA3), SCA1 or SCA2 and other related disorders, such as congenital anomalies, Alzheimer's disease, amyotrophic lateral sclerosis, ataxia, telangiectasia ataxia, cerebellar ataxia, cerebellar diseases, chorea, cleft palate, cystic fibrosis, psychiatric depression, depressive disorders, dystonia, esophageal neoplasms, exotropia, cardiac arrest, Huntington's disease, Machado-Joseph disease, motor disorders, muscular dystrophy, myotonic dystrophy, narcolepsy, neurodegeneration, neuroblastosis Tumor, Parkinson's disease, peripheral neuropathy, restless leg syndrome, retinal degeneration, retinitis pigmentosa, schizophrenia, Shy-Drager syndrome, sleep disorders, hereditary spastic paraplegia, thromboembolism, generalized rigidity syndrome, spinocerebellar ataxia, esophageal cancer, polyneuropathy, effects of fever, muscle spasms, extrapyramidal signs, ataxia, neurological symptoms, cerebral atrophy, Parkinson's disease-like disorders, protein S deficiency, cerebellar degeneration, familial amyloid neuropathy (Portuguese type), spastic syndrome, vertical nystagmus, extreme nystagmus, anti Thrombin III deficiency, atrophic hereditary spastic paraplegia, multiple system atrophy, pallidoluysian degeneration, dystonia, pure autonomic dysfunction, thrombogenic tendency, protein C deficiency, congenital myotonic dystrophy, motor symptoms, neuropathy, neurodegenerative disorders, malignant neoplasms of the esophagus, visual impairment, activated protein C resistance, terminal illness, myokymia, central nervous system blastoma, sleep disorders, ataxia, narcolepsy / cataplexy syndrome, Machado-Joseph disease type I, Machado-Joseph disease type II, Machado-Joseph disease These include Jad Joseph disease type III, dentatorubral-pallidoluysian atrophy, gait ataxia, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 6 (disorder), spinocerebellar ataxia type 7, spinal and bulbar muscular atrophy, genomic instability, paroxysmal ataxia type 2 (disorder), X-linked spinal and bulbar atrophy, fragile X tremor ataxia syndrome, thrombogenic tendency due to activated protein C resistance (disorder), amyotrophic lateral sclerosis type 1, intranuclear inclusion body disorder, hereditary antithrombin III deficiency, and late-onset Parkinson's disease.

[0277] In embodiments, the disease is a cancer or non-cancer-related indication disease associated with the expression of tumor antigens, such as acute lymphoblastic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, Hodgkin lymphoma, or non-Hodgkin lymphoma. In embodiments, the target may be a sequence located within a TET2 intron, a TET2 intron-exon junction, or a genomic region of chr4.

[0278] In some embodiments, neurodegenerative diseases can be treated. In detailed embodiments, the target is alpha-synuclein (SNCA). In certain embodiments, the disorders to be treated are pain-related disorders, including congenital insensitivity to pain, compressive neuropathy, paroxysmal severe pain, high-grade atrioventricular block, small-diameter fiber neuropathy, and familial paroxysmal pain syndrome type 2. In certain embodiments, the target is sodium channel, voltage-opening, type X alpha subunit (SCNIOA).

[0279] In certain embodiments, hematopoietic stem cells and progenitor stem cells are modified, including for the treatment of lysosomal storage disorders, glycogen storage diseases, mucopolysaccharidosis, or any disease in which protein secretion would improve the condition. In one embodiment, the disease is sickle cell disease (SCD). In another embodiment, the disease is β-thalassemia.

[0280] The method and system can target myotonic dystrophy protein kinase (DMPK). DMPK-related disorders or diseases include atherosclerosis, azoospermia, hypertrophic cardiomyopathy, celiac disease, congenital chromosomal disorders, diabetes mellitus, focal segmental glomerulosclerosis, Huntington's disease, hypogonadism, muscular atrophy, myopathy, muscular dystrophy, myotonia, myotonic dystrophy, neuromuscular diseases, optic nerve atrophy, paresis, schizophrenia, cataracts, spinocerebellar ataxia, muscle weakness, adrenoleukodystrophy, and central nucleus myopathy. These include interstitial fibrosis, myotonic muscular dystrophy, abnormal mental states, X-linked Charcot-Marie-Tooth disease type 1, congenital myotonic dystrophy, bilateral cataracts (impairment), congenital muscle fiber type inequality, myotonic disorders, multiple system disorders, 3-methylglutaconic aciduria type 3, cardiac events, cardiac syncope, congenital structural myopathy, intellectual disability, adrenal spinal neuropathy, myotonic dystrophy type 2, and intellectual disability.

[0281] In embodiments, the disease is a congenital metabolic disorder. The disease may be selected from disorders of carbohydrate metabolism (glycostored storage disease, G6PD deficiency), disorders of amino acid metabolism (phenylketonuria, maple syrup urine disease, glutaric acidemia type 1), urea cycle disorders or urea cycle abnormalities (carbamoyl phosphate synthase I deficiency), disorders of organic acid metabolism (alkaptonuria, 2-hydroxyglutaric aciduria), disorders of fatty acid oxidation / mitochondrial metabolism (medium-chain acyl-coenzyme A dehydrogenase deficiency), disorders of porphyrin metabolism (acute intermittent porphyria), disorders of purine / pyrimidine metabolism (Lesch-Nyhan syndrome), disorders of steroid metabolism (lipoid congenital adrenal hyperplasia, congenital adrenal hyperplasia), disorders of mitochondrial function (Kerns-Sayre syndrome), disorders of peroxisome function (Zellweger syndrome), or disorders of lysosome storage (Gaucher disease, Niemann-Pick disease).

[0282] In embodiments, the targets include recombinant activator gene 1 (RAG1), BCL11 A, PCSK9, laminin, alpha 2 (lama2), ATXN3, alanine-glyoxylate aminotransferase (AGXT), type vii collagen alpha 1 chain (COL7a1), spinocerebellar ataxia type 1 protein (ATXN1), angiopoietin-like protein 3 (ANGPTL3), frataxin (FXN), superoxide dismutase 1, soluble (SOD1), synuclein, alpha (SNCA), sodium channel, voltage-opening, type X alpha subunit (SCN10A), spinocerebellar ataxia type 2 protein (ATXN2), myotonic dystrophy protein kinase (DMPK), and the beta-globin gene on chromosome 11. It may contain, medium-chain fatty acid acyl coenzyme A dehydrogenase (ACADM), long-chain fatty acid long-chain 3-hydroxyl coenzyme A dehydrogenase (HADHA), very long-chain fatty acid acyl coenzyme A dehydrogenase (ACADVL), apolipoprotein C3 (APOCIII), transthyretin (TTR), angiopoietin-like 4 (ANGPTL4), voltage-opening sodium channel alpha subunit 9 (SCN9A), interleukin-7 receptor (IL7R), glucose-6-phosphatase, catalytic (G6PC), hemochromatosis (HFE), SERPINA1, C9ORF72, β-globin, dystrophin, and γ-globin.

[0283] In certain embodiments, the disease or disorder is related to apolipoprotein C3 (APOCIII), which can be targeted for editing. In embodiments, the disease or disorder may be dyslipidemia, hyperalphalipoproteinemia type 2, lupus nephritis, Wilms tumor type 5, morbid obesity and spermatogenesis, glaucoma, diabetic retinopathy, arthralgia, renal dysfunction, cholestatic syndrome, cognitive impairment, altered response to myocardial infarction, glucose intolerance, positive regulation of triglyceride biosynthesis, chronic renal insufficiency, hyperlipidemia, chronic renal failure, apolipoprotein C-III deficiency, coronary artery disease, neonatal diabetes mellitus with congenital hypothyroidism, autosomal dominant hypercholesterolemia type 3, hyperlipoproteinemia type 3, hyperthyroidism, coronary artery disease, renal artery occlusion, metabolic syndrome X, familial combined hyperlipidemia, insulin resistance, transient childhood hypertriglyceridemia, diabetic nephropathy, diabetes mellitus (type 1), nephrotic syndrome type 5 with or without ocular abnormalities, and hemorrhagic fever with renal syndrome.

[0284] In certain embodiments, the target is angiopoietin-like 4 (ANGPTL4). Diseases or disorders associated with ANGPTL4 that can be treated include dyslipidemia, low plasma triglyceride levels, regulation of angiogenesis and tumorigenesis, and severe diabetic retinopathy, both proliferative and nonproliferative, in which ANGPTL4 is associated.

[0285] The binding protein induces phosphorylation from the kinase, thus binding to the target protein even if the target protein is not a substrate of that kinase. One such protein belongs to the bromodomain protein family. Bromodomains are a family of structurally and evolutionarily conserved protein interaction modules (approximately 110 amino acids) that specifically recognize substrate proteins, particularly acetylated lysine present in histones. Bromodomains exist as components of large, multi-domain nucleoproteins involved in chromatin remodeling, cell signaling, and transcriptional regulation. Examples of bromodomain-containing proteins with known functions include (i) histone acetyltransferases (HATs), including CREBBP, GCN5, PCAF, and TAFII250; (ii) methyltransferases such as ASH1L and MLL; (iii) components of chromatin remodeling complexes such as Swi2 / Snf2; and (iv) several transcription factors (Florence et al. Front. Biosci. 2001, 6, D1008-1018, which is incorporated herein by reference as a whole).

[0286] Bromodomain-mediated or BET-mediated disorders or conditions, such as BRD2-mediated, BRD3-mediated, BRD4-mediated, and / or BRDT-mediated disorders or conditions, may be any disease or other adverse condition in which one or more bromodomain-containing proteins, such as BET proteins including BRD2, BRD3, BRD4, and / or BRDT, or their mutants, are known to play a role. Accordingly, another embodiment of the present disclosure relates to treating or reducing the severity of one or more diseases in which one or more bromodomain-containing proteins, such as BET proteins including BRD2, BRD3, BRD4, and / or BRDT, or their mutants, are known to play a role. For example, diseases or conditions in which the biological function of a bromodomain affects the development and / or course of the disease or condition, and / or where the regulation of a bromodomain alters the development, course, and / or symptoms. Bromodomain-mediated diseases or conditions include those in which bromodomain inhibition provides therapeutic benefits, such that treatment with bromodomain inhibitors, including, for example, the compounds described herein, provides therapeutic benefits to subjects suffering from or at risk of such diseases or conditions. Bromodomain-inhibiting compounds or bromodomain inhibitors are typically compounds that inhibit the binding of bromodomains to acetylated proteins of their cognitive molecules, for example, bromodomain inhibitors are compounds that inhibit the binding of bromodomains to acetylated lysine residues.

[0287] A method for modifying a target protein is also provided, which involves contacting the target protein with a compound disclosed herein in an environment containing one or more activators. A method for treating a disease, disorder, or condition in a subject in need may include administering a molecule disclosed herein to that subject.

[0288] A method for producing a polyfunctional conjugation molecule is also provided, which includes attaching a localization moiety and an activator moiety to different ends of a linker molecule, the localization moiety and the activator moiety being optionally attached to the linker molecule via an orientation adapter, the linker molecule linking the activator molecule such that both the activator molecule and the localization moiety are active in the cell. Exemplary methods include, for example, those described in the examples of this specification, as depicted in Figures 5, 57, 58, 78, 80, 84C, and 84D.

[0289] The present invention is further illustrated in the following embodiments, which do not limit the scope of the present invention as described in the claims. [Examples]

[0290] Example 1- While small molecules have classically been used to inhibit (i.e., cause loss of function) enzymes, several novel classes of small molecules are emerging that impart new functions to enzymes through proximity-mediated effects. Here, we describe a novel class of molecules, which we call phosphorylation-inducing chimeric small molecules (PHICS), that enable two kinases (AMPK and PKC) to phosphorylate a target protein (BRD4) that is not a substrate of those kinases. PHICS are formed by linking small molecule activators of these kinases with a BRD4-binding agent (+)-JQ1 and a linker, and exhibit several bifunctional molecular characteristics, including a "hook effect," turnover, isoform specificity, and proximity (i.e., linker length) dependence of activity. This study provides yet another example that expands the range of chemical dimerization inducers that induce post-translational modifications in proteins by rewiring enzyme specificity. Site-specific, biologically relevant phosphorylation and neophosphorylation mediated by PHICS are expected to find utility in basic research and pharmaceuticals.

[0291] For many proteins, the addition of a phosphoryl group significantly impacts their structure and function. Naturally, small molecules that block protein phosphorylation through kinase inhibition have had a transformative impact on basic science and medicine. We hypothesize that small molecules that induce phosphorylation of any given target protein as needed will also be useful in countless scenarios. For example, inducing specific and biologically relevant phosphorylation can trigger signaling events, while protein neophosphorylation can affect its structure, induce immune responses, induce phase separation in cells, or influence the interaction between proteins and other biomolecules, particularly RNA / DNA with negatively charged phosphate diester skeletons. In designing such phosphorylation-inducing molecules, we have developed chemical dimerization inducers. 5~6 and ubiquitination-inducing small molecules (e.g., PROTAC) that increase the effective molar concentration of ubiquitin ligase around the target protein, thereby inducing ubiquitination even when the target protein is not a substrate of the ligase. 7 This was inspired by [the previous work]. This specification describes a novel class of bifunctional molecules called phosphorylation-inducible chimeric small molecules (PHICS), which are formed by conjugating a kinase activator with a small molecule binder for a target protein. Specifically, this specification demonstrates that induction of PHICS rewires the specificity of two kinases, AMP-activated protein kinase (AMPK) and protein kinase C (PKC), to induce phosphorylation of bromodomain-containing protein 4 (BRD4), which is not a substrate of AMPK or PKC (Figure 1A). The kinase specificity is rewired using an adapter protein. 8~9 These studies are considered to provide the first examples of kinase specificity rewiring using small molecules.

[0292] To create PHICS for AMPK and PKC, the applicants designed small molecule kinase conjugates having functional groups for linker bonding. For AMPK, the applicants modified its allosteric activator (14) PF-06409577 by replacing the cyclobutyl ring with a synthetically more suitable aminoethyl handle (Figure 55A), while for PKC, they used a 9-(4-aminomethylbenzyloxy)-substituted benzolactam activator (15). When evaluated by the ADP-Glo® assay, which measures the amount of ADP produced by the kinase reaction (16), the ability of these conjugates to activate AMPK or PKC was not disrupted by these chemical modifications (Figure 56). Next, the applicants created PHICS by conjugating these conjugates with the BRD4 conjugate (+)-JQ1 with linkers of various lengths (Figures 57 and 58). AMPK PHICS was synthesized using convergent synthesis based on modular click chemistry (17) (Figure 57), while PKC PHICS was constructed using two consecutive amidation steps (Figure 58).

[0293] The applicants evaluated both the ternary complex formation and BRD4 phosphorylation levels induced by these PHICS (Figures 59 and 60) and identified PHICS1 and PHICS2 as optimal for AMPK and PKC, respectively (Figures 8A and 11). The applicants synthesized iPHICS1 and iPHICS2 using (-)-JQ1(18), an inactive enantiomer of the BRD4 binder, and used them as negative controls. Using the ADP-Glo® assay, the applicants confirmed that iPHICS1 and iPHICS2 still activated AMPK and PKC, respectively, with the kinase binding moiety remaining unchanged (Figures 61A-61C). Ternary complex formation was evaluated using the AlphaScreen assay (amplified luminescence proximity homogeneity assay) (19-20) with BRD4 and AMPK (α1β1γ1 isoform) or PKC (α isoform). PHICS1 and PHICS2 showed bell-shaped curves consistent with the ternary complex equilibrium, but iPHICS1 and iPHICS2 did not (Figures 39B and 39C) (21). Here, when the bifunctional molecule is at a high concentration, the kinase-PHICS and BRD4-PHICS species become dominant in equilibrium, eliciting a "hook effect" (22). The applicants observed that BRD4 phosphorylation occurs only in the presence of PHICS, BRD4, and kinase using phospho-AMPK- or PKC-substrate motif antibodies (Figures 2C and 51A) (23-24). BRD4 phosphorylation was observed independently of the tag's properties (i.e., GST vs. His tag) (Figures 61D-61E). BRD4 phosphorylation levels also increased in an AMPK-dependent / PKC-dependent manner with PHICS, but not with iPHICS (Figures 61F-61G). The applicants also observed a hook effect in BRD4 phosphorylation with increasing PHICS concentration (Figure 62), reaching the highest phosphorylation level at 1 μM PHICS1, which corresponds to the concentration of the maximum signal in the AlphaScreen assay for ternary complex formation (Figure 39B).The applicants observed, using the pSer484 / 488 antibody, that phosphorylation by AMPK at a site on (25)BRD4, which is phosphorylated by casein kinase II (CK2) in the natural environment, was induced by PHICS1 (Figure 51B).

[0294] Next, the applicants confirmed using mass spectrometry that PHICS can induce neophosphorylation of truncated BRD4 (49-460aa). For AMPK PHICS, the statistically significant phosphorylation sites were T169, T186, T221, S324, and S325, while for PKC, these sites were T229, S324, and S338 (Figures 9 and 14). The phosphorylation at sites T169, T186, T221, and T229 is neophosphorylation, as has not been previously reported (26). To confirm that AMPK does not have a specific preference for phosphorylation of these BRD4 sites and that these sites are not part of an unknown substrate motif, the applicants tested peptides derived from BRD4 sequences carrying these residues. In the ADP-Glo ​​assay, these peptides showed a phosphorylation preference of 1 / 250th that of AMPK's native ACC substrate-SAMS peptide (Figure 65) (27), further confirming that proximity between AMPK and BRD4, induced by PHICS, is critically important for the rewiring of AMPK specificity. Finally, the applicants note that sites T186, S324, and S325 are in the most preferred AMPK consensus substrate recognition motif, preferably with the basic residue at position -3 (RXXpS / pT), but T169 and T221 are absent; however, AMPK can phosphorylate proteins lacking this motif as well (28-30).

[0295] Because PHICS was designed based on a reversible binder, the applicants hypothesized that PHICS would exhibit turnover, with each PHICS molecule phosphorylating multiple BRD4 molecules. Using the ADP-Glo® assay and iPHICS1 and iPHICS2 as negative controls (Figures 51C and 51D), the calculated ADP production in the presence of PHICS1 (324±22nM) and PHICS2 (740±31nM) was higher than the critical AMPK (20nM) and PKC (50nM) concentrations, respectively, suggesting that PHICS exhibits turnover. Another characteristic of the bifunctional molecule is isoform specificity, which arises not only from differences in the binding affinity of PHICS to various isoforms, but also from the inherently different interactions between the enzyme and the target isoform during ternary complex formation. (31). In AMPK isoforms (α1β2γ1) not activated by PF-06409577 (Figure 66) (14), the applicants did not observe induction of BRD4 phosphorylation by PHICS1 (Figure 52A). In addition, PHICS2 also exhibited isoform specificity, with the highest BRD4 phosphorylation occurring in PKCα, moderate phosphorylation in PKCβI and II, and only slight phosphorylation in PKCγ and δ isoforms (Figure 52B) (32). Isoform specificity was also observed for the target protein BRD(2 / 3 / 4), with BRD4 showing the highest phosphorylation level (Figures 2H, 3E, and 67).

[0296] The applicants were unable to observe PHICS-mediated BRD4 phosphorylation in cells, possibly due to differences in the localization of kinases and BRD4 (the former is almost entirely in the cytosol, while the latter is mainly in the nucleus). For several reasons, the applicants selected Bruton's tyrosine kinase (BTK), a protein widely expressed in B cells (33-34). Firstly, BTK is a cytoplasmic protein and is therefore available for interaction with cytoplasmic AMPK. Secondly, BTK can interact with PKC, but is not known to interact with AMPK (35). Thirdly, high-quality chemical probes of BTK and their co-crystal structures are available, allowing for the rational design of PHICS and inactive controls through engineering of BTK or PHICS (36). Fourthly, BTK possesses a phosphorylation site (S180), which is not only present in the substrate-like motif of AMPK but also plays a crucial role in the negative regulation of BTK (35). Finally, since BTK is undetectable in HEK293T cells, it is possible to evaluate PHICS' ability to induce BTK phosphorylation in a non-native cell environment.

[0297] The applicants linked an AMPK binder to a non-covalent ibrutinib analog with various linkers (37) (Figure 68) and found that an eight-carbon alkyl linker (PHICS3, Figure 53A) was optimal for in vitro phosphorylation (Figure 69), which was monitored using a phosphoBTK (Ser180) antibody. To demonstrate intracellular ternary complex formation, the applicants transfected HEK293T with BTK-Flag and performed immunoprecipitation of AMPK with BTK in the presence of PHICS3 (Figure 53B). Furthermore, the applicants were able to detect PHICS-mediated BTK phosphorylation at Ser180 (Figures 53C and 70A), and the applicants verified this phosphorylation site using the S180A mutant, where no phosphorylation was detected (Figure 53D). It should be noted that the potent AMPK activator, PF-06409577, did not induce the same level of BTK phosphorylation as PHICS3, even at a four-fold higher concentration, when used alone (Figure 70B). Finally, the applicants were able to control PHICS-inducible S180 phosphorylation rapidly and in a dose-dependent manner (Figures 70C and 70D).

[0298] To confirm that BTK phosphorylation observed in non-natural environments was mediated by PHICS, the applicants employed several chemogenetic techniques. First, pretreatment of cells with the covalent BTK binder ibrutinib disrupted PHICS3's ability to induce BTK phosphorylation (Figures 54A and 71A). Second, the applicants mutated residues (Thr474, Lys430, and Asp539) that are thought to be involved in PHICS binding to BTK. In particular, Thr474 forms a hydrogen bond with the four amino groups of ibrutinib (Figure 54B)(38), and the applicants observed a dramatic decrease in BTK phosphorylation in the T474A mutant (Figure 54C). Changes in the ATP binding pocket due to D539N and K430R mutations also significantly reduced PHICS3-induced phosphorylation (Figures 71B and 71C). Finally, the applicants designed an inactive analog of PHICS3 by placing a bulk pivaloyl (Piv) group on the azapurine nitrogen (which should sterically hinder binding to BTK). Indeed, PHICS supported by pivaloyl (Piv-PHICS3, Figure 53A) failed to induce significant BTK phosphorylation (Figure 54D). In summary, these studies support the idea that Ser180 phosphorylation of BTK occurs due to proximity effects induced by PHICS.

[0299] In this specification, the applicants successfully rewired both AMPK and PKC kinases using chimeric small molecules to induce novel phosphorylation events, including BRD4 neophosphorylation and signaling-related BTK phosphorylation. PHICS exhibited typical bifunctional molecular characteristics, including hook effect, turnover, proximity (linker length) dependence, isoform specificity, and dose and time control of phosphorylation. Turnover may arise from the reversible binding of PHICS to kinases and target proteins, which is also observed with PROTAC. While kinase specificity has previously been achieved using adapter proteins (12-13), the applicants focused on small molecules because they are cell-permeable and non-immunogenic. Furthermore, small molecules offer easy dose and time control, exhibit rapid reaction rates and turnover, and possess a molecular design that allows for rapid construction. While these studies focus on serine / threonine kinases, the applicants are investigating PHICS-mediated rewiring of tyrosine kinases. In future research, PHICS will be deployed to induce PHICS-mediated negative charge delivery to the DNA-binding domains (39) of transcription factors (which are often considered chemically undrugable) that may negatively affect signaling-related phosphorylation and neophosphorylation of oncogenic proteins that may induce immune responses against tumors or their ability to bind to DNA (these are often considered chemically undrugable). Overall, PHICS expands the toolkit of chimeric small molecules that can be used to induce a variety of post-translational modifications.

[0300] Materials and methods 2.1 Materials and General Procedure AMPK α1β1γ1-His tag (P47-10H), AMPK α1β2γ1-His tag (P50-10H), PKCα-GST (P61-18G), PKCβI-GST (P62-18G), PKCβII-GST (P63-18G), PKCγ-GST (P66-18G), PKCδ-GST (P64-18G), kinase assay buffer III 5× (K03-09), SAMStide peptide, HMRSAMSGLHLVKRR (S07-58), and CREBtide peptide, KRREILSRRPSYR (C50-58) were all purchased from SignalChem. BRD4-GST ((BD1 and BD2 (49-460))(31044) and BRD4-His tags ((BD1 and BD2 (49-460))(31045), BRD3-GST ((BD1 and BD2 (29-417))(31035), and BRD2-GST ((BD1 and BD2 (65-459))(31024)) were purchased from BPS Bioscience. The ADP-Glo® kinase assay kit (V6930) was from Promega Corporation. OptiPlate-384 (white opaque 384-well microplate, 6007290), nickel chelate AlphaLISA acceptor beads (AL108C), and α-glutathione donor beads (6765300) were all purchased from Perkin Elmer. NuPAGE The 4-12% bis-tris protein gel (NP0336 or NP0335) was from ThermoFisher, and the Ni-NTA agarose beads were purchased from Qiagen.

[0301] For chemical synthesis, all reagents were purchased and used as received from commercial suppliers without further purification. Reactions were carried out in round-bottom flasks with stirring using a Teflon®-coated magnetic stirrer. Reactions sensitive to moisture and air were carried out under a dry nitrogen / argon atmosphere. Liquids or solutions sensitive to moisture and air were transferred using syringes flushed with nitrogen. Where necessary, organic solvents were degassed by bubbling the liquid with nitrogen / argon. The progress of the reactions was monitored by thin-layer chromatography (TLC) and ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS). Flash column chromatography was performed using a Teledyne Isco combiflash Rf system with silica gel (60 Å mesh, 20–40 μm). Analytical TLC was performed using Merck silica gel 60 F254 pre-coated plates (0.25 mm); 254 nm illumination enabled visualization of UV-active substances, and phosphomolybdic acid (PMA) staining was used to visualize UV-inactive substances. UPLC-MS was performed using a Waters ACQUITY UPLC I-Class PLUS system equipped with an ACQUITY SQ detector 2. Nuclear magnetic resonance (NMR) spectra were collected at room temperature using a Bruker AVANCE III HD 400 MHz spectrometer at MIT and the Broad Institute at Harvard University. 1 1H NMR, 400MHz; 13 C, 101MHz). 1 H and 13 ¹³C chemical shifts were expressed in parts per million (ppm), with the residual solvent signal used as an internal standard. NMR solvents were purchased from Cambridge Isotope Laboratories, Inc., and NMR data were obtained in CDCl3 and DMSO-d6. 1The 1H NMR data are reported as follows: chemical shift values ​​in ppm, multiplicity (s=single line, br s=broad single line, d=double line, t=triple line, dd=double line of double lines, and m=multiline), integral value, and coupled constant value in Hz. Tandem liquid chromatography-mass spectrometry (LCMS) was performed using a Waters 2795 separation module with a 3100 mass detector.

[0302] 2.2 Molecular docking Docking was performed using a standard precision protocol with Schroedinger Maestro v11.6. Ligands were prepared by generating a feasible state at pH 7.0 ± 2.0 using Epik, desalting, and subjecting them to the force field of OPLS3e. A two-dimensional ligand interaction map was generated from the docking results to predict the linker binding site.

[0303] 2.3 ADP-Glo ​​kinase assay to verify kinase activation by PHICS molecules ADP-Glo ​​assay in the presence of PHICS molecules with various linkers 1By performing this procedure, we verified its potential ability to activate kinases (AMPKα1β1γ1 or PKCα) and confirmed its binding to the kinases. In a 96-well plate (white, flat-bottomed), concentration series of PHICS (1.1, 1.2, 1.3, 1.4, and 1.5) molecules were prepared using kinase assay buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.1 mg / ml BSA, 50 μM DTT, 1% DMSO) and incubated with 5 ng of AMPK and 0.2 μg / μL SAMStide peptide in the presence of 150 μM ATP. After incubation at room temperature for 2 hours, ADP-Glo ​​reagent was added to the kinase reaction mixture in a 1:1 ratio, and the reaction mixture was maintained at room temperature for a further 40 minutes. Finally, kinase detection reagent was added to this mixture in a 1:2 ratio, and after incubation at room temperature for a further 30 minutes, luminescence was recorded using an Envision 2104 plate reader (PerkinElmer). After removing background signals from AMPK-free controls, kinase activation by PHICS molecules was calculated, and the normalized data against the DMSO control was plotted using GraphPad PRISM version 8.1.1.

[0304] Similarly, the ADP-Glo ​​assay was performed using PHICS (PKC), with the following modifications. In 96-well plates (white, flat-bottomed), similar concentration series of PHICS (2.1, 2.2, 2.3, 2.4, and 2.5) were prepared using the same kinase assay buffer. These were incubated with 2 ng of PKC, 0.2 μg / μL CREBtide peptide, and 50 μM ATP at room temperature for 1 hour, and then incubated with the ADP-Glo ​​assay reagent.

[0305] 2.4 ADP-Glo ​​kinase assay to determine catalytic properties between kinases and PHICS molecules The enzyme turnover efficiency by PHICS (1.2 or 2.3, ternary complex formation promoter) was determined by performing an ADP-Glo ​​assay with a mixture of kinase, BRD4-GST (BD1 and BD2), and PHICS molecules, compared to PHICS ((R)-1.2 or (R)-2.3, ternary complex formation inhibitor). First, 1 μM PHICS1.2 and (R)-PHICS1.2 were prepared in a 96-well plate (white, flat-bottom) using kinase assay buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.1 mg / ml BSA, 50 μM DTT, 1% DMSO) and incubated with 20 nM AMPK and 700 nM BRD4-GST (BD1 and BD2) in the presence of 150 μM ATP for 2 hours at room temperature. Next, ADP-Glo ​​assay reagents were added in the same manner, and luminescence was recorded using an Envision 2104 plate reader (PerkinElmer). The actual signal from the PHICS1.2-mediated kinase reaction (20 nM AMPK, 1 μM PHICS1.2, 700 nM BRD4-GST, and 150 μM ATP) was calculated by subtracting the luminescence signal from the inactive control (20 nM AMPK, 1 μM (R)-PHICS1.2, 700 nM BRD4-GST, and 150 μM ATP). ADP production during the kinase reaction was determined by a calibration curve (luminescence (RLU) vs. %ATP to ADP conversion rate) plotted according to the Promega specifications.

[0306] The same protocol was followed for PHICS2.3. First, 1 μM PHICS2.3 and (R)-PHICS2.3 were prepared in a 96-well plate (white, flat-bottom) using kinase assay buffer, incubated at room temperature for 1 hour in the presence of 50 μM ATP with 50 nM PKC and 700 nM BRD4-GST (BD1 and BD2), and then ADP-Glo ​​reagent was added.

[0307] 2.5 ADP-Glo ​​kinase assay to evaluate BRD4 peptide phosphorylation The ADP-Glo assay was performed with several peptides synthesized from the BRD4 protein to determine the preference of AMPK for the phosphorylation of these peptide substrates. Based on mass spectrometry, the peptide regions shown below were selected and purchased from GenScript.

[0308] S325 = GQRRESSRPVKPPRR (SEQ ID NO: 2) (confirmed as a phosphorylation target by mass spectrometry)

[0309] T169 = ELPTEETEIMIVQRR (SEQ ID NO: 3) (confirmed as a phosphorylation target by mass spectrometry)

[0310] S338 = KKDVPDSQQHPAPRR (SEQ ID NO: 4) (phosphorylation site candidate on BRD4)

[0311] S358 = EQLKCCSGILKEMRR (SEQ ID NO: 5) (phosphorylation site candidate on BRD4)

[0312] After incubating each peptide at 0.2 μg / μL with 20 nM AMPK for 2 hours at room temperature in the presence of 150 μM ATP, the kinase reaction was similarly carried out by adding the ADP-Glo reagent. The ADP-Glo kinase assay with 0.2 μg / μL SAMStide peptide and 20 nM AMPK was used as a positive control, and the preference of AMPK for the phosphorylation of these BRD4 peptides was determined by comparing the luminescence signals generated from each BRD4 peptide with this positive control.

[0313] 2.6 AlphaScreen assay for determining PHICS-induced protein dimerization AlphaScreen assay 2The following procedure was performed to verify the formation of a PHICS-inducible ternary complex between kinase (AMPK or PKC):PHICS molecule:BRD4 (BD1 and BD2). First, concentration series of PHICS with various linkers (1.1, 1.2, 1.3, 1.4, and 1.5) or (R)-PHICS1.2 control with DMSO were prepared in a white opaque 384-well microplate using diluted assay buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1% w / v BSA, 0.01% v / v Tween 20) as 1% DMSO in the final mixture. Next, 7 nM AMPK (6 × His tag) and 67 nM BRD4-GST were added to this mixture and incubated at room temperature for 1 hour. Then, nickel chelate AlphaLISA acceptor beads (PerkinElmer) and glutathione donor beads (PerkinElmer) were added to this mixture at a final concentration of 20 μg / mL. After incubation at room temperature for 1 hour, luminescence was recorded using an Envision 2104 plate reader (PerkinElmer). After removing background signals from DMSO control or protein-free wells, normalized luminescence values ​​were calculated. Data were analyzed and plotted using GraphPad PRISM version 8.1.1. The AlphaScreen assay was performed in the same manner to determine ternary complex formation between AMPK:PHICS1.2:BRD3 (BD1 and BD2) or BRD2 (BD1 and BD2). First, a concentration series of PHICS1.2 was prepared using dilution buffer and incubated with 7 nM AMPK and 67 nM BRD3-GST or BRD2-GST at room temperature for 1 hour, after which acceptor and donor beads were added.

[0314] Following a similar procedure, the formation of PHICS2-inducible ternary complexes between PKC:PHICS2:BRD4-GST (BD1 and BD2) was determined. First, concentration series of PHICS (2.1, 2.2, 2.3, 2.4, and 2.5) with various linkers and (R)-PHICS2.3 with a DMSO control were prepared in a white, opaque 384-well microplate using diluted assay buffer as 1% DMSO in the final mixture. Next, 10 nM PKC-GST and 100 nM BRD4 (6 × His tag) were added to this mixture and incubated at room temperature. After incubation for 1 hour, acceptor beads and donor beads were added to the system.

[0315] 2.7 Pull-down assay to determine ternary complex formation The interaction between AMPK and BRD4 mediated by PHICS1.2 was determined by an in vitro pull-down assay. His-tagged AMPK (70 nM) was immobilized on Ni-NTA agarose beads by incubation at 4°C in binding buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1% w / v BSA, 0.01% v / v Tween 20, 10 mM imidazole). After 1 hour, the beads were washed twice and incubated with BRD4-GST (300 nM) and PHICS1.2 or (R)-PHICS1.2 (1 μM) at 4°C for 4 hours. The beads were then thoroughly washed, mixed with SDS loading buffer, and heated at 95°C for 5 minutes. Western blot analysis was performed using an antibody against AMPK-α (Cell Signaling, catalog no. 5832) and BRD4 (Biovision, catalog no. 6644).

[0316] 2.8 Immunoblotting analysis to confirm BRD4 phosphorylation The phosphorylation of Ser / Thr on BRD4, driven by proximity, was confirmed by PHICS induction using Western blotting with a phospho-(Ser / Thr) kinase substrate antibody. First, PHICS1 (1.1, 1.2, 1.3, 1.4, and 1.5), (R)-PHICS1.2, and a DMSO control were prepared using 1× kinase assay buffer as 1% DMSO, with various linkers at concentrations of 10 μM, 5 μM, and 1 μM. Next, the kinase reaction was carried out at room temperature for 2 hours in the presence of 20 nM AMPK, 700 nM BRD4-GST, and 150 μM ATP. In another experiment, the kinase reaction was carried out similarly with diluted concentrations of PHICS1.2. In addition to BRD4-GST, the same kinase reaction was carried out with 700 nM BRD3-GST (BD1 and BD2) and 700 nM BRD3-GST (BD1 and BD2) in or without PHICS1.2. The importance of the GST tag on BRD4 for PHICS-induced phosphorylation was investigated using the same kinase assay with BRD4-(6×His tag)(BD1 and BD2)(700nM) versions. After incubation for 2 hours, the kinase reaction was quenched by adding SDS loading buffer. The protein was separated by NuPAGE 4-12% Bistrice protein gel and transferred to a PVDF membrane. The membrane was then incubated at room temperature for 1 hour in blocking buffer (0.1% tween 20 and 5% BSA-containing TBS). Subsequently, the membrane was incubated with the phospho-AMPK substrate motif [LXRXX(pS / pT) (Cell Signaling, catalog no. 5759) (1:1000)] primary antibody to detect Ser / Thr phosphorylation on BRD4, and with the anti-BRD4 primary antibody (Biovision, catalog no. 6644) (1:1000) to detect loading levels. After incubation at 4°C overnight, the membrane was washed three times with TBST buffer (TBS containing 0.1% tween 20).After washing three times with TBST buffer, protein bands were visualized either by chemiluminescence with an appropriate HRP-conjugated secondary antibody (rabbit / mouse, Cell Signaling, catalog numbers 7074 / 7076) using an Azure Biosystems C600 imager, or by NIR fluorescence with an IRDye 800CW / IRDye 680RD secondary antibody (rabbit / mouse, LI-COR, catalog numbers 926-32211 / 926-68070) using an LI-COR Odyssey imager.

[0317] PHICS-induced phosphorylation on BRD4 by PKC was also investigated using similar conditions. A kinase reaction was carried out at room temperature for 1 hour using 1 μM PHICS (2.1, 2.2, 2.3, 2.4, 2.5, and (R)-2.3), 50 nM PKC, 700 nM BRD4-GST, and 50 μM ATP, followed by quenching with SDS loading buffer. Proteins were separated on a NuPAGE 4-12% Bistrice protein gel and transferred to a PVDF membrane. After incubation of the membrane with blocking buffer, the phospho-PKC substrate motif [(R / K)XpSX(R / K)] (Cell Signaling, catalog number 6967) (1:1000) primary antibody was added to the membrane, and it was incubated further overnight at 4°C. Protein bands were visualized using an HRP conjugate secondary antibody or an IRDye 800CW / IRDye 680RD secondary antibody.

[0318] Following a similar methodology, the phosphorylation of BRD4 in various AMPK and PKC isoforms, as well as the phosphorylation of various BRD proteins, was determined.

[0319] 2.9 Mass spectroscopy to identify phosphorylated sites in BRD4 The phosphorylation sites on BRD4, driven by proximity, were determined by performing kinase reactions in the presence of AMPK, BRD4, and PHICS, followed by mass spectrometry studies. Initially, kinase reactions were carried out similarly using 20 nM AMPK and 700 nM BRD4-GST (BD1 and BD2) in the presence of 1 μM PHICS1.2 or (R)-PHICS1.2. After incubation for 2 hours, the reaction was quenched by adding 6× loading dye. Proteins in each reaction mixture with PHICS1.2, (R)-PHICS1.2, and DMSO control were separated by NuPAGE 4-12% Bistrice protein gel. After digestion with trypsin or elastase, gel sections containing BRD4-GST were submitted to the Taplin Mass Spectrometry Facility at Harvard Medical School to determine the phosphorylation sites.

[0320] 2.10 Synthesis of PHICS1 analogs (+)-JQ1 PA was purchased from MedChemExpress, and its inert analog, (-)-JQ1 PA, was synthesized according to the literature. 3 Compounds 5-9 were either purchased or synthesized by amide coupling with precursor azidic acid and N-hydroxysuccinimide, washed with water, and then used in the next step without further purification. 4 .

[0321] [ka] 5-Bromo-6-chloro-1H-indole-3-carboxylate methyl(1) Following the procedure described in the literature, 5-bromo-6-chloro-1H-indole (2.22 g, 0.63 mmol) was dissolved in 50 mL of DMF and cooled to 0°C in an ice bath. Trifluoroacetic anhydride (5.4 mL, 38.5 mmol) was slowly added to this solution with stirring. After 1 hour, the reaction was quenched with saturated Na₂CO₃ (aq), and the yellowish-brown precipitate was filtered and collected. This crude precipitate was directly treated with 3 M NaOH (aq) and refluxed overnight to form a carboxylic acid. Next, the reactants were acidified to a pH of 1-2, extracted with siRNA (100 mL x 3), dried over Na₂SO₄, and evaporated to dryness under reduced pressure. This crude solid was refluxed in 50 mL of MeOH and 1 mL of concentrated H₂SO₄ to obtain the methyl ester as a reddish solid (2.00 g, 72% yield).

[0322] The data is consistent with the literature report.

[0323] [ka] N-Boc-((4-aminoethoxy)phenyl)boronic acid pinacol ester (2) Following the procedure described in the literature, 4-hydroxyphenylboronic acid (2.00 g, 9.09 mmol), 2-(Boc-amino)ethyl bromide (2.04 g, 9.09 mmol), and oven-dried K2CO3 (3.77 g, 27.26 mmol) were added to 40 mL of DMF, and the resulting suspension was heated overnight at 60°C. Next, water was added to this reaction mixture, and the aqueous layer was extracted with SiO2 (100 mL × 3). After drying over Na2SO4 and concentrating under reduced pressure, the crude oil was purified by silica gel column chromatography (4:1 hexane:SiO2) to obtain a colorless oil (3.04 g, 92% yield). The data are consistent with the literature report. 5 .

[0324] [ka] 5-(4-(2-((N-Boc)amino)ethoxy)phenyl)-6-chloro-1H-indole-3-carboxylate methyl(3) 5-Bromo-6-chloro-1H-indole-3-carboxylate methyl (0.97 g, 3.36 mmol) and N-Boc-((4-aminoethoxy)phenyl)boronic acid pinacol ester (1.22 g, 3.36 mmol) were suspended in 67 mL of toluene. Potassium carbonate (3.02 g, 21.85 mmol) was dissolved in 33 mL of deionized water and added to the toluene mixture, then Pd(dppf)Cl2·DCM (0.247 g, 0.303 mmol) was added. The reaction mixture was quickly degassed and then refluxed for 2 hours. The reaction product was then concentrated under reduced pressure, redissolved in ethyl acetate, filtered through a Celite pad, washed with water and brine, and then dried over Na2SO4 to concentrate. The residue was purified by silica gel column chromatography (1:1 hexane: siRNA) to obtain a white solid (1.40 g, 94%). 1 H NMR(400MHz,DMSO-d6)δ 12.05(s,1H),8.17(s,1H),7.91(s,1H),7.64(s,1H),7.35(d,J=8.6Hz,2H),7.02(d, J=8.7Hz,2H),4.03(t,J=5.9Hz,2H),3.80(s,3H),3.34(t,J=5.8Hz,2H)1.40(s,9H).

[0325] [ka] 5-(4-(2-aminoethoxy)phenyl)-6-chloro-1H-indole-3-carboxylic acid(4) 5-(4-(2-((N-Boc)amino)ethoxy)phenyl)-6-chloro-1H-indole-3-carboxylate methyl (1.40 g, 3.17 mmol) was first hydrolyzed to the corresponding carboxylic acid by treatment with 3 equivalents of 1 M NaOH, and the mixture was microwaved at 100°C for 2 hours in a 1:1:1 THF:MeOH:water solution. The solution was then concentrated under reduced pressure, the aqueous layer was acidified to pH=3-4, and extracted three times with siRNA. The combined organic layers were dried over Na2SO4, filtered, and concentrated to obtain a white residue, which was sent to the next step without further purification. To the white solid dissolved in 25 mL of DCM at 0°C, TFA (8 mL) was slowly added as a 50% solution in DCM. The reaction mixture was stirred at room temperature for 2 hours, then concentrated under reduced pressure to obtain a reddish-purple oil. This oil was treated with several 20 mL aliquots of ether, evaporated, and dried under high vacuum to obtain a reddish-yellowish-brown solid (0.94 g, 90%). 1 H NMR(400MHz,DMSO-d6)δ 11.99(s,1H),8.08(s,1H),7.93(s,1H),7.63(s,1H),7.39(d,J=8.7Hz,2H),7.07(d,J=8.7Hz,2H),4.22(t,J=5.1Hz,2H),3.26(t,J=5.1Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ 165.6,157.1,136.0,133.9,133.1,132.7,130.9,125.8,125.3,122.7,114.2,112.9,107.6,64.5.

[0326] [ka] 5-(4-(2-aminoethoxy)phenyl)-6-chloro-1H-indole-3-acyladides (10-14) Azides of various linker lengths were prepared using the following general method: Compound 5 - 9 (0.22 mmol, 1 equivalent) was added to a solution of 4 (0.20 mmol, 0.9 mmol) in DMF (0.1 M). DIPEA was added under nitrogen (2 equivalents), and the solution was stirred at room temperature for 2 hours under nitrogen and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (1:10 MeOH:DCM) to obtain 10 - 14 as white powders.

[0327]

Chem.

[0328] PHICS1.1: 1H NMR(400MHz,DMSO-d6)δ 11.93(br),11.42(s),8.71(t,J=5.7Hz,1H),8.17(t,J=5.6Hz,1H),8.07(s,1H),7.94(d,J=10.7Hz,2H),7.62(s,1H),7.47(d,J=8.4Hz,2H),7.39(d,J=8.4Hz,2H),7.34(d,J=8.5Hz,2H),7.02(d,J=8.7Hz,2H),4.53(m,J=7.1Hz,1H),4.38-4.32(m,4H),4.05(t,J=5.8Hz,2H),3.47(q,J=5.7Hz,2H),2.60(s,3H),2.40(s,3H),2.15(t,J=7.3Hz,2H),2.05(q,J=7.1Hz,2H),1.62(s,3H). 13 C NMR(101MHz,DMSO)δ 171.3,169.5,165.6,163.0,157.6,155.0,149.8,144.9,136.7,135.9,135.2,133.8,133.7,132.8,132.5,132.2,130.8,130.6,130.2,129.9,129.5,128.4,125.8,125.2,122.8,122.7,114.0,112.8,66.3,53.9,48.9,38.3,37.5,31.9,25.9,14.0,12.7,11.3.

[0329] PHICS1.2: 11H NMR (400 MHz, DMSO-d6) δ 12.19 - 12.00 (broad, 1H), 11.92 (singlet, 1H), 8.71 (triplet, J = 5.7 Hz, 1H), 8.05 (singlet, 2H), 7.93 (singlet, 2H), 7.61 (singlet, 1H), 7.45 (doublet, J = 8.6 Hz, 2H), 7.38 (doublet, J = 8.6 Hz, 2H), 7.34 (doublet, J = 8.6 Hz, 2H), 7.01 (doublet, J = 8.7 Hz, 2H), 4.52 (multiplet, J = 7.1 Hz, 1H), 4.35 (doublet, J = 5.6 Hz, 2H), 4.28 (triplet, J = 7.2 Hz, 2H), 4.06 - 4.00 (multiplet, 2H), 3.44 (quartet, J = 5.6 Hz, 2H), 3.27 (doublet of doublets, 2H), 2.59 (singlet, 3H), 2.39 (singlet, 3H), 2.09 (triplet, J = 7.4 Hz, 2H), 1.78 (sextet, J = 7.3 Hz, 2H), 1.61 (singlet, 3H), 1.52 (sextet, J = 7.5 Hz, 2H), 1.29 - 1.18 (multiplet, 2H). 13 13C NMR (101 MHz, DMSO-d6) δ 172.3, 169.5, 165.6, 163.0, 157.6, 155.1, 149.8, 144.9, 136.7, 135.9, 135.2, 133.7, 133.7, 132.7, 132.5, 132.3, 130.8, 130.7, 130.2, 129.8, 129.5, 128.4, 125.8, 125.3, 122.7, 122.7, 114.0, 112.8, 66.4, 53.9, 49.1, 38.2, 37.5, 35.0, 34.3, 29.5, 25.5, 24.6, 14.0, 12.7, 11.3. <(R)-PHICS1.2: The NMR data is consistent with PHICS1.2.

[0331] PHICS1.3: 1 ​​H NMR(400MHz,DMSO-d6)δ 11.93(s,1H),8.72(t,J=5.7Hz,1H),8.06(s,2H),7.93(d,J=6.8Hz,2H),7.61(s,1H),7.45(dd,J=8.6,1.6Hz,2H),7.41-7.31(m,4H),7.01(d,J=7.8Hz,2H),4.52(m,J=7.2Hz,1H),4.35(d,J=5.6Hz,2H),4.27(t,J=7.2Hz,2H),4.03(s,2H),3.44(d,J=5.7Hz,2H),3.29-3.22(m,2H),2.59(s,3H),2.40(s,3H),2.09(t,J=7.1Hz,2H),1.61(s,3H),1.49(q,J=7.5Hz,2H),1.25-1.18(br,8H). 13 C NMR(101MHz,DMSO)δ 172.5,169.6,165.6,163.0,157.6,155.1,149.8,144.9,136.7,135.9,135.2,133.8,133.8,132.8,132.5,132.3,130.8,130.7,130.2,129.8,129.5,128.4,125.8,125.3,122.7,122.7,114.0,112.9,66.4,53.9,50.6,49.2,38.2,37.5,35.2,34.3,29.7,28.4,28.1,25.7,14.1,12.7,11.3.

[0332] PHICS1.4: 1H NMR(400MHz,DMSO-d6)δ 11.92(s,1H),8.72(t,J=5.7Hz,1H),8.05(d,J=7.6Hz,2H),7.92(d,J=8.4Hz,2H),7.61(s,1H),7.45(d,J=8.3Hz,2H),7.35(dd,J=11.0,8.4Hz,4H),7.01(d,J=8.5Hz,2H),4.52(m,J=8.1,6.2Hz,1H),4.35(t,J=5.8Hz,2H),4.26(t,J=7.2Hz,2H),4.03(t,J=5.6Hz,2H),3.44(q,J=5.6Hz,2H),3.28-3.20(m,2H),2.59(s,3H),2.40(s,3H),2.10-2.06(m,2H),1.61(s,3H),1.51-1.46(m,2H),1.21(s,12H). 13 C NMR(101MHz,DMSO)δ 172.7,169.6,165.6,163.0,157.6,155.1,149.8,144.9,136.7,135.9,135.2,133.8,133.7,132.8,132.5,132.3,130.7,130.7,130.2,129.8,129.5,128.4,125.8,125.2,122.7,114.0,112.8,107.6,66.4,53.9,49.3,38.2,37.5,35.3,34.3,29.7,28.7,28.6,28.4,25.8,25.2,25.1,14.1,12.7,11.3.

[0333] PHICS1.5: 1H NMR(400MHz,DMSO-d6)δ 11.92(s,1H),8.72(t,J=5.7Hz,1H),8.05(d,J=7.6Hz,2H),7.92(d,J=8.4Hz,2H),7.61(s,1H),7.4 5(d,J=8.3Hz,2H),7.35(dd,J=11.0,8.4Hz,4H),7.01(d,J=8.5Hz,2H),4.52(m,J=8.1,6.2Hz,1H),4 .35(t,J=5.8Hz,2H),4.26(t,J=7.2Hz,2H),4.03(t,J=5.6Hz,2H),3.44(q,J=5.6Hz,2H),3.28-3.20 (m,2H),2.59(s,3H),2.40(s,3H),2.10-2.06(m,2H),1.61(s,3H),1.51-1.46(m,2H),1.21(s,12H). 13 C NMR(101MHz,DMSO)δ 172.95,170.03,163.48,158.12,137.17,136.38,135.66,134.32,133.24,132. 94,132.77,131.21,131.14,130.65,130.28,129.99,128.87,126.27,125.70,1 23.14, 114.50, 113.33, 66.90, 54.38, 49.73, 38.68, 38.01, 35.78, 34.75, 30.21, 29.39, 29.36, 29.30, 29.27, 29.10, 28.86, 26.34, 25.73, 14.52, 13.13, 11.78.

[0334] 2.11 Synthesis of PHICS2 analogs (S)-(1-hydroxy-3-(4-hydroxy-2-nitrophenyl)propan-2-yl)methyl carbamate (20) was synthesized according to the reported procedure. 6 . [ka] Synthesis of (S)-(1-(4-((4-(((tert-butoxycarbonyl)amino)methyl)benzyl)oxy)-2-nitrophenyl)-3-hydroxypropan-2-yl)carbamate methyl (21)

[0335] To a solution of 20 (1.2 g, 4.4 mmol) and K2CO3 (1.2 g, 8.7 mmol) in 33 mL of DMF, tert-butyl (4-(bromomethyl)benzyl)carbamate (1.4 g, 4.7 mmol) was added dropwise in 3 mL of DMF. The reaction mixture was heated overnight at 50 °C, and the progress of the reaction was monitored by LC-MS. When the signal for the starting material VS-3 disappeared, the solvent was removed under reduced pressure. The solid residue was partitioned between 220 mL of ethyl acetate and 40 mL of water. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. This crude product was purified by column chromatography (gradient from 20:80 ethyl acetate / petroleum ether to 50:50 / petroleum ether as the eluent) to provide 1.5 g (70%) of the desired product as a yellow solid, which was obtained. 1 H NMR (400MHz, CDCl3): δ 7.50(s,1H),7.38-7.36(m,2H),7.31-7.29(m,2H),7.15-7.12(m,1H),5.32(br s,1H),5.06(s,2H),4.92(br s,1H),4.31(s,2H),3.96(s,1H),3.73-3.62(m,2H),3.57(s,3H),3.14-3.11(m,1H),2.99-2.95(m,1H),2.39(br s,1H),1.45ppm(s,9H); 13 C NMR(101MHz,CDCl3)δ 157.8,157.2,156.1,150.2,139.4,134.9,133,7,128.0,127.9,125.5,120.7, 110.6,79.81,70.43,64.58,54.32,52.31,33.46,28.52ppm.LS-MS[M+Na]=512.

[0336] [ka] Synthesis of (5-((4-(((tert-butoxycarbonyl)amino)methyl)benzyl)oxy)-2-((S)-3-hydroxy-2-((methoxycarbonyl)amino)propyl)phenyl)-D-benzyl valate (22) Step 1. To a solution of 21 (1.5 g, 3.1 mmol) in MeOH (52 mL), a saturated solution of copper(II) acetate (17 mL) was added. While cooling the resulting solution with ice water, NaBH4 (1.8 g, 47.4 mmol) was added in small amounts over 1 hour. The progress of the reaction was monitored by LC-MS (if the reaction did not reach completion, more NaBH4 had to be added). Once the nitro group was completely reduced, the reaction mixture was passed through a short silica gel column. The filtrate was concentrated under reduced pressure, and the solid residue was partitioned between 180 mL of RINKAN and 35 mL of water. The organic layer was washed with brine, dried over Na2SO4, and concentrated to provide crude aniline, which was used in the next step without further purification.

[0337] Step 2: To the product from the previous step, 1.1 g of triflate obtained from benzyl (R)-3-methyl-2-hydroxybutanoate was added. The mixture was degassed, dissolved in 35 mL of dichloromethane and added under a nitrogen atmosphere, and treated with 600 μL of 2,6-lutidine, and stirred at 70°C. The progress of the reaction was monitored by LC-MS, and when the starting materials were completely consumed (approximately 72 hours), the solvent was removed under reduced pressure, and the crude mixture was purified by LC-MS (gradient from 20:80 ethyl acetate / petroleum ether to 70:30 ethyl acetate / petroleum ether as the eluent). 970 mg (48%) of the desired product was obtained as a white solid. 1H NMR(400MHz,CDCl3):δ 7.32(d,J=8.0Hz,2H),7.26-7.23(m,7H),6.97(d,J=8.4Hz,1H),6.30(d,J=8.4Hz,1H),6.22(s,1H),5.3 4(d,J=8.0Hz,1H),5.10(s,2H),4.90-4.83(m,3H),4.29(d,J=5.6Hz,2H),3.85(d,J=5.6Hz,1H),3.74(br s,1H),3.65(s,3H),3.52(d,J=12.8Hz,1H),3.44(d,J=14.4Hz,1H),2.82-2.78(m,1H),2.72- 2.66(m,1H),2.15-2.10(m,1H),1.44(s,9H),1.03(d,J=6.4Hz,3H),0.98ppm(d,J=6.8Hz,3H); 13 C NMR(101MHz,CDCl3)δ 174.7,159.0,157.0,156.0,146.4,138.7,136.5,135.4,132.1,128.7,128.6,127.9,127.8,116.2,10 3.9,99.2,79.7,69.7,67.1,62.6,61.8,53.4,52.2,44.6,31.7,28.5,19.4,19.2ppm.LS-MS[M+H]=651.

[0338] [ka] Synthesis of (4-((((2S,5S)-5-(hydroxymethyl)-2-isopropyl-3-oxo-1,2,3,4,5,6-hexahydrobenzo[e][1,4]diazosin-9-yl)oxy)methyl)benzyl)carbamate tert-butyl(23) Mixture 22 (220 mg, 0.34 mmol), 5.8 mL of methanol, and 2.9 mL of 2N KOH (aqueous solution) were heated overnight at 70°C. The progress of the reaction was monitored by LC-MS. After the reaction was complete, the mixture was cooled to room temperature, neutralized to pH 7 with 2N HCl, and then concentrated under reduced pressure. The solid residue was dried under vacuum for 1 day and then dissolved in 34 mL of DMF. To this solution, triethylamine (102 μL) and DPPA (90 μL) were added at 0°C. After stirring for 1 hour at 0°C and 17 hours at room temperature, the DMF was evaporated under reduced pressure. The residue was partitioned between 200 mL of ethyl acetate and 35 mL of water. The organic layer was washed with brine, dried over Na2SO4, concentrated, and purified by flash column chromatography (gradient from 20:80 ethyl acetate / petroleum ether to 70:30 ethyl acetate / petroleum ether as eluent). 120 mg (74%) of the desired product was obtained as a yellow oil. 1 H NMR(400MHz,CDCl3):δ 7.34(d,J=8.0Hz,2H),7.29-7.26(m,3H),6.84(d,J=8.0Hz,1H),6.75-6.72(br s,1H),6.46(d,J=8.0Hz,1H),6.42(s,1H),5.01(t,J=6.0Hz,1H),4.93(s,2H),4.30(d,J=5.6Hz,1H),3.82(d,J=8.4Hz,1H),3.70-3.66(m,1H) ),3.62-3.59(m,1H),3.14-3.07(m,1H),2.80-2.76(m,1H),2.14-2.02(m,1H),1.47(s,9H),1.09(d,J=6.8Hz,3H),0.96ppm(d,J=6.8Hz,3H);δ 13 ¹³C NMR (101 MHz, CDCl3): 175.3, 158.4, 156.1, 147.7, 138.8, 136.2, 132.8, 127.8, 127.7, 118.4, 108.1, 106.7, 79.6, 69.6, 66.8, 66.0, 55.46, 44.4, 35.4, 30.5, 28.5, 20.3, and 19.2 ppm. LS-MS [M+H] = 485.

[0339] [ka] Synthesis of (4-((((2S,5S)-5-(hydroxymethyl)-2-isopropyl-1-methyl-3-oxo-1,2,3,4,5,6-hexahydrobenzo[e][1,4]diazosin-9-yl)oxy)methyl)benzyl)carbamate tert-butyl(24) To a solution of 23 (120 mg, 0.25 mmol) in 5 mL of CH3CN, formaldehyde (0.2 mL, 37% wt in water), NaCNBH3 (47 mg), and HOAc (25 μM) were sequentially added at 0°C. The reaction was monitored by LC-MS, and when the signal from the starting materials disappeared, the reactants were partitioned into 50 mL of ethyl acetate and 10 mL of water. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (gradient from 10:90 ethyl acetate / petroleum ether to 40:60 ethyl acetate / petroleum ether as the eluent) to obtain 100 mg (80%) of the desired product. 1 H NMR(400MHz,CDCl3):δ 7.39(d,J=8.4Hz,2H),7.30(d,J=8.4Hz,2H),6.93(d,J=8.4Hz,1H),6.59(d,J=2.8Hz,1H),6.55(br s,1H),6.48(dd,J=8.4&2.8Hz,1H),5.00(s,2H),4.88(br s,1H),4.31(s,2H),3.91(br s,1H),3.69(d,J=3.8Hz&7.0Hz,1H),3.52-3.46(m,2H),3.03(dd,J=16.8&8.0Hz,1H),2.7 5(s,3H),2.45-2.36(m,1H),1.46(s,9H),1.04(d,J=6.4Hz,3H),0.85ppm(d,J=6.8Hz,3H); 13 ¹³C NMR (101 MHz, CDCl3) δ 174.0, 158.7, 156.18, 156.15, 152.9, 139.0, 136.4, 132.6, 128.1, 127.9, 123.7, 107.3, 107.1, 79.9, 70.1, 66.1, 54.6, 54.5, 36.9, 35.3, 28.7, 28.5, 20.7 and 20.1 ppm. LS-MS [M+Na] = 521.

[0340] [ka] Synthesis of (4-((((2S,5S)-5-(hydroxymethyl)-2-isopropyl-1-methyl-3-oxo-1,2,3,4,5,6-hexahydrobenzo[e][1,4]diazosin-9-yl)oxy)methyl)phenyl)methaneaminium trifluoroacetate (25) To an ice-cold solution of VS-43 (12 mg, 24.0 μmol) in DCM (300 μL), 300 μL of trifluoroacetic acid was added, and the reaction mixture was allowed to warm to room temperature and stirred for 30 minutes. The reaction mixture was concentrated to obtain 12.2 mg (99%) of crude VS-35, which was used directly in the next step. 1 H NMR(400MHz,CD3OD):δ 7.52(d,J=8.0Hz,2H),7.46(d,J=8.0Hz,2H),6.96(d,J=8.0Hz,1H),6.75( d,J=2.6Hz,1H),6.59(dd,J=8.4&2.6Hz,1H),5.09(s,2H),4.11(s,2H),3. 59(d,J=4.8Hz&11.0Hz,1H),3.51-3.46(m,2H),2.97-2.83(m,2H),2.74,( s,3H),2.44-2.35(m,1H),1.09(d,J=6.6Hz,3H),0.92ppm(d,J=6.8Hz,3H); 13 C NMR(101MHz,CD3OD)δ 175.6,159.7,154.3,140.2,133.9,133.5,130.3,130.1,129.34,129.31,129.27,126.5,11 0.5,109.1,98.2,70.4,65.7,55.3,40.1,37.7,29.7,20.8,19.9ppm.LS-MS[M-CF3CO2]=398.

[0341] [ka] Synthesis of (S)-4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-yl)acetamide)butanoate tert-butyl(26) Freshly prepared free acid of (+)-JQ-1 (20 mg, 50 μmol) 7 The compound was dissolved in 1.0 mL of DMF and treated with tert-butyl 4-aminobutanoate (9.6 mg, 60 μmol), PyBOP (32 mg, 30 μmol), and Et3N (40 μL). The reaction mixture was stirred overnight and purified by HPLC to obtain 19.5 mg (72%) of VS-327. 1 H NMR(400MHz,CD3OD):δ 7.48(d,J=8.4Hz,2H),7.43(d,J=8.4Hz,2H),4.71(dd,J=5.2&9.2Hz,1H),3.46-3.41(m,1H),3.35-3.24(m ,3H),2.75(s,3H),2.46(s,3H),2.32(t,J=7.4Hz,2H),1.82(t,J=7.2Hz,2H),1.71(s,3H),1.44ppm(s,9H); 13 C NMR(101MHz,CD3OD)δ 174.3,172.5,166.7,156.8,152.6,138.4,134.0,133.3,132.3,132.1,131.5,129. 9,81.6,54.9,39.8,38.4,33.7,28.4,26.1,14.4,13.0,11.5ppm.LS-MS[M+H]=542.

[0342] [ka] Synthesis of (S)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-yl)acetamide)tert-butyl(27) Freshly prepared free acid of (+)-JQ-1 (10 mg, 25 μmol) 7The compound was dissolved in 0.5 mL of DMF and treated with tert-butyl 6-aminohexanoate (5.6 mg, 30 μmol), PyBOP (16 mg, 30 μmol), and Et3N (20 μL). The reaction mixture was stirred overnight and purified by HPLC to obtain 12 mg (85%) of VS-359. 1 H NMR(400MHz,CD3OD):δ 7.47(d,J=8.6Hz,2H),7.42(d,J=8.6Hz,2H),4.71(dd,J=5.4&9.0Hz,1H),3.43(dd,J=9.2 and 15.2Hz,1H),3.32-3.1 9(m,3H),2.75(s,3H),2.45(s,3H),2.24-2.20(m,2H).1.70(s,3H),1.64-1.54(m,4H),1.49-1.37ppm(s and m,13H); 13 C NMR(101MHz,CD3OD)δ 174.8,172.3,166.7,156.8,152.6,138.4,137.45,134.01,133.3,132.3,132.06,131.5,129. 9,81.4,54.9,40.3,38.3,36.3,30.2,28.4,27.4,25.9,14.4,13.0,11.5ppm.LS-MS[M+H]=571.

[0343] [ka] Synthesis of (S)-4-(4-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-yl)acetamide)butanamide)tert-butyl butanoate (28) Freshly prepared free acid of (+)-JQ-1 (10 mg, 25 μmol) 7 The compound was dissolved in 0.5 mL of DMF and treated with tert-butyl 4-(4-aminobutanamide)butanoate (7.3 mg, 30 μmol), PyBOP (16 mg, 30 μmol), and Et3N (20 μL). The reaction mixture was stirred overnight and purified by HPLC to obtain 12.4 mg (79%) of VS-329. 1H NMR(400MHz,CD3OD):δ 7.46(d,J=8.4Hz,2H),7.42(d,J=8.4Hz,2H),4.69(dd,J=5.8&9.0Hz,1H),3.42(dd,J=8.4&15.2Hz,1H),3.36-3.27(m,3H),3.2(t ,J=7.0Hz,2H),2.76(s,3H),2.45(s,3H),2.29-2.23(m,4H),1.89-1.82(m,2H),1.78-1.72(m,2H),1.70(s,3H),1.44ppm(s,9H); 13 C NMR(101MHz,CD3OD)δ 175.4,174.2,172.6,166.6,156.9,152.4,138.2,137.7,133.7,133.5,132.2,132.0,131.4,129.9 ,81.5,55.0,39.9,39.7,38.5,34.4,33.7,28.3,26.9,25.9,14.4,13.0,11.6ppm.LS-MS[M+H]=628.

[0344] [ka] Synthesis of (S)-6-(6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-yl)acetamide)hexanamide)tert-butyl hexanoate (29) Freshly prepared free acid of (+)-JQ-1 (20 mg, 50 μmol) 7 The mixture was dissolved in 1.0 mL of DMF and treated with tert-butyl 6-(6-aminohexanamide)hexanoate (18.0 mg, 60 μmol), PyBOP (32 mg, 30 μmol), and Et3N (40 μL). The reaction mixture was stirred overnight and purified by HPLC to obtain 27.3 mg (80%) of VS-355. 1H NMR(400MHz,CD3OD):δ 7.47(d,J=8.4Hz,2H),7.43(d,J=8.4Hz,2H),4.69(dd,J=5.6&8.8Hz,1H),3.46-3.40(m,1H),3.35-3.26(m,3H),3.16(t,J=7. 0Hz,2H),2.76(s,3H),2.46(s,3H),2.22-2.18(m,4H),1.71(s,3H),1.67-1.48(m,8H),1.43(s+m,11H),1.37-1.28ppm(m,2H); 13 ¹³C NMR (101 MHz, CD3OD) δ 176.0, 174.9, 172.3, 166.7, 156.8, 152.6, 138.4, 137.5, 134.0, 133.3, 132.3, 132.0, 131.5, 129.91, 129.87, 81.3, 54.9, 40.3, 40.2, 38.3, 37.0, 36.3, 30.09, 30.11, 28.4, 27.6, 27.4, 26.7, 25.8, 14.4, 13.0 and 11.5 ppm. LS-MS [M+H] = 684.

[0345] [ka] Synthesis of (R)-6-(2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-yl)acetamide)tert-butyl(30) Freshly prepared free acid of (-)-JQ-1 (6 mg, 15 μmol) 7 The mixture was dissolved in 0.25 mL of DMF and treated with tert-butyl 6-aminohexanoate (2.8 mg, 15 μmol), PyBOP (8 mg, 15 μmol), and Et3N (10 μL). The reaction mixture was stirred overnight and purified by HPLC to obtain 5.7 mg (80%) of VS-361. 1H NMR(400MHz,CD3OD):δ 7.47(d,J=8.4Hz,2H),7.42(d,J=8.4Hz,2H),4.66(dd,J=5.2&9.1Hz,1H),3.45-3.39(m,1H),3.27-3.21(m,3H) ),2.72(s,3H),2.45(s,3H),2.23(t,J=7.4Hz,2H),1.71(s,3H),1.65-1.55(m,4H),1.44-1.29ppm(s and m,12H); 13 C NMR(101MHz,CD3OD)δ 174.9,172.5,166.4,156.9,152.3,138.3,137.9,133.6,133.4,132.1,132.0,131.4,129.8, 81.4,55.1,40.3,38.6,36.3,30.2,28.4,27.4,25.9,14.4,13.0,11.6ppm.LS-MS[M+H]=571.

[0346] [ka] Synthesis of PHICS2.1(31) Freshly prepared free acid of (+)-JQ-1 (5 mg, 12.5 μmol) 7 The mixture was dissolved in 0.35 mL of DMF and treated with freshly prepared 25 (6.4 mg, 12.5 μmol), followed by PyBOP (8.8 mg) and Et3N (25 μL). The reaction mixture was stirred overnight and purified by HPLC to obtain 5.5 mg of the desired product PHICS2.1 (56% yield) as an off-white solid. 1H NMR(400MHz,CD3OD):7.49-7.32(m,8H),6.97(d,J=8.4Hz,1H),6.72(d,J=2.4Hz,1H),6.58(dd,J=8.4 &2.4Hz,1H),5.04(s,2H),4.68(dd,J=9.6&4.2Hz,1H),4.60(d,J=14.8Hz,1H),4.35(d,J=14.8Hz,1H) ,4.24(s,1H),3.59(dd,J=11.2&4.8Hz,1H),3.53-3.45(m,3H),2.95-2.87(m,2H),2.74(s,3H),2.72( s,3H),2.45(s,3H),2.45-2.35(m,1H),1.68(s,3H),1.07(d,J=6.4Hz,3H),0.91ppm(d,J=6.4Hz,3H); 13 ¹³C NMR (101 MHz, CD3OD) δ 172.6, 166.5, 159.9, 156.9, 154.2, 139.8, 138.1, 137.9, 137.8, 133.6, 133.5, 133.4, 132.2, 132.1, 131.4, 129.8, 128.9, 128.8, 110.3, 110.9, 70.8, 65.8, 55.4, 55.2, 43.9, 38.7, 37.8, 36.7, 29.6, 24.2, 20.7, 20, 14.4, 12.9 and 11.5 ppm. LS-MS [M+H] = 780.

[0347] [ka] Synthesis of PHICS2.2(32) A solution of 26 (13.5 mg, 25 μmol) in 1.25 mL of DCM was cooled to 0°C and treated with trifluoroacetic acid (0.35 mL). The reaction mixture was heated to room temperature and stirred for 3 hours, after which the solvent was concentrated under reduced pressure. The freshly prepared 25 (12.8 mg, 25 μmol) was added to 0.7 mL of DMF, followed by the addition of PyBOP (17.5 mg) and Et3N (50 μL). The reaction mixture was stirred overnight and purified by HPLC to obtain 14.7 mg of the desired product PHICS2.2 (68% yield) as an off-white solid. 1H NMR(400MHz,CD3OD):7.45(d,J=8.6Hz,2H),7.40(d,J=8.4Hz,2H),7.36(d,J=8.0Hz,2H),7.27(d,J=8.0Hz,2H),6. 95(d,J=8.4Hz,1H),6.71(d,J=2.4Hz,1H),6.55(dd,J=8.4&2.4Hz,1H),5.01(s,2H),4.69(dd,J=8.4&5.6Hz,1H),4 .37(dd,J=14.8&6.4Hz,1H),4.24(s,1H),3.59(dd,J=10.8&6.4Hz,1H),3.50-3.39(m,3H),2.94-2.87(m,2H),2.73 (s,3H),2.70(s,3H),2.43(s,3H),2.36-2.32(m,2H),1.68(s,3H),1.07(d,J=6.4Hz,3H),0.91ppm(d,J=6.4Hz,3H); 13 ¹³C NMR (101 MHz, CD3OD) δ 175.3, 166.6, 159.8, 154.2, 139.6, 138.2, 138.1, 137.8, 137.7, 133.6, 133.5, 132.2, 131.9, 131.4, 129.9, 128.9, 128.8, 128.6, 126.1, 110.2, 108.9, 70.7, 65.8, 55.4, 55.0, 43.9, 39.9, 38.5, 37.7, 36.8, 34.4, 29.6, 26.9, 20.7, 20.0, 14.4, 13.0 and 11.6 ppm. LS-MS [M+H] = 866.

[0348] [ka] Synthesis of PHICS2.3(33) A solution of 27 (11.4 mg, 20 μmol) in 1 mL of DCM was cooled to 0°C and treated with trifluoroacetic acid (0.3 mL). The reaction mixture was heated to room temperature and stirred for 3 hours, after which the solvent was concentrated under reduced pressure. The freshly prepared 25 (10.2 mg, 20 μmol) was added to 0.5 mL of DMF, followed by the addition of PyBOP (14 mg) and Et3N (40 μL). The reaction mixture was stirred overnight and purified by HPLC to obtain 9 mg of the desired product PHICS2.3 (50% yield) as an off-white solid.1 H NMR(400MHz,CD3OD):7.45-7.36(m,6H),7.27(d,J=7.6Hz,2H),6.94(d,J=8.4Hz,1H),6.71(d,J=2.4 Hz,1H),6.54(dd,J=8.4&び2.4Hz,1H),5.00(s,2H),4.63(dd,J=5.4&8.6Hz,1H),4.36(s,2H),4.23(br s, 1H), 3.61-3.57(m, 1H), 3.50-3.37(m, 3H), 3.28-3.23(m, 2H), 2.91-2.87(m, 2H), 2.72(s, 3H), 2.68(s, 3H), 2.43(s, 3H), 2.26(t, J=7.6Hz, 2H), 1.69(s and σm, 4H), 1.63-1.55(m, 3H), 1.45-1.39(m, 2H), 1.33-1.29(m, 2H), 1.07(d, J=6.6Hz, 3H) and σ0.9(d, J=6.6Hz, 3H); 13 C NMR(101MHz,CD3OD)δ 175.9,175.6,167.0,159.8,154.1,139.7,138.7,137.8,137.1,134.4,133 .5,132.5,132.0,131.6,129.9,128.91,128.87,128.8,128.7,126.1,110. 3,108.9,70.7,65.8,55.4,54.8,44.0,43.8,40.3,38.1,37.7,36.9,36.8, 30.1,29.7,27.5,26.6,20.7,20.0,14.4,13.0,11.5ppm.LS-MS[M+H]=894.

[0349]

change

[0350] [ka] Synthesis of PHICS2.5(35) A solution of 29 (17.1 mg, 25 μmol) in 1.25 mL of DCM was cooled to 0°C and treated with trifluoroacetic acid (0.35 mL). The reaction mixture was heated to room temperature and stirred for 3 hours, after which the solvent was concentrated under reduced pressure. The freshly prepared 25 (12.8 mg, 25 μmol) was added to 0.7 mL of DMF, followed by the addition of PyBOP (17.5 mg) and Et3N (50 μL). The reaction mixture was stirred overnight and purified by HPLC to obtain 15.0 mg of the desired product PHICS2.5 (60% yield) as an off-white solid. 1 1H NMR (400MHz, CD3OD): 1 H NMR(400MHz,CD3OD):7.46(d,J=8.4Hz,2H),7.42(d,J=8.4Hz,2H),7.37(d,J=8.0Hz,2H),7.26(d,J=7.6Hz,2H),6.95(d,J=8. 4Hz,1H),6.72(d,J=2.8Hz,1H),6.56(dd,J=8.4 and 2.8Hz,1H),5.00(s,2H),4.69(dd,J=5.6&8.4Hz,1H),4.34(s,2H),4.25(br s,1H),3.59(dd,J=10.8&4.8Hz,1H),3.49-3.35(m,4H),3.27-3.23(m,3H),3.17-3.13(m,2H),2.91-2.87(m,3H),2.74(s,3H),2.73(s, 3H),2.45(s,3H),2.41-2.34(m,1H),2.25-2.17(m,4H),1.70(s,H),1.67-1.31(m,15H),1.07(d,J=6.8Hz,3H) and 0.90(d,J=6.8Hz,3H); 13 ¹³C NMR (101 MHz, CD3OD) δ 175.5, 166.5, 159.8, 154.2, 139.6, 137.8, 133.5, 132.2, 131.4, 129.9, 128.8, 128.7, 110.2, 108.9, 70.7, 65.8, 55.4, 55.1, 43.9, 39.9, 39.8, 38.6, 37.7, 34.4, 34.3, 29.6, 26.8, 20.7, 20.0, 14.4, 12.9 and 11.6 ppm; 13¹³C NMR (101 MHz, CD3OD) δ 176.0, 175.9, 172.3, 166.7, 159.8, 154.2, 139.7, 138.4, 137.8, 137.5, 134.0, 133.5, 132.3, 132.0, 131.5, 129.9, 128.8, 128.7, 70.7, 65.8, 55.4, 54.9, 43.8, 40.3, 40.2, 38.3, 37.7, 37.0, 36.9, 30.1, 29.7, 27.5, 26.71, 26.66, 20.7, 20.0, 14.4, 13.0 and 11.6 ppm. LS-MS [M+H] = 1007.

[0351] [ka] Synthesis of (R)-PHICS2.3(36) A solution of 30 (5.7 mg, 12 μmol) in 0.6 mL of DCM was cooled to 0°C and treated with trifluoroacetic acid (0.18 mL). The reaction mixture was heated to room temperature and stirred for 3 hours, after which the solvent was concentrated under reduced pressure. The freshly prepared 25 (6.1 mg, 12 μmol) was added to 0.3 mL of DMF, followed by the addition of PyBOP (8.4 mg) and Et3N (24 μL). The reaction mixture was stirred overnight and purified by HPLC to obtain 6.8 mg of the desired product (R)-PHICS2.3 (63% yield) as an off-white solid. 1H NMR(400MHz,CD3OD):7.45(d,J=8.6Hz,2H),7.41(d,J=8.6Hz,2H),7.37(d,J=8.0Hz,2H)7.27(d,J=8.0Hz,2H),6.94(d,J=8. 4Hz,1H),6.71(d,J=2.5Hz,1H),6.54(dd,J=8.4 and 2.5Hz,1H),5.00(s,2H),4.66(dd,J=5.5&8.7Hz,1H),4.36(s,2H),4.24(br s,1H),3.61-3.57(m,1H),3.50-3.47(m,2H),3.27-3.24(m,2H),2.91-2.8 7(m,2H),2.72(s,3H),2.70(s,3H),2.43(s,3H),2.41-2.34(m,1H),2.26( t,J=7.4Hz,2H),1.72-1.67(s and m,4H),1.63-1.55(m,2H),1.45-1.39(m,2 H),1.33-1.29(m,2H),1.07(d,J=6.5Hz,3H) and 0.9ppm(d,J=6.7Hz,3H).13C NMR(101MHz,CD3OD)δ 175.9, 175.6, 172.6, 166.4, 159.8, 158.4, 154.2, 152.3, 139.7, 138.1, 137.9, 137.8, 133.4, 132.1, 132.0, 131.4, 131.1, 129.9, 128.8, 128.7, 128.7, 126.1, 124.3, 12 1.7,111.8,110.2,108.9,70.7,65.8,56.2,55.4,55.1,45.3,43.8,40.3,38.7,37. 7,37.0,30.1,29.7,27.5,26.7,20.7,20.0,14.4,13.0,11.6ppm.LS-MS[M+H]=894.

[0352] Synthesis of PHICS3 and Piv-PHICS3 [ka] Synthesis of (R)-10-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)-10-oxodecanoate 2,5-dioxopyrrolidine-1-yl(37)

[0353] To a solution of 39.6 mg (0.1 mmol, 1 equivalent) of bis(N-succinimidyl) sebacate in 1 mL of DMF, 38.6 mg (0.1 mmol, 1 equivalent) of (R)-3-(4-phenoxyphenyl)-1-(1-piperidine-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine was added. The reaction mixture was stirred at room temperature for 1 hour, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH gradient from 100:0 to 95:5) to obtain 20 mg (30%) of the desired product 37 as a white solid. ¹H NMR (400 MHz, CD3Cl): 8.31(br s,1H),7.62(t,J=8Hz,2H),7.40-7.37(m,2H),7.19-7.13(m,3H),7.08(d,8Hz,2H),4.87-4.76( m,1.5H),4.58(d,J=12Hz,0.5H),4.05(dd,J=12&4Hz,0.5H),3.89(d,J=16Hz,0.5H),3.68-3.62( m,0.5H),3.31-3.25(m,0.5H),3.17-3.10(m,0.5H),2.87-2.78(m,3H),2.74-2.67(m,2H),2.61- 2.55(m,2H),2.42-2.18(m,2H),2.00-1.92(m,1H),1.77-1.55(m,5H),1.40-1.25ppm(m,9H).13C NMR(101MHz,CD3Cl)δ 173.22, 171.96, 171.90, 169.29, 168.69, 158.69, 158.59, 157.74, 156.30, 156.24, 154.85, 154.59, 153.96, 153.76, 144.41, 130.02, 130.00, 129.97, 129.94, 127.50, 127.34, 124.15, 124.08, 119.59, 119.56, 119.52, 119. 16,118.98,53.53,52.66,49.96,45.63,41.69,33.51,33.46,30.97,30.93,30.34,29.99,29.70,29.36,29.32 ,29.19,29.12,29.09,28.93,28.70,25.62,25.51,25.34,25.30,25.15,24.55,24.06ppm.LS-MS[M+H]=668.5.

[0354] [ka] Synthesis of PHICS3 A solution of 3 (28 mg, 65 μmol) in 1 mL of DCM was cooled to 0°C and treated with trifluoroacetic acid (0.1 mL). The reaction mixture was heated to room temperature, stirred for 2 hours, and then concentrated under reduced pressure. The freshly prepared 37 (40 mg, 60 μmol) was added to 1 mL of DMF, followed by the addition of DIPEA (40 μL). The reaction mixture was stirred overnight, concentrated under reduced pressure, and purified by HPLC to obtain 30 mg (57%) of the desired product PHICS3 as a white solid.1H NMR(400MHz,DMSO-d6):11.91(br s,1H),8.25(d,J=8Hz,1H),8.04(br s,2H),7.94(s,1H),7.66-7.62(m,2H),7.61(s,1H),7.45-7.41(m,2H),7.36-7.31(m,2H),7.21-7.09(m,4H), 7.04-6.97(m,2H),4.77-4.68(m,0.5H),4.65-4.56(m,0.5H),4.53-4.46(m,0.5H),4.18-4.11(m,0.5H),4.06 -3.96(m,2.5H),3.89-3.81(m,0.5H),3.61-3.55(m,0.5H),3.48-3.40(m,2H),3.15-3.06(m,1.5H),2.93-2.8 3(m,0.5H),2.36-2.20(m,3H),2.15-2.04(m,3H),1.93-1.83(m,1H),1.65-1.37(m,5H),1.24-1.15(m,8H).13C NMR(100MHz,DMSO-d6):172.55,170.82,170.71,170.31,165.61,162.29,158.19,157.65,157.12,156.30,155.62,154.04,153.90,143.23, 143.15,135.93,133.75,132.79,132.50,130.75,130.09,130.05,127 .95,127.80,125.84,125.27,123.76,122.71,118.95,114.02,112.86, 107.61,97.48,97.37,73.54,66.41,59.75,52.71,52.11,49.31,45.3 0,45.00,41.08,38.23,35.77,35.34,32.38,30.77,29.60,29.22,28. 79,28.72,28.64,25.28,24.94,24.84,24.69,23.46,20.74,14.07ppm .HRMS (ESI-TOF): C49H52ClN8O6 calculated value (M+H): 883.3693, measured value: 883.3690.

[0355]

change

[0356] Step 2: A solution of 3 (13 mg, 30 μmol) in 0.5 mL of DCM was cooled to 0°C and treated with trifluoroacetic acid (0.1 mL). The reaction mixture was heated to room temperature, stirred for 2 hours, and then concentrated under reduced pressure. 38 (30 μmol) of product 3 from Step 1 was added to 1 mL of DMF, followed by DIPEA (20 μL). The reaction mixture was stirred overnight, concentrated under reduced pressure, and purified by HPLC to obtain 7 mg (24%) of the desired product Piv-PHICS3 as an off-white solid. ¹H NMR (400 MHz, CDCl3 / CD3OH 1:1): 8.61 (br s, ¹H), 7.98 (br s ¹H), 7.87 (br s,1H),7.55-7.67(m,2H),7.43-7.50(m,1H),7.34-7.24(m,4H),7.11-7.08(m,1H),7. 04-6.97(m,4H),6.85(m,2H),4.90-4.77(m,1H),4.59-4.48(m,1H),4.32-4.19(m,1H) ,4.09-3.99(m,2H),3.58-3.50(m,2H),3.29-3.24(m,2H),2.35-2.26(m,2H),2.22-2. 10(m,3H),2.03-1.90(m,1H),1.67-1.46(m,5H),1.31-1.17(m,10H),1.07(s,9H).13C NMR(100MHz, CDCl3 / CD3OH 1:1): 174.68, 172.42, 157.74, 157.35, 156.07, 153.97, 152.76, 133.62, 133.07, 132.79, 132.56, 130.25, 129.39, 129.10, 127.97, 126.47, 124.75, 123.10, 122.46, 118.49, 117.79, 113.03, 111.83, 65.7 0, 52.95, 52.12, 49.12, 45.14, 44.90, 41.14, 38.29, 35.21, 32.46, 29.01, 28.76, 28.42, 28.17, 25.64, 25.07, 24.61, 23.97, 22.88 ppm. HRMS (ESI-TOF): Calculated value (M+H) for C54H60ClN8O7: 967.4268, Measured value: 967.4265.

[0357] 2.12. Verification of ternary complex (AMPK:PHICS3:BTK) formation in HEK293T cells Immunoprecipitation was performed to overexpress BTK, which is not normally present in HEK293T cells, and then the formation of the ternary complex (AMPK:PHICS3:BTK) was assessed. HEK293T cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 units / mL), and streptomycin (100 μg / mL). The cells were maintained at 37°C in a 5% CO2 humidified atmosphere.

[0358] After transfecting HEK 293T cells with the pcDNA3.1(+)BTK-Flag plasmid for 36 hours using TransIT®-293 transfection reagent, the cells were maintained overnight in serum-free medium and then incubated with the compound. Subsequently, the cells were treated for 4 hours in fresh serum-free medium with a 5 μM concentration of AMPK activator or BTK binder or PHICS3 or DMSO control, and then lysed on ice in lysis buffer (M-PER® mammalian protein extractant, Halt® protease and phosphatase inhibitor cocktail (2×), and 50 mM NaF). Next, the protein concentration of the cell lysate was measured using the Pierce® BCA protein assay kit. Equal volumes of cell lysate were incubated overnight at 4°C with anti-FLAG® M2 magnetic beads. The beads were then washed three times with TBS buffer (50 mM Tris HCl, 150 mM NaCl, pH 7.4), and the proteins were eluted in SDS loading buffer by heating at 95°C for 5 minutes. The eluted proteins were analyzed by Western blotting using AMPK-α rabbit antibody (Cell Signaling, catalog number 5832), Flag mouse antibody (Cell Signaling, catalog number 8146) (1:2000), or β-actin mouse antibody (Cell Signaling, catalog number 3700) (1:2000).

[0359] 2.13. Immunoblotting analysis to confirm BTK phosphorylation in HEK293T cells Immunoprecipitation was performed to overexpress BTK, which is not normally present in HEK293T cells, and then the formation of the ternary complex (AMPK:PHICS3:BTK) was assessed. HEK293T cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 units / mL), and streptomycin (100 μg / mL). The cells were maintained at 37°C in a 5% CO2 humidified atmosphere.

[0360] HEK 293T cells were transfected with pcDNA3.1(+)BTK-Flag plasmid for 36 hours using TransIT®-293 transfection reagent, then maintained overnight in serum-free medium and incubated with the compound. The cells were then treated for 4 hours in fresh serum-free medium with 5 μM AMPK activator or BTK binder or PHICS3 or DMSO control, and then lysed on ice in lysis buffer (M-PER® mammalian protein extractant, Halt® protease and phosphatase inhibitor cocktail (2×), and 50 mM NaF). Next, the protein concentration of the cell lysate was measured using the Pierce® BCA protein assay kit. Equal volumes of cell lysate were incubated overnight at 4°C with anti-FLAG® M2 magnetic beads. The beads were then washed three times with TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4), and the proteins were eluted in SDS loading buffer by heating at 95°C for 5 minutes. The eluted proteins were analyzed by Western blotting using AMPK-α rabbit antibody (Cell Signaling, catalog number 5832), Flag mouse antibody (Cell Signaling, catalog number 8146) (1:2000), or β-actin mouse antibody (Cell Signaling, catalog number 3700) (1:2000).

[0361] Following a similar protocol, the linker length, concentration, and dose-dependence of PHICS3-induced phosphorylation in cells were observed. HEK293T cells were maintained overnight in serum-free medium, then overexpressed with Flag-BTK and incubated with the compound. In one experiment, 5 μM concentrations of PHICS3 with various linkers (n=6, n=8, and PEG2) were incubated for 4 hours. In another experiment, PHICS3 at various concentrations (0.1, 0.5, 1, 5, and 10 μM) was incubated for 4 hours, and 5 μM of PHICS3 was incubated with HEK293T cells at various time points (1, 2, 4, and 5 hours) before lysis. Western blotting was then performed with phospho-Btk(Ser180)(3D3) antibody (Cell Signaling, catalog no. 3537) (1:1000) and Flag mouse antibody (Cell Signaling, catalog no. 8146) (1:2000). To further verify that AMPK activation alone does not contribute to the induction of significant Ser180 phosphorylation in cells, HEK293T cells were incubated with various concentrations (5, 10, and 20 μM) of a potent commercially available AMPK activator (PF-06409577) or 5 μM PHICS3 or 5 μM AMPK activator or DMSO control for 4 hours. After lysing the cells, Western blotting was performed using phospho-Btk(Ser180)(3D3) antibody and Flag mouse antibody.

[0362] Next, the applicants confirmed the target association of PHICS3 in cells by competitive experiments using chemically modified PHICS3 (Piv-PHICS3) in the presence of ibrutinib. HEK293T cells were maintained overnight in serum-free medium, then overexpressed with Flag-BTK, and incubated with the compound. Next, the cells were treated with 5 μM PHICS3 or Piv-PHICS3 for 4 hours, after which the cells were lysed and Western blotting was performed using phospho-Btk(Ser180)(3D3) antibody and Flag mouse antibody. In this competitive experiment, HEK293T cells were incubated for 4 hours in serum-free medium with DMSO or AMPK activator (5 μM) or PHICS3 (5 μM) or PHICS3 (5 μM) + ibrutinib (1 μM) or ibrutinib (1 μM). In this experiment, ibrutinib was administered 1 hour before the addition of the PHICS3 molecule. After cell lysis, Western blotting was performed using phospho-Btk(Ser180)(3D3) antibody and Flag mouse antibody.

[0363] 2.14. Confirmation of BTK phosphorylation sites and PHICS3 target association through mutation studies To identify the PHICS3-induced phosphorylation site in HEK293T cells, we performed an S180A mutation. In addition, since ibrutinib's reversible analog is an ATP-competitive inhibitor of BTK, we performed several mutations in the ATP-binding pocket of BTK to verify PHICS3 target association. These mutant constructs were created using the Q5® site-directed mutagenesis kit with the following primers: BTK Prepared using S180A (forward: 5'-ACCTGGGAGTGCTCACCGGA-3' (SEQ ID NO: 6), reverse: 5'-TTTAAGCTTCCATTCCTGTTCTCC-3' (SEQ ID NO: 7)), K430R (forward: 5'-CGTGGCCATCAGGATGATCAAAG-3' (SEQ ID NO: 8), reverse: 5'-TCGTACTGGCCTCTCCAT-3' (SEQ ID NO: 9)), T474A (forward: 5'-CTTCATCATCGCTGAGTACATGGCCAATG-3' (SEQ ID NO: 10), reverse: 5'-ATGGGGCGCTGCTTGGTG-3' (SEQ ID NO: 11)), and D539N (forward: 5'-TAAAGTATCTAATTTCGGCCTGTC-3' (SEQ ID NO: 12), reverse: 5'-ACAACTCCTTGATCGTTTAC-3' (SEQ ID NO: 13)). The entire open reading frame (ORF) of all prepared plasmids was verified by DNA sequencing.

[0364] Next, HEK293T cells were transfected with wild-type (WT) or mutant BTK-Flag plasmids using TransIT®-293 transfection reagent. After 36 hours of transfection, the cells were kept overnight in serum-free medium and then incubated with the compounds. Subsequently, the cells were treated with 5 μM AMPK activator or PHICS3 or DMSO control in fresh serum-free medium for 4 hours, and then lysed on ice in lysis buffer (M-PER® mammalian protein extractant, Halt® protease and phosphatase inhibitor cocktail (2×), and 50 mM NaF). Western blotting was then performed with phospho-Btk(Ser180)(3D3) antibody (Cell Signaling, catalog no. 3537) (1:1000) and Flag mouse antibody (Cell Signaling, catalog no. 8146) (1:2000). 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28.Hardie,D.G.;Schaffer,B.E.;Brunet,A.,AMPK:An Energy-Sensing Pathway with Multiple Inputs and Outputs.Trends in cell biology 2016,26(3),190-201. 29.Weekes,J.;Ball,K.L.;Caudwell,F.B.;Hardie,D.G.,Specificity determinants for the AMP-activated protein kinase and its plant homologue analyzed using synthetic peptides.FEBS letters 1993,334(3),335-9. 30.Schaffer,B.E.;Levin,R.S.;Hertz,N.T.;Maures,T.J.;Schoof,M.L.;Hollstein,P.E.;Benayoun,B.A.;Banko,M.R.;Shaw,R.J.;Shokat,K.M.;Brunet,A.,Identification of AMPK Phosphorylation Sites Reveals a Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction.Cell metabolism 2015,22(5),907-21. 31.Gadd,M.S.;Testa,A.;Lucas,X.;Chan,K.H.;Chen,W.;Lamont,D.J.;Zengerle,M.;Ciulli,A.,Structural basis of PROTAC cooperative recognition for selective protein degradation.Nat Chem Biol 2017,13(5),514-521. 32.Kozikowski,A.P.;Wang,S.;Ma,D.;Yao,J.;Ahmad,S.;Glazer,R.I.;Bogi,K.;Acs,P.;Modarres,S.;Lewin,N.E.;Blumberg,P.M.,Modeling,Chemistry,and Biology of the Benzolactam Analogs of Indolactam V(ILV).2.Identification of the Binding Site of the Benzolactams in the CRD2 Activator-Binding Domain of PKCδ and Discovery of an ILV Analog of Improved Isoenzyme Selectivity.J.Med.Chem.1997,40(9),1316-1326. 33.Pal Singh,S.;Dammeijer,F.;Hendriks,R.W.,Role of Bruton’s tyrosine kinase in B cells and malignancies.Mol Cancer 2018,17(1),57. 34.Burger,J.A.;Wiestner,A.,Targeting B cell receptor signalling in cancer:preclinical and clinical advances.Nat Rev Cancer 2018,18(3),148-167. 35.Kang,S.W.;Wahl,M.I.;Chu,J.;Kitaura,J.;Kawakami,Y.;Kato,R.M.;Tabuchi,R.;Tarakhovsky,A.;Kawakami,T.;Turck,C.W.;Witte,O.N.;Rawlings,D.J.,PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization.The EMBO journal 2001,20(20),5692-702. 36.Liang,C.;Tian,D.;Ren,X.;Ding,S.;Jia,M.;Xin,M.;Thareja,S.,The 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Bender, AT; Gardberg, A.; Pereira, A.; Johnson, T.; Wu, Y.; Grenningloh, R.; Head, J.; Morandi, F.; of Fc Receptor but not B-Cell Receptor Signaling.Mol Pharmacol 2017,91(3),208-219. 39.Koehler,AN,A complex task? Direct modulation of transcription factors with small molecules.Curr. Opin.Chem.Biol.2010,14(3),331-340.

[0365] Example 2 - In vitro enhancement of AMP by phosphorylation An in vitro kinase assay was performed for BRD4 phosphorylation by AMPK (1 μM compound). The compounds tested, MS231 and VS806, are shown below. [ka]

[0366] Compounds MS231, VS806, and VS804 were tested with and without 100 μM AMP. Adding AMP enhanced the phosphorylation signal, as shown in Figure 63A. ADP glo was performed on BRD4 phosphorylation by AMPK with and without AMP. ADP generation was measured over 2 hours with 20 nM AMPK, and the phosphorylation signal was enhanced with AMP. [Table 3]

[0367] Example 3 - Design of Abl-associated PHICS molecules To design an Abl-associated PHICS molecule, the applicants selected two known Abl kinase activators: DPH1 and a dihydropyrazole activator (2). Based on previously reported crystal structures of Abl and their activators, the solvent-exposed phenyl and amino groups at positions 1 and 3 of the DPH and dihydropyrazole rings, respectively, were selected as linker binding sites. Firstly, for proof-of-concept experiments, Halotag-linked chloroalkyl chains were attached to both activators via three linkers of varying lengths and polarities. Next, the applicants used adenosine 5'-[γ-thio]triphosphate (ATP-γ-S) as a phosphate donor and probed phosphorylation with an anti-thiophosphate antibody to determine whether Abl-PHICS induces phosphorylation on the HaloTag protein (3). In vitro phosphorylation revealed that dihydropyrazole-derived bifunctional molecules (PHICS9.1–9.3, Figures 64A–64B) significantly increased Halo-tag phosphorylation levels by Abl kinase compared to phosphorylation levels with Abl binders alone. Interestingly, PHICS9.1, containing a nearly nonpolar linker, exhibited the highest phosphorylation level (Figure 64B). Simultaneously, under the tested conditions, DPH-derived PHICS with the same linker did not result in detectable levels of phosphorylation (data not shown). Both DPH and dihydropyrazole-derived PHICS efficiently labeled the Halotag protein, as confirmed by the suppression of TMR labeling4 (Figure 64B). While DPH and dihydropyrazole activators bound to Abl with similar affinity (100–200 nM), the choice of linker binding sites provided different exit vectors, potentially leading to two distinct ternary complexes. The orientation of the protein in the ternary complex with the DPH-based exit vector may be unproductive. This could explain the observed difference in the phosphorylation levels of Halotag by the bifunctional molecules tested. However, further experiments (e.g., AlphaScreen, design of DPH analogs by altering the exit vector position as well as dihydropyrazole) will be conducted to better understand the two systems.

[0368] Following validation of the dihydropyrazole Abl binder in Halotag targeting experiments, the applicants decided to design a PHICS molecule for one of the proposed targets—BRD4. Similar to the Halo-Abl PHICS design, the BRD4 protein binder—(+)-JQ1—was attached to dihydropyrazole via various linkers (PHICS 10.1-10.5, Figure 64A). The resulting molecules were tested in vitro according to the same thio-ATP-based protocol, demonstrating various BRD4 phosphorylation levels compared to the Abl binder alone (Figure 64C). Interestingly, for BRD4, PHICS 10.3 with a more polar linker of average length showed the highest phosphorylation level. As a control, the applicants prepared an enantiomer of PHICS 10.3—PHICS i10.3 (derived from (-)-JQ1)—which should not have been able to bind to BRD4. As expected, in the presence of PHICS i10.3, the phosphorylation levels were the same as with DMSO or the Abl binder alone, demonstrating that the signal observed in the presence of PHICS i10.3 is a result of productive PHICS-inducible ternary complex formation. Furthermore, phosphorylation was not detected in the absence of any of the three components of the ternary complex or thio-ATP. Finally, the applicants observed ternary complex formation in the presence of PHICS i10.3 by Alpha Screen assay, but not with PHICS i10.3 (data not shown). The following document pertains to Example 3. 1. Yang, J.; Campobasso, N.; Biju, M. P.; Fisher, K.; Pan, X. Q.; Cottom, J.; Galbraith, S.; Ho, T.; Zhang, H.; Hong, X.; Ward, P.; Hofmann, G.; Siegfried, B.; Zappacosta, F.; Washio, Y.; Cao, P.; Qu, J.; Bertrand, S.; Wang, D. Y.; Head, M. S.; Li, H.; Moores, S.; Lai, Z.; Johanson, K.; Burton, G.; Erickson-Miller, C.; Simpson, G.; Tummino, P.; Copeland, R. A.; Oliff, A., Discovery and characterization of a cell-permeable, small-molecule c-Abl kinase activator that binds to the myristoyl binding site. Chem Biol 2011, 18 (2), 177-86. 2. Simpson, G. L.; Bertrand, S. M.; Borthwick, J. A.; Campobasso, N.; Chabanet, J.; Chen, S.; Coggins, J.; Cottom, J.; Christensen, S. B.; Dawson, H. C.; Evans, H. L.; Hobbs, A. N.; Hong, X.; Mangatt, B.; Munoz-Muriedas, J.; Oliff, A.; Qin, D.; Scott-Stevens, P.; Ward, P.; Washio, Y.; Yang, J.; Young, R. J., Identification and Optimization of Novel Small c-Abl Kinase Activators Using Fragment and HTS Methodologies. Journal of Medicinal Chemistry 2019, 62 (4), 2154-2171. 3. Allen, J. J.; Li, M.; Brinkworth, C. S.; Paulson, J. L.; Wang, D.; Hubner, A.; Chou, W. H.; Davis, R. J.; Burlingame, A. L.; Messing, R. O.; Katayama, C. D.; Hedrick, S. M.; Shokat, K. M., A semisynthetic epitope for kinase substrates. Nature methods 2007, 4 (6), 511-6. 4. Los, GV; Encell, LP; McDougall, MG; Hartzell, DD; Karassina, N.; Zimprich, C.; Wood, MG; Learish, R.; Ohana, RF; Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris, G.; DH; Bulleit, RF; Wood, KV, HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS chemical biology 2008, 3 (6), 373-82.

[0369] Example 4 - Diversification of the properties of kinases and their binders used in PHICS preparation As described above, the applicants have developed two proof-of-concept PHICS using AMPK and PKC kinases, both of which phosphorylate Ser and Thr residues. However, the range of cellular phosphorylation is much broader: there are over 500 kinases, about one-fifth of which belong to the Tyr kinase family, which is involved in the phosphorylation of Tyr residues. Taking into account the fact that there is considerable variability in kinase abundance and localization across different cell types, the applicants propose to extend the range of PHICS beyond AMPK / PKC to include other kinases.

[0370] Firstly, PHICS for Tyr phosphorylation had to be constructed. In addition, to date, the applicants have only used validated kinase activators, which have a very limited range. Reversible allosteric inhibitors are also likely to be able to create functional PHICS. Thalidomide, bestatin, and pomalidomide are inhibitors of E3 ligases; nevertheless, PROTACs containing these compounds lead to the efficient degradation of POIs. The primary purpose of a bifunctional molecule (PROTAC or PHICS) is to bring the appropriate enzyme and the target protein closer together. Since binding to the enzyme by a non-covalent inhibitor is reversible, when the E3 ligase or kinase dissociates from the bifunctional molecule, its conformation changes to the active form, which can lead to ubiquitination (or phosphorylation in the case of PHICS) of the adjacent protein.

[0371] To expand the range of kinases utilized by PHICS, the applicants will explore two relatively abundant tyrosine kinases with different localization sites: membrane-bound insulin receptor (IRTK) and Ebelson (ABL) Tyr kinase, which can be found in the nucleus, cytoplasm, and mitochondria. To construct PHICS molecules for these kinases, the applicants will utilize their well-characterized activators, DPH and kojic acid (16-18). Regarding allosteric inhibitors, borcertib and trametinib are present in relatively high abundances (8.6 × 10⁶ per U2OS cell, respectively). 3 and 1.2 × 10 5 It is a validated chemical that targets RAC-α-serine / threonine-protein kinase (AKT) and mitogen-activated protein kinase (MEK), which are enzymes with individual molecules (19) and various cellular localizations (cytoplasm, membrane, and nucleus) (20).

[0372] To determine the four proposed kinases, the applicants are designing PHICS molecules for nuclear targets (BRD4) and cytoplasmic targets (AR). PROTACs derived from their binders, (+)-JQ1 and enzalutamide, have successfully degraded both proteins, one of which (ARV-110) has even received Fast Track designation from the FDA for the treatment of patients with metastatic castration-resistant prostate cancer. Starting with binders for six kinases and two targets, the applicants plan to link them with three different types of linkers, thus resulting in multiple combinations. To simplify the synthesis of these molecules, a rapid modular approach will be employed: as the six kinase activators are functionalized with alkynes (six unique molecules, Figure 80), (+)-JQ1 and enzalutamide will be attached (by amide and ether bonds, respectively) to three different linkers containing azides at their terminals (six unique molecules). The resulting building blocks will be linked together using bioorthogonal click chemistry. All combinations will be synthesized and evaluated in vitro using the assays described above and in cellulose using U2OS and HEK293 cell lines. The most promising kinase will be used for transcription factor targeting in Example 5. The applicant notes that if the designed PHICS does not induce phosphorylation of BRD4 and AR in the biochemical assay, other kinases for which several kinase conjugates exist (e.g., Lyn(21), BTK(22), Src(23), GSK(24), and LIMK(25)) will be used.

[0373] Example 5 - Design and in vitro determination of PHICS for regulating various transcription factors Approximately 1600 human transcription factors (TFs) are known, representing 8% of all genes and 20% of oncogenes (26). The applicants selected four TFs that differ in subclass assignment, targeted interaction modes, and intended tumor suppression mechanisms (Figures 81A-81D). The applicants' primary goal is to disrupt protein-protein interactions in the oncogenic Myc-Max pair (a subclass of latent cytoplasmic factors). Recently, Koehler's laboratory discovered compound KI-MS2-008 (Figure 81A) using a small molecule microarray screening assay, which can disrupt the heterodimer Myc-Max and stabilize the Max-Max homodimer in vivo, thereby suppressing tumor growth (27). Building on this discovery, the applicants plan to construct a PHICS capable of phosphorylating Max using KI-MS2-008. It is expected that the introduction of phosphate groups onto the surface of Max will prevent the formation of the Max-Myc heterodimer, shifting the equilibrium towards unbound Myc.

[0374] The applicants' second objective is to disrupt the protein-DNA interaction of the estrogen receptor (ER, a nuclear commensal factor subclass). The PHICS molecule will be designed using a known ER inhibitor, raloxifene (Figure 81B) (28). When raloxifene is used alone, a certain ligand saturation should be maintained so that the ER does not interact with DNA. The PHICS strategy relies on the catalysis of ER phosphorylation by a bifunctional small molecule, which has the potential to exhibit increased therapeutic effects at lower doses. The applicants' third objective, the p53 protein, is even more interesting because, depending on the site and valency of phosphorylation, either protein-protein interactions or protein-DNA interactions can be disrupted. It has been found that deficiency of p53 phosphorylation contributes to the acquisition of p53 resistance in oral squamous cell carcinoma because p53 cannot dissociate from its degradation factor MDM2 (29). Therefore, the applicants aim to restore phosphorylation and disrupt the protein-protein p53-MDM2 interaction by PHICS derived from 2,5-bis(5-hydroxymethyl-2-thienyl)furan or RITA (30). Simultaneously, phosphorylation of p53 in the DNA-binding domain leads to interference with protein-DNA interactions, which could be extremely beneficial in types of cancer involving p53 overexpression (31-32). The ultimate goal is to improve the stability of protein-protein interactions: it is known that β-catenin, upon phosphorylation, forms a stable degradation complex that interferes with downstream signaling (33). For this purpose, PHICS derived from UU-T02 will be designed (34).

[0375] Using the kinases identified above, the applicants will design several PHICS molecules for each target and determine their ability to induce phosphorylation in vitro. Preliminary data will be generated in U2OS and HEK293 cell lines. Cells will be treated with active or inactive PHICS, and target phosphorylation will be monitored after immunoprecipitation with phosphoSer / Thr or Tyr-specific antibodies and subsequent immunoblotting. Immunoprecipitation of kinases and targets will be attempted after treatment of cells with active or inactive PHICS to further confirm complex formation. Secondly, mass spectrometry studies will be conducted to identify phosphorylation sites and determine whether any changes in PHICS design affect the target phosphorylation sites and levels. Thirdly, the effects of the designed PHICS on various cancer cell models will be determined. More specifically, studies involving the Myc-Max pair will use P493-6, ST486, and Myc-induced T-cell acute lymphoblastic leukemia (T-ALL) cell lines. In the case of ER, PHICS molecules will be identified in MCF-7 and T47D ER+ cells. Finally, the effects of p53 and β-catenin phosphorylation will be studied using SW480, HCT116, HT29, MDA-MB-231 Daoy MB, and Rh36 cell lines.

[0376] If the proposed target cannot be directly phosphorylated, the applicants would modulate the transcription factor by phosphorylating its binding partner. For example, HSP90, which stabilizes MDM2 or HIF-α, whose binding to p53 labels it for degradation, can be targeted with MI-1061 or deguerin-derived PHICS, respectively (35-37).

[0377] The following references pertain to Examples 4 and 5. 1. Klaeger, S.; Heinzlmeir, S.; Wilhelm, M.; Polzer, H.; Vick, B.; Koenig, P.-A.; Reinecke, M.; Ruprecht, B.; Petzoldt, S.; Meng, C.; Zecha, J.; Reiter, K.; Qiao, H.; Helm, D.; Koch, H.; Schoof, M.; Canevari, G.; Casale, E.; Depaolini, S. R.; Feuchtinger, A.; Wu, Z.; Schmidt, T.; Rueckert, L.; Becker, W.; Huenges, J.; Garz, A.-K.; Gohlke, B.-O.; Zolg, D. P.; Kayser, G.; Vooder, T.; Preissner, R.; Hahne, H.; Tonisson, N.; Kramer, K.; Goetze, K.; Bassermann, F.; Schlegl, J.; Ehrlich, H.-C.; Aiche, S.; Walch, A.; Greif, P. A.; Schneider, S.; Felder, E. R.; Ruland, J.; Medard, G.; Jeremias, I.; Spiekermann, K.; Kuster, B., The target landscape of clinical kinase drugs. Science (Washington, DC, U. S.) 2017, 358 (6367), 1148. 2. Wu, P.; Nielsen, T. E.; Clausen, M. H., FDA-approved small-molecule kinase inhibitors. Trends Pharmacol. Sci. 2015, 36 (7), 422-439. 3. Bhullar, K. S.; Lagaron, N. O.; McGowan, E. M.; Parmar, I.; Jha, A.; Hubbard, B. P.; Rupasinghe, H. P. V., Kinase-targeted cancer therapies: progress, challenges and future directions. Mol. Cancer 2018, 17, 48 / 1-48 / 20. 4. Koehler, A. N., A complex task? Direct modulation of transcription factors with small molecules. Curr. Opin. Chem. Biol. 2010, 14 (3), 331-340. 5. Hagenbuchner, J.; Ausserlechner, M. J., Targeting transcription factors by small compounds-Current strategies and future implications. Biochem. Pharmacol. (Amsterdam, Neth.) 2016, 107, 1-13. 6. Choudhary, A.; Wu, P.; Ding, E. A. Compositions and methods for inducing protein phosphorylation. March 31 2016, 2016. 7. Fegan, A.; White, B.; Carlson, J. C. T.; Wagner, C. R., Chemically Controlled Protein Assembly: Techniques and Applications. Chem Rev 2010, 110 (6), 3315-3336. 8. Spencer, D.; Wandless, T.; Schreiber, S.; Crabtree, G., Controlling signal transduction with synthetic ligands. Science 1993, 262 (5136), 1019-1024. 9. Guilinger, J. P.; Pattanayak, V.; Reyon, D.; Tsai, S. Q.; Sander, J. D.; Joung, J. K.; Liu, D. R., Broad Specificity Profiling of TALENs Results in Engineered Nucleases With Improved DNA Cleavage Specificity. Nat Meth 2014, 11 (4), 429-435. 10. Lu, J.; Qian, Y.; Altieri, M.; Dong, H.; Wang, J.; Raina, K.; Hines, J.; Winkler, J. D.; Crew, A. P.; Coleman, K.; Crews, C. M., Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4. Chem. Biol.(Oxford, U. K.) 2015, 22 (6), 755-763. 11. Zengerle, M.; Chan, K.-H.; Ciulli, A., Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4. ACS Chem. Biol. 2015, 10 (8), 1770-1777. 12. Salami, J.; Alabi, S.; Willard, R. R.; Vitale, N. J.; Wang, J.; Dong, H.; Jin, M.; McDonnell, D. P.; Crew, A. P.; Neklesa, T. K.; Crews, C. M., Androgen receptor degradation by the proteolysis-targeting chimera ARCC-4 outperforms enzalutamide in cellular models of prostate cancer drug resistance. Commun. Biol. 2018, 1 (1), 1-9. 13. Neklesa, T. K.; Winkler, J. D.; Crews, C. M., Targeted protein degradation by PROTACs. Pharmacol. Ther. 2017, 174, 138-144. 14. Mach, U. R.; Lewin, N. E.; Blumberg, P. M.; Kozikowski, A. P., Synthesis and pharmacological evaluation of 8- and 9-substituted benzolactam-V8 derivatives as potent ligands for protein kinase C, a therapeutic target for Alzheimer's disease. ChemMedChem 2006, 1 (3), 307-314. 15. Cameron, K. O.; Kung, D. W.; Kalgutkar, A. S.; Kurumbail, R. G.; Miller, R.; Salatto, C. T.; Ward, J.; Withka, J. M.; Bhattacharya, S. K.; Boehm, M.; Borzilleri, K. A.; Brown, J. A.; Calabrese, M.; Caspers, N. L.; Cokorinos, E.; Conn, E. L.; Dowling, M. S.; Edmonds, D. J.; Eng, H.; Fernando, D. P.; Frisbie, R.; Hepworth, D.; Landro, J.; Mao, Y.; Rajamohan, F.; Reyes, A. R.; Rose, C. R.; Ryder, T.; Shavnya, A.; Smith, A.C.; Tu, M.; Wolford, A. C.; Xiao, J., Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy. J. Med. Chem. 2016, 59 (17), 8068-8081. 16. Yang, J.; Campobasso, N.; Biju, M. P.; Fisher, K.; Pan, X.-Q.; Cottom, J.; Galbraith, S.; Ho, T.; Zhang, H.; Hong, X.; Ward, P.; Hofmann, G.; Siegfried, B.; Zappacosta, F.; Washio, Y.; Cao, P.; Qu, J.; Bertrand, S.; Wang, D.-Y.; Head, M. S.; Li, H.; Moores, S.; Lai, Z.; Johanson, K.; Burton, G.; Erickson-Miller, C.; Simpson, G.; Tummino, P.; Copeland, R. A.; Oliff, A., Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site. Chem. Biol. (Cambridge, MA, U. S.) 2011, 18 (2), 177-186. 17. Simpson, G. L.; Bertrand, S. M.; Borthwick, J. A.; Campobasso, N.; Chabanet, J.; Chen, S.; C...

Claims

1. A polyfunctional chemical conjugation molecule comprising a localization portion, a chemical linker portion, an activator portion, a first orientation adapter that interconnects the chemical linker portion with the activator portion at one end, and optionally a second orientation adapter that interconnects the chemical linker molecule with the localization portion at a different end.

2. Formula I-A Loc-L-(V １ -Act) ｎ (I-A) (In the formula, Loc includes the localized portion, L is the chemical linker portion, and V １ (where is the first orientation adapter, and Act is the activator portion, and n is at least 1); or formula I-B Loc-V ２ -L-(V １ -Act) ｎ (I-B) (In the formula, Loc includes the localized portion, L is the chemical linker portion, and V １ is the first orientation adapter, V ２ (wherein is the second orientation adapter, and Act is the activator portion.) The molecule according to claim 1, represented by [the given expression].

3. The molecule according to claim 1 or 2, wherein the first and second orientation adapters are independently selected from Table 2.

4. The molecule according to claim 1, wherein the activating agent portion binds to an enzyme that modifies a target substrate associated with the localization portion and activates it.

5. The molecule according to claim 4, wherein, if the target substrate is not a native substrate of the enzyme, or if the activation of the enzyme by the activator molecule is not activated by binding to the activator moiety, the enzyme modifies the target substrate by the enzyme at one or more new modification sites that would otherwise remain unmodified by the enzyme.

6. Linker, 【Chemistry 1】 (In the formula, n is between 1 and 50.) A molecule selected from the following, according to claim 1.

7. The molecule according to claim 1, wherein the linker is a PEG molecule, an alkyl, a heterocycloalkyl, a cycloalkyl, an aryl, an alkylene, an alkenyl, a heteroaryl, an amide, an amine, a thiol, or a derivative thereof.

8. The molecule according to claim 1, wherein the linker is a polyfunctional linker.

9. The molecule according to claim 8, wherein the linker is a polyfunctional PEG linker.

10. The molecule according to claim 2, wherein n is 2 to 5.

11. The molecule according to claim 1, wherein the activator portion has the ability to locate and activate an enzyme.

12. The molecule according to claim 11, wherein the enzyme is a kinase, phosphatase, transferase, or ligase.

13. The molecule according to claim 12, wherein the kinase is a serine / threonine kinase, a tyrosine kinase, or a bispecific protein kinase that phosphorylates protein serine / threonine and protein tyrosine.

14. The molecule according to claim 13, wherein the kinase is AMP-activated protein kinase (AMPK), glucokinase (GK), or AGC kinase.

15. The molecule according to claim 1, wherein the activator portion binds to protein kinase C (PKC) and activates it.

16. The molecule according to claim 15, wherein the activator portion binds to a PKC isoform selected from PKC-α, PKC-βI, PKC-βII, PKC-γ, PKC-ε, PKC-δ, PKC-η, or PKC-ζ and activates it.

17. The activator portion is the molecule according to claim 15, selected from Table 2.

18. The molecule according to claim 1, wherein the localization portion targets nucleic acids, polypeptides, or polysaccharides.

19. The molecule according to claim 1, wherein the localization portion is a target polypeptide binding portion.

20. The molecule according to claim 19, wherein the target polypeptide binding portion binds to a target polypeptide comprising a bromodomain and an extra-terminal motif (BET).

21. The molecule according to claim 20, wherein the target polypeptide is bromodomain-containing protein 4 (BRD4), BRD3, BRD2, and BRDT.

22. The molecule according to claim 21, wherein the target polypeptide binding portion is (+)-JQ1.

23. formula 【Chemistry 2】 (In the formula, n = 3, 5, 7, 9, 11 [PHICS 1.1 to 1.5]) The molecule according to claim 1, relating to the present invention.

24. formula 【Transformation 3】 (In the equation, n=0 and m=0, n=1 and m=1, n=2 and m=3, n=2 and m=1, and n=2 and m=3) The molecule according to claim 1, relating to the present invention. 【Request Item 25】 【Chemistry 4】 A molecule selected from the molecules described in claim 24.

26. The molecule according to claim 1, wherein the localization portion is independently selected from Table Y, the activation portion is independently selected from Table Z, the first and second orientation adapters are independently selected from Table 1, and the linker is independently selected from Table 2.

27. formula 【Transformation 5】 (wherein X is (CH ２ ), (CH ｎ ), (CH ２ ), (CH ｎ NHCO(CH ２ ), (CH ｎ ), (CH ２ ), (CH ｎ )(OCH ２ H[[ID=十九]] ４ ), (CH ｎ ), (CH ２ ), (CH ｎ NHCOCH ２ )(OCH ２ H ４ ), and (CH ｎ ), and (CH ２ ), and (CH ｎ NHCO(CH ２ )(OCH ｎ H ２ ), and (CH ４ ), and (CH ｎ selected from, where each n is independently selected from 0, 1, 2, 3, 4, 5, 6 or 7, and R is 【Transformation 6】 (is) The molecule according to claim 1, relating to the present invention.

28. formula 【Transformation 7】 The molecule according to claim 1, relating to the present invention.

29. formula 【Transformation 8】 (In the formula, R １ This is selected from JQ1, ibrutinib, dasatinib, MRTX, MI-1061, gefitinib, palbociclib, or foretinib, and R ２ X is selected from PF-06409577, benzolactam, or DPH, and X is CH ２ or (CH ２ ) ２ O is O, and X = CH ２ When this is the case, n = 1 or 5 or m = 0 or 4, and X = (CH ２ ) ２ When the value is O, then n = 3 or m = 3. The molecule according to claim 1, relating to the present invention.

30. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 29 and one or more pharmaceutically acceptable salts, carriers, or diluents.

31. The pharmaceutical composition according to claim 30, further comprising AMP.

32. A method for modifying a target substrate of a cell, comprising contacting the cell with a molecule described in any one of claims 1 to 32.

33. The method according to claim 33, wherein the modification includes post-translational modification.

34. The method according to claim 33, wherein the post-translational modification includes phosphorylation, hydroxylation, acetylation, methylation, glycosylation, prenylation, amidation, elimination, lipidation, acylation, lipoylation, deacetylation, formylation, S-nitrosylation, S-sulfenylation, sulfonylation, sulfinylation, succinylation, sulfation, carbonylation, or alkylation.

35. The method according to claim 34, wherein the modification includes inducing the phosphorylation of a protein in the cell.

36. A method for phosphorylating a protein, comprising contacting the protein with a molecule described in any one of claims 1 to 32, wherein the protein is in close proximity to a kinase specific to the activator portion of the molecule.

37. The method according to claim 36, wherein the phosphorylation of the protein comprises the phosphorylation of a plurality of proteins that are not substrates of the kinase.

38. The method according to claim 36, wherein the protein is BRD4.

39. The method according to claim 38, wherein the protein is phosphorylated between BD1 and BD2 of BRD4.

40. A method for modifying a target substrate in a subject that requires such modification, comprising administering a molecule according to any one of claims 1 to 32 to the subject.

41. The method according to claim 40, wherein the subject has cancer.

42. A method for modifying a target protein, comprising contacting the target protein with a compound according to any one of claims 1 to 32 in an environment containing one or more activators.

43. A method for treating a disease, disorder, or condition in a subject in need thereof, comprising administering a molecule described in any one of claims 1 to 32 to the subject.

44. A method for producing a polyfunctional conjugation molecule, comprising attaching a localization portion and an activator portion to different ends of a linker molecule, wherein the localization portion and the activator portion are optionally attached to the linker molecule via an orientation adapter, and the linker molecule links the activator molecule such that both the activator molecule and the localization portion are active in cells.

45. The method according to claim 42, wherein the compound is selected from compounds in Table 4, Table 5, Table 6, Table 8, Table 9, Table 10, Table 11, Table 12, or Table 14.

46. formula 【Chemistry 9】 The composition according to claim 1, relating to the present invention.

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