Sumoylation-targeting chimeras (sutacs)

Abstract

The present disclosure relates to SUMOylation-targeting chimeras (SUTACs) comprising a SUMOylation enzyme binder group attached to a target protein binder group via a linker and to uses thereof for the treatment of diseases.

Description

[0001] SUMOYLATION-TARGETING CHIMERAS (SUTACS)

[0002] SEQUENCE LISTING

[0003]

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 2, 2025, is entitled “P-629947-PC_SL.xml”, and is 8,263 bytes in size.

[0004] FIELD OF THE DISCLOSURE

[0005]

[0002] The present disclosure relates to SUMOylation-targeting chimeras (SUTACs) comprising a SUMOylation enzyme binder group attached to a target protein binder group via a linker and to uses thereof for the treatment of diseases.

[0006] BACKGROUND

[0007]

[0003] Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) constitutes a critical molecular regulator for numerous proteins involved in myriad cellular processes (Gareau, J. R. & Lima, C. D. The SUMO pathway: Emerging mechanisms that shape specificity, conjugation and recognition. Nat Rev Mol Cell Biol 11, 861-871, 2010).

[0008]

[0004] SUMO is reversibly conjugated to substrate proteins to regulate a variety of fundamental cellular processes, such as proteolysis, signal transduction, cell division, gene expression and differentiation (Hay, R. T. SUMO: A history of modification. Molecular Cell vol. 18 1-12, 2005). The conjugation of SUMO onto substrates is facilitated by a multi-step enzymatic cascade involving El activating, E2 conjugating and E3 ligating enzymes. SUMOylation was shown to be a robust protein modification able to modulate protein stability, activity, solubility, localization and interaction with other proteins in context- and substrate-specific manners. Previous studies identified antagonistic interactions between ubiquitination and SUMOylation for specific targets by competition over the same acceptor lysine residue. In other cases, SUMOylation was shown to play agonistic interactions with ubiquitination, which result in the degradation of proteins by the proteasome. Beyond protein stability, SUMOylation was shown to be a critical regulator in controlling the activity and functionality of key proteins by either promoting or abrogating their binding to their protein partners.

[0009]

[0005] The concept of altering the modification state of proteins of interest (POIs) to regulate their proper levels or functions has gained recognition as a promising therapeutic strategy with the advent of heterobifunctional chimeras (Modell, A. E., Lai, S., Nguyen, T. M. & Choudhary, A. Bifunctional modalities for repurposing protein function. Cell Chemical Biology vol. 28, 2021), including PROteolysis-TArgeting chimeras (PROTACs) (Bekes, M., Langley, D. R. & Crews, C. M. PROTAC targeted protein degraders: the past is prologue. Nature Reviews Drug Discovery vol. 21, 2022). Modalities for selectively triggering the SUMOylation of proteins, have not been created thus far. Unlike PROTACs, which are designed to result in the degradation of the protein, the SUTAC modality may be utilized to alter protein function. It is therefore desirable to develop SUMOylation-targeting chimeras (SUTACs) to induce SUMOylation of proteins.

[0010] SUMMARY OF THE DISCLOSURE

[0011]

[0006] In one aspect, disclosed herein is a SUMOylation-targeting chimera (SUTAC) comprising a SUMOylation enzyme binder group attached to a target protein binder group via a linker.

[0012]

[0007] In certain embodiments, the SUMOylation enzyme binder group is capable of binding a SUMO E3 ligase. In certain embodiments, the SUMO E3 ligase is selected from the group consisting of PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, TOPORS, Triml9, Trim28, PML, ZNF451, and RANBP2.

[0013]

[0008] In certain embodiments, the SUTAC is represented by the structure of formula I or salt thereof: wherein,

[0014] E is H, amide, acetyl amide, or an electrophilic group;

[0015] Z is O, S and NH; each of R6, R7are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, - C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl; or R6, R7form together a 5-8 membered ring, wherein the ring is substituted or unsubstituted;

[0016] R11 is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0017] L2is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and W is a target protein binder group.

[0018]

[0009] In certain embodiments, Z is S.

[0019]

[0010] In certain embodiments, the SUTAC is represented by the structure of formula I’ or salt thereof: wherein R6, R7, R11, L1, L2, Z and W are as defined for formula I.

[0020]

[0011] In certain embodiments, the SUTAC is represented by the structure of formula IB or salt thereof:

[0021] wherein: the represents a saturated or non-saturated bond; wherein if is a saturated bond, then X is C, N, O or S; wherein if is a non-saturated bond, X is C or N; each of R8, R9, R10are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, - C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl;

[0022] R11 is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; n is an integer 1, 2 or 3; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0023] L2 is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and W is a target protein binder group.

[0024]

[0012] In certain embodiments, the target protein binder group comprises a Bromodomaincontaining protein 4 (BRD4)-targ eting binder to form a BRD4-targeting SUTAC.

[0025]

[0013] In certain embodiments, the BRD4-targeting binder comprises JQ1 or an acid or ester thereof.

[0026]

[0014] In certain embodiments, the BRD4-targeting SUTAC is represented by one of the following structures:

[0027] Ċ

[0028] IA-7 (=l-24)(Figure 8 A)

[0029] ID-3 (Figure 17 A).

[0030]

[0015] In certain embodiments, the target protein binder group comprises a P300-targeting binder to form a P300-targeting SUTAC.

[0031]

[0016] In certain embodiments, the P300-targeting binder comprises GNE-207 or a derivative thereof.

[0032]

[0017] In certain embodiments, the P300-targ eting SUTAC is represented by one of the following structures:

[0033] ID-4 (Figure 18 A)

[0034]

[0035] ID-5 (Figure 18D)

[0036]

[0018] In certain embodiments, the target protein binder group comprises an Androgen receptor (AR)-targeting binder to form an AR-targeting SUTAC.

[0037]

[0019] In certain embodiments, the AR-targeting SUTAC is represented by one of the following structures:

[0038] IA-2 (Figure 10 A)

[0039] IA-3.

[0040]

[0020] In another aspect, the present disclosure provides a pharmaceutical composition comprising the SUTAC described herein and a suitable acceptable carrier.

[0041]

[0021] In another aspect, the SUTAC described herein is for use in treating, improving the condition, inhibiting the decline of a subject afflicted with cancer. In certain embodiments, the cancer is selected from the group consisting of: kidney cancer, lung cancer, endometrial / uterine cancer, esophageal cancer, breast cancer, cervical cancer, liver cancer, gastric cancer, esophageal cancer, head and neck cancer, ovarian cancer, skin cancer, bile duct cancer, leukemia, lymphoma, rhabdoid, brain cancer, colon / colorectal cancer, pancreatic cancer, myeloma, prostate cancer, neuroblastoma, gastric cancer, sarcoma, thyroid cancer, bladder cancer, bone cancer or eye cancer.

[0022] In another aspect, the SUTAC described herein is for use in treating, improving the condition, inhibiting the decline of a subject afflicted with inflammation, neuro-inflammation, allergy, aging, autoimmunity, viral infections, bacterial infections, obesity, neurodegenerative diseases, fibrosis, cardiovascular diseases, diabetes, Crohn’s disease, Colitis, osteoporosis, Multiple Sclerosis (MS), SLE, or non-alcoholic fatty liver disease.

[0042]

[0023] In certain embodiments, the diabetes is type II diabetes.

[0043]

[0024] In certain embodiments, administering the SUTAC to a subject decreases the degradation of the target protein compared to a non -treated subject. In certain embodiments, administering the SUTAC to a subject increases the degradation of the target protein compared to a non -treated subject.

[0044]

[0025] In certain embodiments, administering the SUTAC to a subject decreases the stabilization of the target protein compared to a non-treated subject. In certain embodiments, administering the SUTAC to a subject increases the stabilization of the target protein compared to a non-treated subject.

[0045]

[0026] In certain embodiments, administering the SUTAC to a subject decreases the activation of the target protein compared to a non-treated subject. In certain embodiments, administering said SUTAC to a subject increases the activation of the target protein compared to a non-treated subject.

[0027] In certain embodiments, administering the SUTAC to a subject reduces the repression of the target protein compared to a non-treated subject. In certain embodiments, administering said SUTAC to a subject increases the repression of the target protein compared to a non-treated subject.

[0046]

[0028] In certain embodiments, administering the SUTAC to a subj ect increases the binding of the target protein to other protein(s) or DNA compared to a non-treated subject. In certain embodiments, administering said SUTAC to a subject decreases the binding of the target protein to other protein(s) or DNA compared to a non-treated subject.

[0047]

[0029] In certain embodiments, administering the SUTAC to a subj ect decreases the solubilization of the target protein compared to a non-treated subject. In certain embodiments, administering the SUTAC to a subject increases the solubilization of the target protein compared to a non-treated subject.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049]

[0023] Figures 1A-1C Figure 1A shows a schematic overview highlighting differences between PROTAC and SUTAC modalities; Figure IB schematically shows the Electrophile screen against a recombinant domain derived from a SUMO E3 ligase; Figure 1C shows initial hits from the screen of PIAS1 binders.

[0050]

[0024] Figure 2 shows the binding potency (PIAS 1 -binding IC50 values) and thiol reactivity characterization of derivatives of molecules 2 and 3.

[0051]

[0025] Figures 3A-3C showing the binding potency (PIAS 1 -binding IC50 values) and thiol reactivity characterization of derivatives of molecule 1. Figure 3A derivatives of the amide position; Figure 3B thiophene ring substituents; Figure 3C sulfamate acetamide derivatives of the chloroacetamide.

[0052]

[0026] Figures 4A-4C showing cellular engagement of 1-12 with PIAS proteins. Figure 4A structure of 1-34 used for proteomics characterization of cellular targets; Figure 4B Gel -based ABPP experiment where Daudi cells were incubated with 1-34 (6 hours), 1-12 (6 hours) or preincubated with 1-12 for 2 hours followed by incubation with 1-34 (6 hours). The cells were lysed, and the lysates were clicked to TAMRA azide and imaged on a gel; Figure 4C Proteomic characterization of targets where Daudi cells were treated with 1 μM 1-34 (6 hours) with or without preincubation with 10 μM 1-12 (2 hours). The cells were lysed, clicked to a biotin- functionalized, trypsin-cleavable azide-containing peptide, and the labeled proteins were enriched on streptavidin beads, trypsinized and analyzed using LC-MS / MS.

[0027] Figures 5A-5D showing BRD4-targeting SUTAC lowers the protein levels of BRD4 and c-MYC. Figure 5A shows the structure of BRD4-targeting SUTACs (left) and their IC50 binding to PIAS1, and the structure JQ1 (right); Figure 5B western blot with the indicated antibodies of Daudi cells treated with increasing concentrations of the indicated SUTAC molecules for 20 hours; Figure 5C shows the structure of 1-5, 1 PIAS1 binder and a PEG linker (left), and the structure of JQ1 -ester (right), which is cell permeable; Figure 5D western blot with the indicated antibodies of Daudi cells treated with increasing concentrations of the indicated molecules for 20 hours.

[0053]

[0028] Figures 6A-6B showing BRD4-targeting SUTAC reduces the levels of BRD4 in SUMOylation and PIAS-dependent manners. Figure 6A western blot with the indicated antibody of Daudi cells treated with increasing concentrations of the indicated SUTAC molecules for 20 hours. Cells were either preincubated for 1 hour with 50μM 2D08 (Sigma, SML1052) or DMSO as control; Figure 6B western blot with the indicated antibody of TK6 cells with the indicated genetic background treated with the indicated SUTAC for 20 hours.

[0054]

[0029] Figure 7 showing that recombinant BRD4 gets modified by SUTAC under SUMOylation-promoting conditions. Recombinant (Glu49-Glu460)-FLAG-BRD4 was incubated in active protein extracts derived from A549 cells for 1 hour at 30C, with increasing concentrations of SUTAC or DMSO control, under conditions that promote SUMOylation preservation (SUMO 1 -aldehyde and SUMO2-aldehyde) or control. This was followed by a flagtag pulldown and the eluates were subjected to western blot with the indicated antibodies.

[0055]

[0030] Figures 8A-8B showing that optimized binders result in enhancing the effects of SUTAC. Figure 8A the structure of BRD4-targeting SUTACs and their IC50 binding to PIAS1; Figure 8B western blot with the indicated antibodies of Daudi cells treated with increasing concentrations of the indicated SUTAC molecules for 20 hours.

[0056]

[0031] Figures 9A-9C showing that the BRD4-targeting SUTAC is efficient in attenuating the proliferation of hematological cancer cells. Figure 9A Daudi cells were treated with increasing doses of BRD4-targeting SUTACs or JQ1 and the relative proliferation was determined by Cell Titer Glow assay 72 hours of treatment; Figure 9B OCI-AML2 cells were treated with increasing doses of 1-26 BRD4-targeting SUTAC or JQ1 or the 1-12 PIAS binder and the relative proliferation was determined by Cell Titer Glow assay 72 hours of treatment; Figure 9C Daudi cells were treated with increasing doses of 1-26 BRD4-targeting SUTAC or JQ1 or the 1-12 PIAS binder and the relative proliferation was determined by Cell Titer Glow assay 72 hours of treatment.

[0057]

[0032] Figures 10A-10C showing that the AR-targeting SUTAC can lower AR levels. Figure 10A shows the structure of the AR-targeting SUTAC, Figure 10B LNCaP cells treated with increasing concentrations of the indicated SUTAC molecules for 20 hours, were subjected to western blot with the indicated antibodies; Figure 10C Upon 48 hours, transiently transfected 293T cells with the indicated plasmids were treated with increasing concentrations of the indicated SUTAC molecules for an additional 20 hours and were then subjected to western blot with the indicated antibodies.

[0058]

[0033] Figures 11A-11C enhanced SUMOylation of BRD4 by ID-1 (=1-26) SUTAC and chromatin eviction of BRD4. Figure 11A PIAS4 or vehicle were pre-incubated with 1-26 SUTAC or DMSO control for 4 hours at 25C. After which, they were incubated with SUMO 1,2 and SUMO-E1 (SAE1 / 2), with or without SUMO-E2 enzyme (UBC9), with or without recombinant BRD4 for 1 hour at 30C. The samples were then subjected to western blot with the indicated antibodies; Figure 11B OCI-AML2 were treated with increasing concentrations of ID- 1 for about 20 hours and then were harvested and analyzed using western blotting with the indicated antibodies. Figure 11C OCI-AML2 cells were treated with 1 μM of either ID-1 SUTAC, JQ 1 -ester and MZ 1 (PROTAC) for about 20 hours. After which, cells were fractionated into different cellular fractions and were subsequently analyzed using western blotting with antibodies against His, BRD4, UBC9, MYC-tag.

[0059]

[0034] Figure 12 Chloroacetamide fragment library screen for PIAS1 and PIAS4. Thiophenebased hits containing an amide (left), ester (middle) and nitrile (right) are shown.

[0060]

[0035] Figures 13A-13B showing Medicinal Chemistry campaign for optimization of PIAS4 binders. Figure 13A compounds 1-10, 1-11, 1-12, 1-16, 1-17, 1-18, 1-19, 1-40 having expansion around thiophene, compounds 1-36, 1-37, 1-38, 1-39 with optimization of amide substituents and a combined structure of compounds 1-40 and 1-39 (compound 1-41); Figure 13B Addition of linkers and recruiters for target proteins.

[0061]

[0036] Figures 14A-14B Identification of PIAS4 binding site. Figure 14A MS / MS spectra of the modified peptide from PIAS4(322-330). Y ions indicated in blue and B ions in magenta: modified by carb amidomethyl (top) and modified by compound 1-41 (bottom); Figure 14B left: overlay of AlphaFold structure of unbound PIAS4 (gray) and bound PIAS (protein: salmon, compound 1-41 : blue) and right: model of compound 1-41 bound to PIAS4.

[0062]

[0037] Figure 15 Cellular engagement of compound 1-43 with PIAS proteins. Proteomic characterization of targets where Daudi cells were pre-incubated with 1 μM of compound 1-43 (or DMSO) for 2 hours, followed by an incubation with 0.1 μM of compound 1-42 (or DMSO) for 1 hour. The cells were lysed, clicked to a biotin-functionalized, trypsin-cleavable azide- containing peptide, and the labeled proteins were enriched on streptavidin beads, trypsinized and analyzed using LC-MS / MS.

[0038] Figures 16A-16E BRD4-targeting SUTAC (ID-2) enhances the SUMOylation of recombinant BRD4 and its downstream target c-MYC. Figure 16A PIAS4 or vehicle were preincubated with ID-2 (SUTAC) or DMSO control for 1.5 hours at 25C. After which, they were incubated with SUMO1,2 and SUMO-E1 (SAE1 / 2), with or without SUMO-E2 enzyme (UBC9), with or without recombinant BRD4 for 1 hour at 30C. The samples were then subjected to western blot with anti-BRD4 antibody; Figure 16B Cancer cell lines Daudi, OCI-AML2, RAMOS and RPMI-8226 were treated with increasing concentrations of the ID-2 for 20 hours. Cells were lysed and were subjected to western blot with antibodies against BRD4, cMYC and Actin; Figure 16C Daudi cells were treated with increasing concentrations ID-2 or JQ1 -ester for 24 hours. Cells were then lysed and subjected to a western blot analysis with antibodies, against BRD4, cMYC and Actin; Figure 16D Daudi cells were pre-treated with 10 μM MG132 or DMSO and then were treated with ID-2 for 4 hours. Cells were then lysed and subjected to a western blot analysis with antibodies against BRD4, Ubiquitin, and Actin; Figure 16E PIAS1 and PIAS4 double knock outs (DKO) TK6 cells were treated with: (left) 1 μM ID-2 for 4 hours or (right) 0.1 μM ID-2 overnight. Cells were then lysed and subjected to a western blot analysis with antibodies against BRD4, PIAS1, PIAS2, PIAS3, PIAS4 and Actin.

[0063]

[0039] Figures 17A-17B SUMOylation of recombinant BRD4 is enhanced by compound 7 (SUTAC) in an in vitro SUMOylation reaction. Figure 17A Structure of ID-2 and ID-3; Figure 17B PIAS4 or vehicle were pre-incubated with ID-2 (SUTAC) or ID-3 (SUTAC) or DMSO control for 1.5 hours at 25C. After which, they were incubated with SUMO1,2 and SUMO-E1 (SAE1 / 2), with or without SUMO-E2 enzyme (UBC9), with or without recombinant BRD4 for 1 hour at 30C. The samples were then subjected to western blot with anti-BRD4 antibody (abeam).

[0064]

[0040] Figures 18A-18E P300-targeting SUTACs enhance the SUMOylation of P-300 and its downstream target c-MYC. Figure 18A Structure of P300-targeting SUTAC (ID-4); Figure 18B Daudi (right) or OCI-AML2 (left) cancer lines were treated with increasing concentrations of ID-4 for 20 hours. Cells were lysed and were subjected to western blot with antibodies against P300, H2B, cMYC and Actin; Figure 18C OCI-AML2 cells were treated with increasing doses of ID-4 or 1-41 and the relative proliferation was determined by Cell Titer Glow assay 72 hours of treatment; Figure 18D structure of ID-4, ID-5 and P300 inhibitor (compound 10) Figure 18E OCI-AML2 lines were treated with increasing ID-4, ID-5, P300 inhibitor (compound 10) or DMSO control, for 20 hours. Cells were lysed and were subjected to western blot analysis with antibodies against cMYC. DETAILED DESCRIPTION

[0065]

[0041] The present subj ect matter may be understood more readily by reference to the following detailed description which forms a part of this disclosure. It is to be understood that this disclosure is not limited to the specific products, methods, conditions or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure.

[0066]

[0042] The present disclosure shows the development of SUTACs as new drug modalities in the form of modular heterobifunctional molecules containing two functional groups: The first part (part 1) is a novel chemical moiety that selectively binds a specific SUMO E3 ligase (a SUMOylation enzyme binder group). The second part (part 2) is a chemical moiety (a target protein binder group) that specifically binds a Protein-of Interest (POI) (a target protein). The two parts are conjugated through a short chemical linker (part 3) to produce a chimeric molecule. The SUTAC molecules induce the selective SUMOylation of any target POI, on demand. The SUTAC approach can harness other SUMO E3 ligases or the SUMO E2 conjugating enzyme to induce SUMOylation of any POI.

[0067]

[0043] To establish the SUTAC modality, a dedicated electrophile screen was performed against a recombinant domain derived from a well-defined SUMO E3 ligase, namely PIAS1. Three binders able to label PIAS1 at a single digit μM concentration were identified. The binding potency of these molecules was optimized and the binding in cells to PIAS1 and two other PIAS family members (i.e., PIAS4 and PIAS2) was validated. Three of the identified binders were used to generate the first generation of SUTACs.

[0068]

[0044] Using a short PEG linker, the binders were conjugated to JQ1, a potent binder against the transcriptional activator BRD4. BRD4 is a transcriptional activator that is known to promote the expression of critical oncogenes in several cancers and other pathologies. The BRD4-targeting SUTACs were cell-permeable and induced the SUMOylation of a recombinant BRD4 in cellular extracts at -200 nM and in in vitro SUMOylation assay with purfied SUMOylation enzymes. Further, these SUTAC molecules lowered BRD4 activity and / or levels, in SUMOylation- and PIAS- dependent manners. These SUTAC molecules could result in the evection of BRD4 from the chromatin fraction. Moreover, SUTAC treatment significantly reduced the levels of the oncoprotein cMYC, which is characteristically transactivated by BRD4. Notably, these SUTACs were efficient in attenuating the proliferation of hematological cancer cell lines. Moreover, Androgen Receptor (AR)-targeting SUTACs were generated and demonstrated a utility in lowering AR protein levels in a prostate cancer line and under AR-overexpression settings.

[0045] Additionally, broader electrophile screens were conducted against recombinant PIAS1 and PIAS4, identifying an amide-containing thiophene scaffold as a dual binder. Through optimization, its binding to PIAS4 was enhanced by more than 100-fold and its co-structure with PIAS4 was resolved. The binding of these compounds to PIAS was validated in cells and it was demonstrated that BRD4-targeting SUTACs utilizing these binders can induce BRD4 SUMOylation. Furthermore, these BRD4-targeting SUTACs were shown to impact BRD4 activity and / or levels and significantly attenuate cancer cell growth. Notably, when these binders were conjugated to a P300 / CBP binder, the resulting SUTACs effectively downregulated P300 activity and / or levels and significantly attenuated cancer cell growth.

[0069]

[0046] In some embodiments, provided herein a SUMOylation-targeting chimera (SUTAC) comprising a SUMOylation enzyme binder group attached to a target protein binder group via a linker (e.g. Li, L2). In some embodiments the linker comprises one or more polyethylene glycol (PEG) groups. In some embodiments, the linker comprises LI as described herein in detail. In some embodiments, the linker comprises L2 as described herein in detail.

[0070]

[0047] In some embodiments, provided herein a SUMOylation-targeting chimera (SUTAC) comprising a SUMOylation enzyme binder group attached to a target protein binder group via a linker. In some embodiments of the SUTAC described herein, the SUMOylation enzyme binder group is capable of binding a SUMO E3 ligase. In some embodiments of the SUTAC described herein, the SUMOylation enzyme binder group is capable of binding a SUMO E2 conjugating enzyme.

[0071]

[0048] In some embodiments, the term ’’SUMOylation” may encompass the binding of SUMO protein to a target protein, which may affect the activity of the target protein. In some embodiments, the “SUMOylation enzyme binder group” may encompass a compound or molecule which binds or at least interacts with a SUMOylation enzyme. In some embodiments, the SUMOylation enzyme comprises SUMO enzyme El. In some embodiments the SUMOylation enzyme comprises SUMO conjugating enzyme E2. In some embodiments, the SUMOylation enzyme comprises a SUMO E3 ligase. In someembodiments, the SUMO E3 ligase is selected from the group consisting of protein inhibitor of activated STAT1 (PIAS1), PIAS2, PIAS3, PIAS4, NSMCE2, TOPORS, Triml9, Trim28, PML, ZNF451and Ran-binding protein 2 (RanBP2).

[0072]

[0049] In some embodiments, the SUMO E3 ligase is selected from the group consisting of PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, TOPORS, Trim 19, Trim28, PML, ZNF451and RANBP2. In some embodiments, the SUMO E3 ligase comprises PIAS1. In some embodiments, the SUMO E3 ligase comprises PIAS2. In some embodiments, the SUMO E3 ligase comprises PIAS3. In some embodiments, the SUMO E3 ligase comprises PIAS4. In some embodiments, the SUMO E3 ligase comprises NSMCE2. In some embodiments, the SUMO E3 ligase comprises TOPORS. In some embodiments, the SUMO E3 ligase comprises Triml9. In some embodiments, the SUMO E3 ligase comprises Trim28. In some embodiments, the SUMO E3 ligase comprises PML. In some embodiments, the SUMO E3 ligase comprises ZNF451. In some embodiments, the SUMO E3 ligase comprises RANBP2.

[0073]

[0050] In some embodiments, the “target protein binder group” may encompass a polypeptide or protein which binds or at least interacts with a target protein. In some embodiments, the target protein binder group comprises an inhibitor of the target protein. In some embodiments, the target protein binder group comprises JQ1 or an acid or ester thereof. In some embodiments, the target protein binder group comprises GNE-207, or a derivative thereof. In some embodiments, the target protein binder group comprises A-485, or a derivative thereof. In some embodiments, the target protein binder group comprises a kinase inhibitor. In some embodiments, the target protein binder group comprises gefitinib. In some embodiments, the target protein binder group comprises W, as described herein in detail.

[0074]

[0051] In some embodiments, the “target protein” may encompass a polypeptide or protein of interest (POI). In some embodiments, the protein of interest comprises any protein that can undergo SUMOylation, i.e. be covalentely modified by a SUMO protein. In some embodiments, the protein of interest comprises any protein that requires increased degradtion. In some embodiments, the protein of interest comprises any protein that requires decreased degradtion. In some embodiments, the protein of interest comprises any protein that requires increased stabilization. In some embodiments, the protein of interest comprises any protein that requires decreased stabilization. In some embodiments, the protein of interest comprises any protein that requires increased activation. In some embodiments, the protein of interest comprises any protein that requires decreased activation. In some embodiments, the protein of interest comprises any protein that requires increased repression. In some embodiments, the protein of interest comprises any protein that requires decreased repression. In some embodiments, the protein of interest comprises any protein that requires increased solubilization. In some embodiments, the protein of interest comprises any protein that requires decreased solubilization.

[0075]

[0052] In some embodiments, the protein of interest is selected from proteases, cellulases, amylases, carbohydrases, lipases, isomerases, transferases, kinases and phosphatases. In some embodiments, the protein of interest is selected from the group consisting of antibodies, hormones and growth factors.

[0076]

[0053] In some embodiments, the protein of interest is selected from the group consisting of DNA-binding proteins, transcription factors, RNA binding proteins, scaffolding proteins, GTPases, solute carriers, kinases, phosphatases, chromatin remodellers, transcrtiptional activators, transcriptional repressors, bromodomain- and chromodomain containing proteins, and G-protein coupled receptors.

[0077]

[0054] In some embodiments, the protein of interest is selected from the group consisting of an integrase, recombinase, transcription factor, protease, kinase, and growth factor. In some embodiments, the protein of interest comprises an integrase. In some embodiments, the protein of interest comprises a recombinase. In some embodiments, the protein of interest comprises a protease. In some embodiments, the protein of interest comprises a kinase. In some embodiments, the protein of interest comprises a growth factor.

[0078]

[0055] In some embodiments, the target protein comprises a Bromodomain-containing protein 4 (BRD4). In some embodiments, the target protein comprises an Androgen receptor (AR). In some embodiments, the target protein comprises an Estrogen receptor (ER). In some embodiments, the target protein comprises a P300 / CBP protein(s).

[0079]

[0056] In some embodiments, provided herein a SUMOylation-targeting chimera (SUTAC) represented by the structure of formula I or salt thereof: wherein,

[0080] E is H, amide, acetyl amide, or an electrophilic group;

[0081] Z is O, S and NH; each of R6, R7are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, - C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl; or R6, R7form together a 5-8 membered ring, wherein the ring is substituted or unsubstituted;

[0082] R11 is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0083] L2 is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and

[0084] W is a target protein binder group.

[0085]

[0057] In some embodiments the electrophilic group E of Formula I comprises any known electrophilic group. In some embodiments, the electrophilic group comprises a chloroacetamide, an acrylamide, a methacrylamide, a sulfamate, a propynamide, butanamide, or a vinyl sulfone. In some embodiments, the electrophilic group comprises a chloroacetamide. In some embodiments, the electrophilic group comprises an acrylamide. In some embodiments, the electrophilic group comprises a methacrylamide. In some embodiments, the electrophilic group comprises a sulfamate. In some embodiments, the electrophilic group comprises a propynamide. In some embodiments, the electrophilic group comprises butanamide. In some embodiments, the electrophilic group comprises a vinyl sulfone.

[0086]

[0058] In some embodiments the electrophilic group is represented by the following structures:

[0087]

[0059] In some embodiments, provided herein a SUMOylation-targeting chimera (SUTAC) represented by the structure of formula I’ or salt thereof:

[0088] wherein,

[0089] Z is O, S and NH; each of R6, R7are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; andR” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl; or R6, R7form together a 5-8 membered ring, wherein the ring is substituted or unsubstituted;

[0090] R11 is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0091] L2 is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, -(CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and W is a target protein binder group.

[0092]

[0060] In some embodiments, provided herein a SUMOylation-targeting chimera (SUTAC) represented by the structure of formula IA or salt thereof: wherein, each of R6, R7are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; andR” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl; or R6, R7form together a 5-8 membered ring, wherein the ring is substituted or unsubstituted;

[0093] R11 is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0094] L2is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, -(CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and

[0095] W is a target protein binder group.

[0096]

[0061] In some embodiments, provided herein a SUMOylati on-targeting chimera (SUTAC) represented by the structure of formula IB or salt thereof: wherein: the represents a saturated or non-saturated bond; wherein if is a saturated bond, then X is C, N, O or S; wherein if is a non-saturated bond, X is C or N; each of R8, R9, R10are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; andR” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl;

[0097] R11 is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; n is an integer 1, 2 or 3; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0098] L2 is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, -(CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and

[0099] W is a target protein binder group.

[0100]

[0062] In some embodiments, provided herein a SUMOylation-targeting chimera (SUTAC) represented by the structure of formula IC or salt thereof: wherein, each of R8, R9, R1a0re independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; andR” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl;

[0101] R11 is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; n is an integer 1, 2 or 3; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0102] L2 is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, -(CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and

[0103] W is a target protein binder group.

[0104]

[0063] In some embodiments, provided herein a SUMOylation-targeting chimera (SUTAC) represented by the structure of formula ID or salt thereof: wherein, each of R8, R9, R10are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; andR” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl; R11 is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; n is an integer 1, 2 or 3; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0105] L2 is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, -(CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and

[0106] W is a target protein binder group

[0107]

[0064] In some embodiments, provided herein a SUMOylation-targeting chimera (SUTAC) represented by the structure of formula II or salt thereof: wherein,

[0108] Ri is H, -S-CF3, S-CHF2, S-CH2F, -O-CF3, -O-CHF2, -O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, - NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl;

[0109] R2 is H, -S-CF3, S-CHF2, S-CH2F, -O-CF3, O-CHF2, O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl; n is an integer 1-3; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0110] L2 is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, -(CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and

[0111] W is a target protein binder group.

[0112]

[0065] In some embodiments, provided herein a SUMOylation-targeting chimera (SUTAC) represented by the structure of formula IIA or salt thereof: wherein,

[0113] Ri is H, -S-CF3, -S-CHF2, -S-CH2F, -O-CF3, -O-CHF2, -O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR’ or - NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl;

[0114] R2 is H, -S-CF3, -S-CHF2, -S-CH2F, -0-CF3, -0-CHF2, -0-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR’ and - NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl; n is an integer 1-3; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0115] L2 is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, -(CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and

[0116] W is a target protein binder group.

[0117]

[0066] In some embodiments, provided herein a SUMOylation-targeting chimera (SUTAC) represented by the structure of formula IIB or salt thereof:

[0118] wherein, each of Q1, Q2, Q3 are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;

[0119] L2 is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, -(CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and

[0120] W is a target protein binder group.

[0121]

[0067] In some embodiments, Z of formula I or I’ is O, S and NH. In another embodiment, Z of formula I is O. In another embodiment, Z of formula I is S. In another embodiment, Z of formula l is NH.

[0122]

[0068] In some embodiments Ri of formula II or IIA is H, -S-CF3, S-CHF2, S-CH2F, -O-CF3, - O-CHF2, -O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl. In another embodiment, Ri is H. In another embodiment, Ri is -S-CF3. In another embodiment, Ri is S-CHF2. In another embodiment, Ri is S-CH2F. In another embodiment, Ri is -O-CF3. In another embodiment, Ri is -O-CHF2. In another embodiment, Ri is -O-CH2F. In another embodiment, Ri is thioalkyl. In another embodiment, Ri is thioalkenyl. In another embodiment, Ri is thiohaloalkyl. In another embodiment, Ri is halo. In another embodiment, Ri is alkyl. In another embodiment, Ri is alkenyl. In another embodiment, Ri is alkynyl. In another embodiment, Ri is cycloalkyl. In another embodiment, Ri is aryl. In another embodiment, Ri is haloaryl. In another embodiment, Ri is heteroaryl. In another embodiment, Ri is heterocyclic. In another embodiment, Ri is hydroxy. In another embodiment, Ri is alkoxy. In another embodiment, Ri is aryloxy. In another embodiment, Ri is thioalkoxy. In another embodiment, Ri is cyano. In another embodiment, Ri is nitro. In another embodiment, Ri is azide. In another embodiment, Ri is amino. In another embodiment, Ri is -COOH. In another embodiment, Ri is -C(O)NHR’. In another embodiment, Ri is -NHCOR”-. In another embodiment, R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl. In another embodiment, R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl.

[0123]

[0069] In some embodiments R2 of formula II or IIA is H, -S-CF3, S-CHF2, S-CH2F, -O-CF3, O-CHF2, O-CH2F, thioalkyl, thioalkenyl, thiohaloalkyl, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, cyano, nitro, azide, amino, - COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl. In another embodiment, R2 is H. In another embodiment, R2 is -S-CF3. In another embodiment, R2 is S-CHF2. In another embodiment, R2 is S-CH2F. In another embodiment, R2 is -O-CF3. In another embodiment, R2 is O-CHF2. In another embodiment, R2 is O-CH2F. In another embodiment, R2 is thioalkyl. In another embodiment, R2 is thioalkenyl. In another embodiment, R2 is thiohaloalkyl. In another embodiment, R2 is halo. In another embodiment, R2 is alkyl. In another embodiment, R2 is alkenyl. In another embodiment, R2 is alkynyl. In another embodiment, R2 is cycloalkyl. In another embodiment, R2 is aryl. In another embodiment, R2 is heteroaryl. In another embodiment, R2 is heterocyclic. In another embodiment, R2 is hydroxy. In another embodiment, R2 is alkoxy. In another embodiment, R2 is aryloxy. In another embodiment, R2 is thioalkoxy. In another embodiment, R2 is cyano. In another embodiment, R2 is nitro. In another embodiment, R2 is azide. In another embodiment, R2 is amino. In another embodiment, R2 is -COOH. In another embodiment, R2 is -C(O)NHR’. In another embodiment, R2 is -NHCOR”-. In another embodiment, R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl. In another embodiment, R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl.

[0124]

[0070] In some embodiments each of R6, R7of formula I, I’ or IA are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl; or R6, R7form together a 5-8 membered ring, wherein the ring is substituted or unsubstituted. In another embodiment, each of R6, R7of formula I, I’ or IA are independently H. In another embodiment, each of R6, R7of formula I, I’ or IA are independently halo. In another embodiment, each of R6, R7of formula I, I’ or IA are independently alkyl. In another embodiment, each of R6, R7of formula I, I’ or IA are independently alkenyl. In another embodiment, each of R6, R7of formula I, I’ or IA are independently alkynyl. In another embodiment, each of R6, R7of formula I, I’ or IA are independently cycloalkyl. In another embodiment, R6, R7of formula I, I’ or IA are independently aryl. In another embodiment, each of R6, R7of formula I, I’ or IA are independently haloaryl. In another embodiment, each of R6, R7of formula I, I’ or IA are independently heteroaryl. In another embodiment, R6, R7of formula I, I’ or IA are independently heterocyclic. In another embodiment, R6, R7of formula I, I’ or IA are independently hydroxy. In another embodiment, each of R6, R7of formula I, I’ or IA are independently alkoxy. In another embodiment, each of R6, R7of formula I , I’ or IA are independently aryloxy. In another embodiment, each of R6, R7of formula I, I’ or IA are independently thioalkoxy. In another embodiment, each of R6, R7of formula I, I’ or IA are independently thioalkyl. In another embodiment, each of R6, R7of formula I, I’ or IA are independently thioalkenyl. In another embodiment, each of R6, R7of formula I, I’ or IA are independently thiohaloalkyl. In another embodiment, each of R6, R7of formula I, I’ or IA are independently cyano. In another embodiment, each of R6, R7of formula I, I’ or IA are independently nitro. In another embodiment, each of R6, R7of formula I, I’ or IA are independently azide. In another embodiment, each of R6, R7of formula I, I’ or IA are independently amino. In another embodiment, each of R6, R7of formula I, I’ or IA are independently -COOH. In another embodiment, each of R6, R7of formula I, I’ or IA are independently -C(O)NHR’ . In another embodiment, each of R6, R7of formula I, I’ or IA are independently -NHCOR”-. In other embodiments, R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl. In other embodiments R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl. In some embodimentRs8, R7of formula I , I’ or IA form together a 5-8 membered ring, wherein the ring is substituted or unsubstituted.

[0125]

[0071] In some embodiments, each of R8, R9, R10of formula IB, IC, or ID are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl. In another embodiment, each ofR8, R9, R10are independently H. In another embodiment, each of R8, R9, R10are independently halo. In another embodiment, each ofR8, R9, R10are independently alkyl. In another embodiment, each ofR8, R9, R10are independently alkenyl. In another embodiment, each of R8, R9, R10are independently alkynyl. In another embodiment, each ofR8, R9, R10are independently cycloalkyl. In another embodiment, each of R8, R9, R10are independently aryl. In another embodiment, each ofR8, R9, R10are independently haloaryl. In another embodiment, each ofR8, R9, R10are independently heteroaryl. In another embodiment, each of R8, R9, R10are independently heterocyclic. In another embodiment, each of R8, R9, R10are independently hydroxy. In another embodiment, each ofR8, R9, R10are independently alkoxy. In another embodiment, each of R8, R9, R10are independently aryloxy. In another embodiment, each ofR8, R9, R10are independently thioalkoxy. In another embodiment, each ofR8, R9, R10are independently thioalkyl. In another embodiment, each of Rs, R9, R10are independently thioalkenyl. In another embodiment, each ofR8, R9, R10are independently thiohaloalkyl. In another embodiment, each ofR8, R9, R10are independently cyano. In another embodiment, each ofR8, R9, R10are independently nitro. In another embodiment, each ofR8, R9, R10are independently azide. In another embodiment, each of R8, R9, R10are independently amino. In another embodiment, each of R8, R9, R10are independently -COOH. In another embodiment, each of R8, R9, R10are independently -C(O)NHR’ . In another embodiment, each of R8, R9, R10are independently -NHCOR”-. In another embodiment, R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl. In another embodiments R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl.

[0126]

[0072] In some embodiments, n of formula IB, IC, ID, II or IIA is an integer 1 , 2 or 3. In another embodiment, n is 1. In another embodiment, n is 2 in another embodiment, n is 3.

[0127]

[0073] In some embodiments each of Q1, Q2, Q3 of formula IIB are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic. In another embodiment, each of Qi, Q2, Q3 is independently H. In another embodiment, each of Q1, Q2, Q3 is independently halo. In another embodiment, each of Qi, Q2, Q3 is independently alkyl. In another embodiment, each of Q1, Q2, Q3 is independently alkenyl. In another embodiment, each of Qi, Q2, Q3 is independently alkynyl. In another embodiment, each of Q1, Q2, Q3 is independently cycloalkyl. In another embodiment, each of Qi, Q2, Q3 is independently aryl. In another embodiment, each of Q1, Q2, Q3 is independently heteroaryl. In another embodiment, each of Q1, Q2, Q3 is independently heterocyclic.

[0128]

[0074] In some embodiments, L1of formula I, I’, IA, IB, IC, ID, II, IIA or IIB is a bond, - (CH2CH2O)q, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10. In another embodiment, L1is a bond. In another embodiment, L1is -(CH2CH2O)q wherein q is an integer 2-10. In another embodiment, L1is substituted or unsubstituted linear or branched alkylene. In another embodiment, L1is substituted or unsubstituted linear or branched alkenylene. In another embodiment, L1is substituted or unsubstituted linear or branched alkynylene. In another embodiment, L1is substituted or unsubstituted cycloalkyl. In another embodiment, L1is substituted or unsubstituted heterocyclic. In another embodiment, Li is substituted or unsubstituted aryl. In another embodiment, L1is substituted or unsubstituted heteroaryl. In another embodiment, L1is an ether group. In another embodiment, L1is an ester. In another embodiment, L1is an amine group. In another embodiment, L1is an amide group. In another embodiment, L1is an amide group. In another embodiment, L1is -(CH2CH2O)q. In another embodiment, L1is -(CH2CH2O)q(CH2CH2)NH(CO)-. In another embodiment, Li is - (CH2CH2O)q(CH2CH2)-. In another embodiment, L1is -(CH2CH2O)q(CH2CH2)NH-. In another embodiment, L1is -NHCH2(CO)NHCH2Ph-. In another embodiment, L1is - N(alkyl)CH2(CO)NHCH2Ph-.

[0129]

[0075] In some embodiments, L2 of formula I, I’ , IA, IB, IC, ID, II, IIA or IIB is a bond, - (CH2CH2O)q, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10. In another embodiment, L2 is a bond. In another embodiment, L2 is -(CH2CH2O)q wherein q is an integer 2-10. In another embodiment, L2 is substituted or unsubstituted linear or branched alkylene. In another embodiment, L2 is substituted or unsubstituted linear or branched alkenylene. In another embodiment, L2 is substituted or unsubstituted linear or branched alkynylene. In another embodiment, L2 is substituted or unsubstituted cycloalkyl. In another embodiment, L2 is substituted or unsubstituted heterocyclic. In another embodiment, L2 is substituted or unsubstituted aryl. In another embodiment, L2 is substituted or unsubstituted heteroaryl. In another embodiment, L2 is an ether group. In another embodiment, L2 is an ester. In another embodiment, L2 is an amine group. In another embodiment, L2 is an amide group. In another embodiment, L2 is -(CH2CH2O)q. In another embodiment, L2 is - (CH2CH2O)q(CH2CH2)NH(CO)-. In another embodiment, L2 is -(CH2CH2O)q(CH2CH2)-. In another embodiment, L2 is -(CH2CH2O)q(CH2CH2)NH-. In another embodiment, L2 is - NHCH2(CO)NHCH2Ph-. In another embodiment, L2is -N(alkyl)CH2(CO)NHCH2Ph-.

[0130]

[0076] In some embodiments, R11 of formula I, I’, IA, IB, IC, ID, is a H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl. In another embodiment R11 of formula I is H. In another embodiment R11 of formula I, I’, IA, IB, IC, ID is substituted or unsubstituted alkyl. In another embodiment R11 of formula I, I’, IA, IB, IC, ID is substituted or unsubstituted alkenyl. In another embodiment R11 of formula I, I’, IA, IB, IC, ID is substituted or unsubstituted aryl. In another embodiment R11 of formula I, I’, IA, IB, IC, ID is substituted or unsubstituted heterocyclic. In another embodiment R11 of formula I, I’, IA, IB, IC, ID is substituted or unsubstituted heteroaryl.

[0131]

[0077] In some embodiments, W of formula I, I’ IA, IB, IC, ID, II, IIA or IIB is a target protein binder group. The target protein binder group is defined based on the target protein. For example, for BRD4 the target protein binder group is JQ1 (structure [a] below) and for kinases the target protein binder group is a kinase inhibitor such as gefitinib (structure [b] below).

[0132]

[0078] In some embodiments, the target protein binder group comprises JQ1. In some embodiments, the target protein binder group comprises a kinase inhibitor. In some embodiments, the target protein binder group comprises gefitinib. In some embodiments, the target protein binder group comprises GNE-207, or a derivative thereof. In some embodiments, the target protein binder group comprises A-485, or a derivative thereof.

[0079] The invention includes SUTAC salts, which may be produced, by reaction of a SUTAC of this invention with an acid or base. Certain SUTACs, particularly those possessing acid or basic groups, can also be in the form of a salt, optionally a pharmaceutically acceptable salt. The term “pharmaceutically acceptable salt” refers to those salts that retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxylic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, -toluenesulfonic acid, salicylic acid, / ' / -acetylcysteine and the like. Other salts are known to those of skill in the art and can readily be adapted for use in accordance with the present invention.

[0133]

[0080] Suitable acceptable salts of amines of the SUTACs of this invention may be prepared from an inorganic acid or from an organic acid. In various embodiments, examples of inorganic salts of amines are bi sulfates, borates, bromides, chlorides, hemisulfates, hydrobromates, hydrochlorates, 2- hydroxyethylsulfonates (hydroxyethanesulfonates), iodates, iodides, isothionates, nitrates, persulfates, phosphate, sulfates, sulfamates, sulfanilates, sulfonic acids (alkylsulfonates, arylsulfonates, halogen substituted alkyl sulfonates, halogen substituted aryl sulfonates), sulfonates and thiocyanates.

[0134]

[0081] In various embodiments, examples of organic salts of amines may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which are acetates, arginines, aspartates, ascorbates, adipates, anthranilates, algenates, alkane carboxylates, substituted alkane carboxylates, alginates, benzenesulfonates, benzoates, bi sulfates, butyrates, bicarbonates, bitartrates, citrates, camphorates, camphorsulfonates, cyclohexylsulfamates, cyclopentanepropionates, calcium edetates, camsylates, carbonates, clavulanates, cinnamates, dicarboxylates, digluconates, dodecyl sulfonates, dihydrochlorides, decanoates, enanthuates, ethanesulfonates, edetates, edisylates, estolates, esylates, fumarates, formates, fluorides, galacturonates gluconates, glutamates, glycolates, glucorate, glucoheptanoates, glycerophosphates, gluceptates, glycollylarsanilates, glutarates, glutamate, heptanoates, hexanoates, hydroxymaleates, hydroxycarboxlic acids, hexylresorcinates, hydroxybenzoates, hydroxynaphthoates, hydrofluorates, lactates, lactobionates, laurates, malates, maleates, methylenebis(beta-oxynaphthoate), malonates, mandelates, mesylates, methane sulfonates, methylbromides, methylnitrates, methylsulfonates, monopotassium maleates, mucates, monocarboxylates, naphthalenesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, napsylates, A -methyl glucamines, oxalates, octanoates, oleates, pamoates, phenylacetates, picrates, phenylbenzoates, pivalates, propionates, phthalates, phenyl acetate, pectinates, phenylpropionates, palmitates, pantothenates, polygalacturates, pyruvates, quinates, salicylates, succinates, stearates, sulfanilate, subacetates, tartrates, theophyllineacetates, -toluenesulfonates (tosylates), trifluoroacetates, terephthalates, tannates, teoclates, trihaloacetates, triethiodide, tricarboxylates, undecanoates and valerates.

[0135]

[0082] In various embodiments, examples of inorganic salts of carboxylic acids or hydroxyls may be selected from ammonium, alkali metals to include lithium, sodium, potassium, cesium; alkaline earth metals to include calcium, magnesium, aluminium; zinc, barium, cholines, quaternary ammoniums.

[0136]

[0083] In some embodiments, examples of organic salts of carboxylic acids or hydroxyl may be selected from arginine, organic amines to include aliphatic organic amines, alicyclic organic amines, aromatic organic amines, benzathines, t-butylamines, benethamines N- benzylphenethylamine), dicyclohexylamines, dimethylamines, diethanolamines, ethanolamines, ethylenediamines, hydrabamines, imidazoles, lysines, methylamines, meglamines, N-methyl-D-glucamines, dibenzylethylenediamines, nicotinamides, organic amines, ornithines, pyridines, picolies, piperazines, procain, tris(hydroxymethyl)methylamines, triethylamines, triethanolamines, trimethylamines, tromethamines and ureas.

[0137]

[0084] In various embodiments, the salts may be formed by conventional means, such as by reacting the free base or free acid form of the product with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble or in a solvent such as water, which is removed in vacuo or by freeze drying or by exchanging the ions of a existing salt for another ion or suitable ion-exchange resin.

[0138]

[0085] The alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and the ring formed with Rs and R7, referred herein may have one or more substituents, whereby each substituent group can independently be, for example, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O- carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, amino halogen, alkylaryloxy, heteroaryloxy, oxo, cycloalkyl, phenyl, heteroaryls, heterocyclyl, naphthyl, amino, alkylamino, arylamino, heteroarylamino, dialkylamino, diarylamino, alkylarylamino, alkylheteroarylamino, arylheteroarylamino, acyl, acyloxy, nitro, carboxy, carbamoyl, carboxamide, cyano, sulfonyl, sulfonyl amino, sulfoneamido, sulfinyl, sulfinylamino, thiol, alkylthio, arylthio, or alkylsulfonyl groups. Any substituents can be unsubstituted or further substituted with any one of these aforementioned substituents.

[0086] Herein, the term “alkyl” describes an unsaturated aliphatic hydrocarbon, including straight chain and branched chain groups. Optionally, the alkyl group has 2 to 20 carbon atoms. More optionally, the alkyl is a medium size alkyl having 2 to 10 carbon atoms. Most optionally, the alkyl is a lower alkyl having 2 to 4 carbon atoms or 1 to 6 carbons. The alkyl group may be substituted or non-substituted.

[0139]

[0087] In some embodiments, unless otherwise specified, a substituted alkyl or alkylene may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, and oxo. In some embodiments, a substituted alkyl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo and cyano.

[0140]

[0088] Herein, the term “alkenyl” describes an unsaturated aliphatic hydrocarbon comprise at least one carbon-carbon double bond, including straight chain and branched chain groups. Optionally, the alkenyl group has 2 to 20 carbon atoms. More optionally, the alkenyl is a medium size alkenyl having 2 to 10 carbon atoms or 2 to 6 carbons. Most optionally, unless otherwise indicated, the alkenyl is a lower alkenyl having 2 to 4 carbon atoms. The alkenyl group may be substituted or nonsubstituted.

[0141]

[0089] Substituted alkenyl or alkenylene may have one or more substituents, whereby each substituent group can independently be, for example, alkynyl, cycloalkyl, alkynyl, aryl, heteroaryl, heteroali cyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfoneamido, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0142]

[0090] In some embodiments, unless otherwise specified, a substituted alkenyl or alkenylene may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, and oxo. In some embodiments, a substituted alkenyl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo and cyano.

[0143]

[0091] Herein, the term “alkynyl” describes an unsaturated aliphatic hydrocarbon comprise at least one carbon-carbon triple bond, including straight chain and branched chain groups. Optionally, the alkynyl group has 2 to 20 carbon atoms. More optionally, the alkynyl is a medium size alkynyl having 2 to 10 carbon atoms. Most optionally, unless otherwise indicated, the alkynyl is a lower alkynyl having 2 to 4 carbon atoms. The alkynyl group may be substituted or non-substituted.

[0144]

[0092] Substituted alkynyl or alkynylene may have one or more substituents, whereby each substituent group can independently be, for example, cycloalkyl, alkenyl, aryl, heteroaryl, heteroali cyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0145]

[0093] In some embodiments, unless otherwise specified, a substituted alkynyl or alkynylene may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, and oxo. In some embodiments, a substituted alkynyl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo and cyano.

[0146]

[0094] As used herein, “alkylene” refers to a linear, branched or cyclic, in certain embodiments linear or branched, divalent aliphatic hydrocarbon group, in one embodiment having from 1 to about 20 carbon atoms, in another embodiment having from 1 to 12 carbons. In a further embodiment alkylene includes lower alkylene. There may be optionally inserted along the alkylene group one or more oxygen, sulfur, including S(=O) and S(=O)2 groups, or substituted or unsubstituted nitrogen atoms including -NR- and -N+RR- groups, where the nitrogen substituent(s) is (are) alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl or COR, wherein each R is independently selected from alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, -OY or -NYY, wherein each Y is independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl. Alkylene groups include, but are not limited to, methylene (-CH2), ethylene (-CH2CH2-), propylene (-(CH2)3), methylenedioxy (-O-CH2- O-) and ethylenedioxy (-O-(CH2)2-O-). The term “lower alkylene” refers to alkylene groups having 1 to 6 carbons. In certain embodiments, alkylene groups are lower alkylene, including alkylene of 1 to 3 carbon atoms.

[0147]

[0095] As used herein, “alkenylene” refers to a linear, branched or cyclic, in one embodiment straight or branched, divalent aliphatic hydrocarbon group, in certain embodiments having from 2 to about 20 carbon atoms and at least one double bond, in other embodiments 1 to 12 carbons. In further embodiments, alkenyl ene groups include lower alkenylene. There may be optionally inserted along the alkenylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl. Alkenylene groups include, but are not limited to, - CH=CH-CH=CH- and -H=CH-CH2. The term “lower alkenylene” refers to alkenylene groups having 2 to 6 carbons. In certain embodiments, alkenylene groups are lower alkenylene, including alkenylene of 3 to 4 carbon atoms.

[0148]

[0096] In some embodiments, unless otherwise specified, a substituted alkenylene may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, and oxo. In some embodiments, a substituted alkenylene may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo and cyano.

[0149]

[0097] As used herein, “alkynylene” refers to a straight, branched or cyclic, in certain embodiments straight or branched, a divalent aliphatic hydrocarbon group, in one embodiment having from 2 to about 20 carbon atoms and at least one triple bond, in another embodiment 1 to 12 carbons. In a further embodiment, alkynylene includes lower alkynylene. There may be optionally inserted along the alkynylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, where the nitrogen substituent is alkyl. Alkynylene groups include, but are not limited to, -C=C- C=C, -C=C- and -C=C-CH2-. The term “lower alkynylene” refers to alkynylene groups having 2 to 6 carbons. In certain embodiments, alkynylene groups are lower alkynylene, including alkynylene of 3 to 4 carbon atoms.

[0150]

[0098] In some embodiments, unless otherwise specified, a substituted alkynylene may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, and oxo. In some embodiments, a substituted alkynylene may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo and cyano.

[0151]

[0099] A “cycloalkyl” group refers to a saturated on unsaturated all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system. Examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. A cycloalkyl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. When a cycloalkyl group is unsaturated, it may comprise at least one carboncarbon double bond and / or at least one carbon-carbon triple bond. The cycloalkyl group can be an end group, as this phrase is defined herein, wherein it is attached to a single adjacent atom, or a linking group, as this phrase is defined herein, connecting two or more moieties.

[0152]

[0100] In some embodiments, unless otherwise specified, a substituted cycloalkyl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, and oxo. In some embodiments, a substituted cycloalkyl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo and cyano.

[0153]

[0101] An “aryl” group refers to an all-carbon monocyclic or fiised-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) end groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroali cyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O- carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. The aryl group can be an end group, as this phrase is defined herein, wherein it is attached to a single adjacent atom, or a linking group, as this phrase is defined herein, connecting two or more moieties.

[0154]

[0102] In some embodiments, unless otherwise specified, a substituted aryl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, and oxo. In some embodiments, a substituted aryl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo and cyano.

[0155]

[0103] A “heteroaryl” group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) end group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroali cyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0156]

[0104] In some embodiments, unless otherwise specified, a substituted heteroaryl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, and oxo. In some embodiments, a substituted heteroaryl may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo and cyano.

[0157]

[0105] A “heterocyclic” group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroali cyclic may be substituted or non-substituted. When substituted, the substituted group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O- carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholine and the like. The heteroalicyclic group can be an end group, as this phrase is defined herein, wherein it is attached to a single adjacent atom, or a linking group, as this phrase is defined herein, connecting two or more moieties.

[0158]

[0106] In some embodiments, unless otherwise specified, a substituted heterocyclic may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo, hydroxy, alkoxy, cyano, and oxo. In some embodiments, a substituted heterocyclic may be substituted with one or more (e.g., one, two, three, or more, as valency allows) groups independently selected from halo and cyano.

[0159]

[0107] Herein, the terms “amine” and “amino” each refer to either a -NRxRyend group, a - NT'R'R' end group, a -NRX- linking group, or a -N+RxRy- linking group, wherein Rx, Ryand Rzare each hydrogen or a substituted or non-substituted alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic (linked to amine nitrogen via a ring carbon thereof), aryl, or heteroaryl (linked to amine nitrogen via a ring carbon thereof), as defined herein. Optionally, Rx, Ryand Rzare hydrogen or alkyl comprising 1 to 4 carbon atoms. Optionally, Rxand Ry(and Rz, if present) are hydrogen. When substituted, the carbon atom of an Rx, Ryor Rzhydrocarbon moiety which is bound to the nitrogen atom of the amine is optionally not substituted by oxo, such that Rx, Ryand Rzare not (for example) carbonyl, C-carboxy or amide, as these groups are defined herein, unless indicated otherwise.

[0160]

[0108] An “alkoxy” or “alkoxyl” group refers to both an -O-alkyl and an -O-cycloalkyl end group, as defined herein, or to an -O-alkylene- or -O-cycloalkyl- linking group, as defined herein.

[0161]

[0109] An “aryloxy” group refers to both an -O-aryl and an -O-heteroaryl end group, as defined herein, or to an -O-aryl ene- linking group, as defined herein.

[0162]

[0110] An “azide” group refers to a -N=N+=N" group.

[0111] A “hydroxy” group refers to a -OH group.

[0163]

[0112] A “nitro” group refers to an -NO2 group.

[0164]

[0113] A “cyano” group refers to a -C=N group.

[0165]

[0114] In some embodiments, the Chimeras are represented by the structures of Formula IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7 (1-24), IC-l(l-27), ID-l(l-26), ID-2, ID-3, ID-4, ID-5:

[0166]

[0167]

[0115] In some embodiments, the SUMOylation enzyme binder group is selected from the groups presented in Table 1. In some embodiments the SUMOylation enzyme binder group binds a SUMO E3 ligase. In some embodiments the SUMOylation enzyme binder group binds PIAS1. In some embodiments the SUMOylation enzyme binder group binds PIAS4. In some embodiments the SUMOylation enzyme binder groups of Table 1 bind PIAS1. In some embodiments the SUMOylation enzyme binder groups of Table 1 bind PIAS4.

[0168] Table 1: SUMOylation compounds which are used as SUMOylation enzyme binder group in the Chimeras of this invention.

[0169]

[0116] In some embodiments, the SUMOylation enzyme binder group in the Chimeras of this invention comprise a N-C(O)-CH2C1 or a N-C(O)-CH=CH2 (acrylamide), wherein, for example a Chimera of formula I (as disclosed above); or its acrylamide derivative presented as follows:

[0170]

[0117] Similarly, the SUMOylation enzyme binder groups presented in Table 1, are part of the SUTAC chimeras of this invention.

[0171]

[0118] In some embodiments, provided herein is a pharmaceutical composition comprising a SUMOylati on-targeting chimera (SUTAC) comprising a SUMOylation enzyme binder group attached to a target protein binder group via a linker and a suitable acceptable carrier.

[0172]

[0119] In some embodiments, provided herein is a pharmaceutical composition comprising a the SUTAC described herein in detail and a suitable acceptable carrier.

[0173] Compound / composition for use

[0174]

[0120] In some embodiments, provided herein is a SUMOylation-targeting chimera (SUTAC) comprising a SUMOylation enzyme binder group attached to a target protein binder group via a linker, for use in treating, improving the condition or inhibiting the decline of a subject afflicted with cancer. In some embodiments, provided herein is a SUMOylation-targeting chimera (SUTAC) as described herein in detail, for use in treating, improving the condition or inhibiting the decline of a subject afflicted with cancer.

[0175]

[0121] In some embodiments, provided herein are methods of treating, improving the condition or inhibiting the decline of a subject afflicted with cancer comprising administering to the subject a SUMOylation-targeting chimera (SUTAC) comprising a SUMOylation enzyme binder group attached to a target protein binder group via a linker. In some embodiments, provided herein are methods of treating, improving the condition or inhibiting the decline of a subject afflicted with cancer comprising administering to the subject a SUMOylation-targeting chimera (SUTAC) as described herein in detail.

[0122] In some embodiments, the target protein comprises a Bromodomain-containing protein 4 (BRD4). In some embodiments, the target protein comprises an Androgen receptor (AR). In some embodiments, the target protein comprises a P300 / CBP protein.

[0176]

[0123] In some embodiments, the target protein comprises an Estrogen receptor (ER).

[0177]

[0124] In some embodiments, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. Thus, in some embodiments, “treating”, has the same meaning as “ameliorating”, and “alleviating” and refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, or a combination thereof.

[0178]

[0125] As used herein, the terms “administering”, “administer”, or “administration” refer to the delivery of the SUTACs or compositions described herein to a subject. In some embodiments, the compositions described herein can be administered either parenterally, enterally, or topically. Illustrative examples of parenteral administration include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. Illustrative examples of enteral administration include, but are not limited to, sublingual, and oral administration.

[0179]

[0126] In some embodiments, the SUTACs or compositions described herein are administered in a therapeutically effective amount. The terms “effective”, “efficacy”, or “effectiveness” are used herein to refer to the ability of a therapy to obtain beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering a SUTAC or a composition that treats cancer, an effective amount of a SUTAC or composition is, for example, an amount sufficient to achieve treatment, as defined herein, as compared to the response obtained without administration of the SUTAC or composition. In some embodiments, a therapeutically effective amount is an amount of a SUTAC or composition to be delivered that is sufficient, when administered to a subject suffering from cancer, to treat, improve the condition or inhibit the decline of a subject afflicted with cancer.

[0180]

[0127] In some embodiments, the cancer or tumor comprises a non-solid tumor. In some embodiments, the non-solid cancer or tumor comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, a multiple myeloma (a plasma cell myeloma), an acute promyelocytic leukemia, an acute lymphoblastic leukemia, an acute myelogenous leukemia, a chronic myelogenous leukemia, a Hodgkin lymphoma, a non-Hodgkin lymphoma, Burkitt’s lymphoma (BL), or plasma cell leukemia.

[0181]

[0128] In some embodiments, the cancer or tumor comprises a solid tumor. In some embodiments, the cancer is selected from the group consisting of: kidney cancer, lung cancer, endometrial / uterine cancer, esophageal cancer, breast cancer, cervical cancer, liver cancer, gastric cancer, esophageal cancer, head and neck cancer, ovarian cancer, skin cancer, bile duct cancer, rhabdoid, brain cancer, colon / colorectal cancer, pancreatic cancer, myeloma, Neuroblastoma, gastric cancer, sarcoma, thyroid cancer, bladder cancer, bone cancer or eye cancer.

[0182]

[0129] In some embodiments, methods of use comprising administering the pharmaceutical composition comprising the SUTAC described herein reduces the number of cancer cells in a subject, reduces the size of a tumor in a subject, or reduces the amount of cancer in the body of a subject, or any combination thereof, compared with a subject not administered the pharmaceutical composition comprising the SUTAC described herein. In some embodiments, treating, improving the condition or inhibiting the decline of cancer comprises reduction of tumor size.

[0183]

[0130] In one embodiment, the term “decreasing the size of the tumor” as used herein is assessed using the “Response Evaluation Criteria in Solid Tumors” (RECIST). In one embodiment, RECIST measures reduction in tumor size by measuring the longest dimension of a target lesion. In one embodiment, the target lesion is selected on the basis of its size (lesion with the longest diameter) and its suitability for accurate repeated measurements (either by imaging techniques or clinically). In one embodiment, all other lesions (or sites of disease) are identified as non-target lesions and are also recorded at baseline. Measurements of these lesions are not required, but the presence or absence of each is noted throughout follow-up.

[0184]

[0131] In some embodiments, treating, improving the condition or inhibiting the decline of cancer comprises a reduction of metastasis. In some embodiments, treating, improving the condition or inhibiting the decline of cancer comprises an elimination of metastasis. In some embodiments, treating, improving the condition or inhibiting the decline of cancer comprises an increased survival rate compared with a subject not receiving the pharmaceutical composition comprising the SUTAC described herein.

[0185]

[0132] In some embodiments of the methods disclosed herein, following administration of the pharmaceutical composition comprising the SUTAC described herein, the subject remains disease free for a time period longer than a subject not administered the pharmaceutical composition comprising the SUTAC.

[0186]

[0133] A skilled artisan would appreciate that the term “disease free” as used herein, may refer to the subject remaining alive, without return of the cancer or tumor, for a defined period of time such as about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, or more from initiation of treatment or from initial diagnosis.

[0187]

[0134] In some embodiments, the subject remains disease free for at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, or 10 years. In some embodiments, the subject remains disease free for at least 1 year. In some embodiments, the subject remains disease free for at least 5 years. In some embodiments, the subject remains disease free for at least 10 years.

[0188]

[0135] In some embodiments, the methods disclosed herein reduce the tumor load, or reduce the incidence of the cancer or tumor in said subject, compared with a subject not administered the pharmaceutical composition comprising the SUTAC. In some embodiments, the methods disclosed herein reduce the minimal residual disease, increase remission, increase remission duration, reduce tumor relapse rate, prevent metastasis of said tumor or said cancer, or reduce the rate of metastasis of said tumor or said cancer, or any combination thereof.

[0189]

[0136] In some embodiments, provided herein is a SUMOylati on-targeting chimera (SUTAC) comprising a SUMOylation enzyme binder group attached to a target protein binder group via a linker, for use in treating, improving the condition, inhibiting the decline of a subject afflicted with obesity, neurodegenerative diseases (e.g. Parkinson’s Disease, Alzheimer’s Disease), fibrosis, cardiovascular diseases, diabetes, Crohn’s disease, osteoporosis, Multiple Sclerosis (MS), SLE, or non-alcoholic fatty liver disease.

[0190]

[0137] In some embodiments, the neurodegenerative disease comprises Parkinson’s Disease. In some embodiments, the neurodegenerative disease comprises Alzheimer’s Disease. In some embodiments, diabetes is type II diabetes.

[0191]

[0138] In some embodiments, administering the SUTAC to a subject decreases the degradation of the target protein compared to a non -treated subject. In some embodiments, administering the SUTAC to a subject increases the degradation of the target protein compared to a non -treated subject.

[0192]

[0139] In some embodiments, administering the SUTAC to a subject decreases the stabilization of the target protein compared to a non-treated subject. In some embodiments, administering the SUTAC to a subject increases the stabilization of the target protein compared to a non-treated subject.

[0193]

[0140] In some embodiments, administering the SUTAC to a subject decreases the activation of the target protein compared to a non-treated subject. In some embodiments, administering the SUTAC to a subject increases the activation of the target protein compared to a non-treated subject.

[0194]

[0141] In some embodiments, administering the SUTAC to a subject decreases the repression of the target protein compared to a non-treated subject. In some embodiments, administering the SUTAC to a subject increases the repression of the target protein compared to a non-treated subject.

[0195]

[0142] In certain embodiments, administering the SUTAC to a subject increases the binding of the target protein to other protein(s) or DNA compared to a non-treated subject. In certain embodiments, administering said SUTAC to a subject decreases the binding of the target protein to other protein(s) or DNA compared to a non-treated subject.

[0196]

[0143] In some embodiments, administering the SUTAC to a subject decreases the solubilization of the target protein compared to a non-treated subject. In some embodiments, administering the SUTAC to a subject increases the solubilization of the target protein compared to a non-treated subject.

[0197]

[0144] In some embodiments, the terms “increased” or “decreased” used for describing the state of a protein may encompass the protein’s state relative to the protein’s state at a different time (which, in some embodiments, may be a time point prior to administration), a different tissue or cell, or in other subjects (such as non-treated subjects), and is presented, for example, as fold change. A skilled artisan would be knowledgeable of the tools and methods available for determining protein levels (amount) and / or their state, i.e. the protein’s level of activation or stabilization.

[0198]

[0145] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0199]

[0146] In the present disclosure the singular forms “a”, “an”, and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment incudes from the one particular and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about”, it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In some embodiments, the term “about”, refers to a deviance of between 0.0001-5% from the indicated number or range of numbers. In some embodiments, the term “about”, refers to a deviance of between 1-10% from the indicated number or range of numbers. In some embodiments, the term “about”, refers to a deviance of up to 25% from the indicated number or range of numbers. The term “comprises” means encompasses all the elements listed, but may also include additional, unnamed elements, and it may be used interchangeably with the terms “encompasses”, “includes”, or “contains” having all the same qualities and meanings. The term “consisting of’ means being composed of the recited elements or steps, and it may be used interchangeably with the terms “composed of’ having all the same qualities and meanings.

[0200]

[0147] It should be understood that the disclosure presented herein is not limited to the particular methodologies, protocols and reagents, and examples described herein. The terminology and examples used herein is for the purpose of describing particular embodiments only, for the intent and purpose of providing guidance to the skilled artisan, and is not intended to limit the scope of the disclosure presented herein.

[0201] EXAMPLES

[0202] EXAMPLE 1: synthesis of SUTACs of Formula IA-1 (AR-targeting SUTAC)

[0203] Scheme 1. presents the synthesis of a SUTAC having the structure of conjugate IA-1,

[0204]

[0148] Scheme 1 : synthetic scheme: (a) Cs2CO3 / Pd(OAc)2 / BINAP / Tol, 110 °C (b)

[0205] HCl / dioxane / DCM / RT (c) EDCI / HOBt / TEA / DMF / 25 °C (d) NaOH / MeOH / 25 °C (e)

[0206] EDCI / HOBt / TEA / DMF / 25 °C (f) NaHCO3 / THF / H2O / 0 °C methyl 4-[ 2-[ 2-[ 2-(tert-butoxycarbonylamino)ethoxy ] ethoxy ] ethylamino ]benzoate:

[0207]

[0149] A mixture of tert-butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate (2 g, 1 eq , methyl 4-iodobenzoate (2.53 g, 1.2 eq , CS2CO3 (7.87 g, 3 eq , Pd(OAc)2 (180.82 mg, 0.1 eq and BINAP (1.00 g, 0.2 eq in Tol. (40 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 110 °C for 6 h under N2 atmosphere. The residue was purified by pre-HPLC (NaHCO3 conditions) to afford compound. The residue was purified by pre- HPLC (FA conditions) to afford compound. The product was obtained as yellow oil (1.3 g, 42.20% yield). LC-MS (m / z): Calculated: 382.46; Found: 381.1 [M-H]+.

[0208]

[0150] 1H NMR (400 MHz, DMSO) δ 7.68 (d, J= 8.8 Hz, 2H), 6.77 (br t, J= 5.5 Hz, 1H), 6.62 (d, J= 8.9 Hz, 2H), 6.53 (t, J= 5.6 Hz, 1H), 3.74 (s, 3H), 3.58 - 3.49 (m, 6H), 3.39 - 3.39 (m, 2H), 3.29 - 3.23 (m, 2H), 3.09 - 3.02 (m, 2H), 1.37 (s, 9H). methyl 4-( (2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)benzoate:

[0209]

[0151] To a solution of methyl 4-[2-[2-[2-(tert- butoxycarbonylamino)ethoxy]ethoxy]ethylamino]benzoate (1.3 g, 1 eq was added HCl / dioxane (2 M, 1.70 mL). The mixture was stirred at 25 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The product was obtained as brown solid (1.2 g, crude, HC1). LC-MS (m / z): Calculated: 282.34; Found: 283.1 [M+H]+. methyl4-[ 2-[ 2-[2-[ (2-aminothiophene-3-carbonyl)amino ] ethoxy ] ethoxy ] ethylamino ]benzoate:

[0210] 6

[0211]

[0152] To a solution of 2-aminothiophene-3 -carboxylic acid (449.08 mg, 1 eq) in DMF (10 mL) was added HOBt (635.78 mg, 1.5 eq) and EDCI (901.99 mg, 1.5 eq), TEA (1.27 g, 1.75 mL, 4 eq). Then added methyl 4-[2-[2-(2-aminoethoxy)ethoxy]ethylamino]benzoate (1 g, 1 eq, HC1) at 25 °C. The mixture was stirred at 25 °C for 2 hr. The residue was purified by pre- HPLC (FA conditions) to afford compound. The product was obtained as yellow oil (560 mg, 43.81% yield). LC-MS (m / z): Calculated: 407.49; Found: 408.1 [M+H]+.

[0212]

[0153] 1H NMR (400 MHz, DMSO) δ 7.78 (d, J= 8.9 Hz, 2H), 6.96 (d, J= 5.9 Hz, 1H), 6.62 (d, J= 8.9 Hz, 2H), 6.23 (d, J= 5.9 Hz, 1H), 4.60 (br s, 1H), 3.83 (s, 3H), 3.72 - 3.59 (m, 8H), 3.53 - 3.47 (m, 2H), 3.36 - 3.32 (m, 5H)

[0213] 4-[ 2-[ 2-[ 2-[ ( 2-aminothiophene-3-carbonyl)amino ] ethoxy ] ethoxy ] ethylamino ]benzoic acid:

[0214]

[0154] To a solution of methyl 4-[2-[2-[2-[(2-aminothiophene-3- carbonyl)amino]ethoxy]ethoxy]ethylamino]benzoate (550 mg, 1 eq) in THF (1 mL), MeOH (1 mL), H2O (1 mL) was added NaOH (215.94 mg, 4 eq). The mixture was stirred at 25 °C for 2 hr. The residue was purified by pre-HPLC (NaHCCL conditions) to afford a yellow solid (133 mg, 25.04% yield). LC-MS (m / z): Calculated: 393.46; Found: 394.0 [M+H]+.

[0215]

[0155] 1H NMR (400 MHz, DMSO) δ 7.76 (t, J= 5.6 Hz, 1H), 7.68 - 7.63 (m, 3H), 7.20 (s, 2H), 7.08 (d, J= 5.9 Hz, 1H), 6.55 (d, J= 8.8 Hz, 3H), 6.25 (d, J= 5.9 Hz, 1H), 6.19 (br t, J= 5.4 Hz, 2H), 3.53 - 3.46 (m, 4H), 3.36 - 3.20 (m, 6H).

[0216] 2-amino-N-[ 2-[ 2-[ 2-[ 4-[[3-( 3-chloro-4-cyano-phenoxy) -2, 2, 4, 4-tetramethyl- cyclobutyl carbamoyl ]n cilino ] ethoxy ] ethoxy ] ethyl ]thiophene-3-carboxamide:

[0217]

[0156] To a solution of 4-[2-[2-[2-[(2-aminothiophene-3 carbonyl)amino]ethoxy]ethoxy]ethylamino]benzoic acid (133 mg, 1 eq) in DMF (2 mL) was added EDCI (97.20 mg, 1.5 eq) and TEA (102.61 mg, 141.15 μL , 3 eq), HOBt (68.51 mg, 1.5 eq). Then added , 4-(3-amino-2,2,4,4-tetramethyl-cyclobutoxy)-2-chloro-benzonitrile (94.23 mg, 1 eq). The mixture was stirred at 25 °C for 2 hr. The residue was purified by pre-HPLC (FA conditions) to afford a yellow solid (150 mg, 67.83% yield). LC-MS (m / z): Calculated: 654.22; Found: 655.1 [M+H]+.

[0218]

[0157] 1H NMR (400 MHz, DMSO) δ 7.95 (d, J= 8.8 Hz, 1H), 7.78 (br t, J= 5.7 Hz, 1H), 7.69 (d, J= 8.8 Hz, 2H), 7.39 (d, J= 9.1 Hz, 1H), 7.28 - 7.20 (m, 3H), 7.11 (d, J= 5.9 Hz, 1H), 7.04 (dd, J= 2.4, 8.8 Hz, 1H), 6.65 (d, J= 8.8 Hz, 2H), 6.29 (d, J= 5.8 Hz, 1H), 6.22 (s, 1H), 4.35 (s, 1H), 4.08 (d, J= 9.1 Hz, 1H), 3.63 - 3.57 (m, 6H), 3.55 - 3.50 (m, 2H), 3.36 (br d, J= 6.0 Hz, 2H), 3.28 (q, J= 5.7 Hz, 2H), 1.25 (s, 6H), 1.16 (s, 6H) 2-[(2-chloroacetyl)amino]-N-[2-[2-[2-[4-[[3-(3-chloro-4-cyano-phenoxy)-2, 2, 4, 4-tetramethyl- cyclobutyl carbamoyl ]n cilino ] ethoxy ] ethoxy ] ethyl ]thiophene-3-carboxamide:

[0219]

[0158] To a solution of 2-amino-N-[2-[2-[2-[4-[[3-(3-chloro-4-cyano-phenoxy)-2, 2,4,4- tetramethyl-cyclobutyl]carbamoyl]anilino]ethoxy]ethoxy]ethyl]thiophene-3-carboxamide (30 mg, 1 eq) in DCM (1 mL) was added TEA (13.92 mg, 19.15 , 3 eq). TμhLen added 2- chloroacetyl chloride (5.18 mg, 3.65 , 1 eq)μL at 0 °C. The mixture was stirred at 0 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by pre-HPLC (TFA conditions) to afford compound. The product was obtained as blue solid (2.52 mg, 7.37% yield). LC-MS (m / z): Calculated: 730.7; Found: 731.1 [M+H]+.

[0220]

[0159] 1H NMR (400 MHz, CDCl3) δ 12.82 (br s, 1H), 7.59 (dd, J= 8.6, 16.2 Hz, 3H), 7.02 (d, J= 5.8 Hz, 1H), 6.98 (d, J= 2.4 Hz, 1H), 6.82 (dd, J= 2.4, 8.8 Hz, 1H), 6.77 (d, J= 5.9 Hz, 1H), 6.70 - 6.63 (m, 1H), 6.60 (d, J= 8.8 Hz, 2H), 6.12 (br d, J= 8.3 Hz, 1H), 4.25 (s, 2H), 4.15 (d, J= 8.3 Hz, 1H), 4.06 (s, 1H), 3.76 - 3.71 (m, 2H), 3.70 - 3.65 (m, 8H), 3.35 (t, J= 5.1 Hz, 2H), 1.27 (s, 6H), 1.22 (s, 6H).

[0221] EXAMPLE 2: Synthesis of SUTACs of Formula IA-2 (AR-targeting SUTAC) Scheme 2, presents the synthesis of a SUTAC having the structure of conjugate IA-2.

[0222]

[0160] Scheme 2: synthetic scheme: (a) DMSO / DIEA / 120 °C (b) HCl / dioxane / DCM / RT (c)

[0223] EDCI / HOBt / TEA / DMF / 25 °C (d) NaOH / MeOH / THF / H2O / 25 °C (e)

[0224] EDCI / HOBt / TEA / DMF / 25 °C (f) DCM / TEA / 0 °C methyl 4- [8-(tert-butoxy carbonylamino) octylamino] benzoate:

[0225]

[0161] To a solution of tert-butyl N-(8-aminooctyl) carbamate (1 g, 4.09 mmol), methyl 4- fluorobenzoate (630.75 mg, 4.09 mmol) in DMSO (2 mL) was added DIEA (1.59 g, 12.28 mmol). The mixture was stirred at 120 °C for 18 hr. The residue was purified by prep-HPLC (NH4HCO3 condition) to afford methyl 4-[8-(tert-butoxy carbonylamino) octylamino] benzoate (500 mg, 32.28% yield) as a white solid. LC-MS (m / z): Calculated: 378.51; Found: 379.5 [M+H]+.

[0226]

[0162] 1H NMR (400 MHz, DMSO) δ 7.67 (d, J = 8.8 Hz, 2H), 6.74 (br t, J = 5.0 Hz, 1H), 6.56 (d, J = 8.8 Hz, 2H), 6.48 (br t, J = 5.2 Hz, 1H), 3.73 (s, 3H), 3.07-3.01 (m, 2H), 2.88 (q, J = 6.5 Hz, 2H), 1.53 (quin, J = 7.0 Hz, 2H), 1.39-1.31 (m, 14H), 1.25 (br s, 6H) methyl 4-(8-aminooctylamino) benzoate:

[0227]

[0163] To a solution of methyl 4-[8-(tert-butoxycarbonylamino)octylamino]benzoate (500 mg, 1.32 mmol) in HCl / di oxane (4 M, 6.06 mL), DCM (5 mL). The mixture was stirred at 15 °C for Ihr. The reaction mixture was concentrated under reduced pressure to remove solvent. The product was obtained as yellow solid (415 mg, 99.78% yield, HC1). LC-MS (m / z): Calculated: 278.40; Found: 279.3 [M+H]+. methyl 4-[8-[(2-aminothiophene-3-carbonyl) amino] octylamino] benzoate:

[0228]

[0164] To a solution of methyl 4-(8-aminooctylamino)benzoate (400 mg, 1.44 mmol), 2- aminothiophene-3 -carboxylic acid (205.70 mg, 1.44 mmol) in DMF (4 mL), DMSO (4 mL) was added HOBt (291.23 mg, 2.16 mmol) and EDCI (413.17 mg, 2.16 mmol), TEA (436.18 mg, 4.31 mmol). The mixture was stirred at 25 °C for 1 hr. The product was obtained as white solid (265 mg, 45.70% yield). LC-MS (m / z): Calculated: 403.54; Found: 404.3 [M+H]+.

[0229] 4-[ 2-[ 2-[ 2-[ ( 2-aminothiophene-3-carbonyl)amino ] ethoxy ] ethoxy ] ethylamino ]benzoic acid:

[0230]

[0165] To a solution of methyl 4-[8-[(2-aminothiophene-3- carbonyl)amino]octylamino]benzoate (260 mg, 644.30 pmol) in MeOH (2 mL), H2O (1 mL), THF (2 mL) was added NaOH (103.08 mg, 2.58 mmol). The mixture was stirred at 25 °C for 1 hr. The residue was purified by prep-HPLC (NH4HCO3 condition) to afford 4-[8-[(2- aminothiophene-3 -carbonyl) amino] octylamino] benzoic acid (45 mg, 17.93% yield) as a yellow solid. LC-MS (m / z): Calculated: 389.51; Found: 389.6 [M+H]+

[0231] 2-amino-N-[8-[4-[[3-(3-chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl- cyclobutyl carbamoyl] anilino ] octyl ]thiophene-3-carboxamide:

[0232]

[0166] To a solution of 4-[8-[(2-aminothiophene-3-carbonyl)amino]octylamino]benzoic acid (40 mg, 102.69 pmol), 4-(3-amino-2,2,4,4-tetramethyl-cyclobutoxy)-2-chloro-benzonitrile (28.63 mg, 102.69 pmol) in DCM (1 mL) was added EDCI (29.53 mg, 154.04 pmol) and HOBt (20.81 mg, 154.04 pmol), TEA (41.57 mg, 410.77 pmol). The mixture was stirred at 25 °C for Ihr. The residue was purified by prep-HPLC (FA condition) to afford 2-amino-N-[8-[4-[[3-(3- chloro-4-cyano-phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]carbamoyl]anilino]octyl]thiophene-3- carboxamide (40 mg, 59.90% yield) as a brown solid. LC-MS (m / z): Calculated: 650.28; Found: 651.1 [M+H]+. 2-[(2-chloroacetyl)amino]-N-[8-[4-[[3-(3-chloro-4-cyano-phenoxy)-2, 2, 4, 4-tetramethyl- cyclobutyl carbamoyl] anilino ] octyl ]thiophene-3-carboxamide:

[0233]

[0167] To a solution of 2-amino-N-[8-[4-[[3-(3-chloro-4-cyano-phenoxy)-2, 2, 4, 4- tetramethyl-cyclobutyl] carbamoyl]anilino]octyl]thiophene-3-carboxamide (15 mg, 23.07 pmol), 2-chloroacetyl chloride (2.61 mg, 23.07 pmol) in DCM (1 mL) was added TEA (7.00 mg, 69.20 pmol). The mixture was stirred at 0 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by prep- HPLC (FA condition) to afford 2-[(2-chloroacetyl) amino]-N-[8-[4-[[3-(3-chloro-4-cyano- phenoxy)-2,2,4,4-tetramethyl-cyclobutyl]carbamoyl]anilino]octyl]thiophene-3-carboxamide (3.23 mg) as a white solid. LC-MS (m / z): Calculated: 726.76; Found: 727.5 [M+H]+.

[0234]

[0168] 1H NMR (400 MHz, CDCh) δ 12.86 (br s, 1H), 7.63 (br d J= 8.6 Hz, 2H), 7.57 (d, J= 8.6 Hz, 1H), 7.00-6.96 (m, 2H), 6.86 (d, J= 5.9 Hz, 1H), 6.81 (dd, J= 2.3, 8.7 Hz, 1H), 6.59 (br d, J= 8.8 Hz, 2H), 6.08 (br d, J= 8.3 Hz, 1H), 5.98 (br s, 1H), 4.26 (s, 2H), 4.15 (d, J= 8.0 Hz, 1H), 4.05 (s, 1H), 3.49 - 3.41 (m, 2H), 3.17 (t, J= 6.9 Hz, 2H), 1.69-1.65 (m, 2H), 1.44- 1.33 (m, 10H), 1.27 (s, 6H), 1.22 (s, 6H).

[0235] EXAMPLE 3: Synthesis of SUTACs of Formula IA-3 (AR-targeting SUTAC) Scheme 3, presents the synthesis of a SUTAC having the structure of conjugate IA-3.

[0236]

[0169] Scheme 3: synthetic scheme: (a) DMSO / DIEA / 110 °C (b) HCl / dioxane / DCM / RT (c)

[0237] EDCI / HOBt / TEA / DMF / 25 °C (d) NaOH, MeOH / THF / H2O / 25 °C (e)

[0238] EDCI / HOBt / TEA / DMF / 25 °C (f) DCM / TEA / 0 °C methyl4-[2-[2-[2-[2-[2-[2-(tert- butoxycarbonylamino) ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethylamino Jbenzoate: :

[0239]

[0170] To a solution of tert-butyl N-[2-[2-[2-[2-[2-(2-aminoethoxy) ethoxy] ethoxy] ethoxy] ethoxy]ethyl]carbamate (1.4 g, 1 eq), methyl 4-iodobenzoate (1.16 g, 1.2 eq) in Tol. (1 mL) was added CS2CO3 (3.60 g, 3 eq), Pd(OAc)2(82.61 mg, 0.1 eq), BINAP (458.24 mg, 0.2 eq). The mixture was stirred at 110 °C for 6 hr. The residue was purified by pre-HPLC (NH4HCO3 conditions) to afford a yellow oil (900 mg, 1.75 mmol, 47.53% yield). LC-MS (m / z): Calculated: 514.29; Found: 515.1 [M+H]+. methyl4-[ 2-[ 2-[2-[2-[ 2-( 2 -aminoethoxy) ethoxy ] ethoxy ] ethoxy ] ethoxy] ethylamino Jbenzoate:

[0171] To a solution of methyl 4-[2-[2-[2-[2-[2-[2-(tert-butoxy carbonylamino) ethoxy] ethoxy] ethoxy] ethoxy]ethoxy]ethylamino]benzoate (700 mg, 1 eq in DCM (5 mL) was added HCl / dioxane (2 M, 14 mL, 20.58 eq). The mixture was stirred at 25 °C for 1 hr. The reaction mixture was concentrated under reduced to give a yellow oil (600 mg, 97.81% yield, HC1). LC- MS (m / z): Calculated: 414.24; Found: 415.3 [M+H]+.

[0240]

[0172] 1H NMR (400 MHz, DMSO) 5 8.11 - 7.79 (m, 3H), 7.68 (d, J= 8.9 Hz, 2H), 6.68 - 6.56 (m, 2H), 3.74 (s, 3H), 3.55 - 3.47 (m, 19H), 3.26 (t, J= 5.7 Hz, 2H), 2.95 (qd, J= 5.3, 10.8 Hz, 2H). methyl 4-[2-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl) amino] ethoxy] ethoxy] ethoxy] ethoxy ] ethoxy ] ethylamino ]benzoate:

[0241]

[0173] To a solution of methyl 4-[2-[2-[2-[2-[2-(2-aminoethoxy) ethoxy] ethoxy] ethoxy] ethoxy] ethylamino]benzoate (600 mg, 1 eq, HCl),2-aminothiophene-3-carboxylic acid (190.48 mg, 1 eq in DMF (1.5 mL) was added HOBt (269.68 mg, 1.5 eq and EDCI (382.59 mg, 1.5 eq), TEA (538.53 mg, 740.76 pL, 4 eq . The mixture was stirred at 25 °C for 2 hr. LCMS (EW42073-456-P1A1) showed 27% peak of desired compound Ms was detected. The residue was purified by pre-HPLC (NH4HCO3 conditions) to afford a yellow oil (400 mg, 55.71% yield). LC-MS (m / z): Calculated: 539.23; Found: 540.0 [M+H]+.

[0242]

[0174] 1H NMR (400 MHz, DMSO) δ 7.75 - 7.65 (m, 3H), 7.19 (s, 2H), 7.07 (d, J= 5.9 Hz, 1H), 6.62 (d, J= 8.9 Hz, 2H), 6.53 (t, J= 5.6 Hz, 1H), 6.25 (d, J= 5.8 Hz, 1H), 3.74 (s, 3H), 3.58 - 3.42 (m, 23H) 4-[ 2-[ 2-[ 2-[ 2-[ 2-[2-[ (2-aminothiophene-3- carbonyl)amino ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethylamino Jbenzoicacid:

[0243]

[0175] To a solution of methyl 4-[2-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino] ethoxy] ethoxy] ethoxy]ethoxy]ethoxy]ethylamino]benzoate (400 mg, 1 eq) in MeOH (0.5 mL), THF (0.5 mL), H2O (0.5 mL) was added NaOH (118.59 mg, 4 eq . The residue was purified by pre-HPLC (NH4HCO3 conditions) to afford a red oil (210 mg, 53.90% yield). LC- MS (m / z): Calculated: 525.21; Found: 526.1 [M+H]+

[0244] 1H NMR (400 MHz, DMSO) δ 7.81 - 7.74 (m, 1H), 7.69 (d, J= 8.8 Hz, 2H), 7.22 (s, 2H), 7.11 (d, J= 5.9 Hz, 1H), 6.62 (d, J= 8.8 Hz, 2H), 6.38 (br t, J= 5.5 Hz, 1H), 6.28 (d, J= 5.8 Hz, 1H), 3.62 - 3.56 (m, 10H), 3.56 - 3.53 (m, 14H).

[0245] 2-amino-N-[ 2-[ 2-[ 2-[ 2-[ 2-[ 2-[ 4-[ [3-( 3-chloro-4-cyano-phenoxy)-2, 2, 4, 4-tetramethyl- cyclobutyl carbamoyl ]n cilino ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethyl ]thiophene-3- carboxamide:

[0176] To a solution of 4-[2-[2-[2-[2-[2-[2-[(2-aminothiophene-3- carbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]benzoic acid (200 mg, 1 eq) in DMF (1 mL) was added (3-amino-2,2,4,4-tetramethyl-cyclobutoxy)-2-chloro-benzonitrile (106.08 mg, 1 eg) 2 mg, 1.5 eq) and TEA (115.51 mg, 158.89 , 3 eq), HOμBLt (77.12 mg, 1.5 eq), EDCI (109.42 mg, 1.5 eq). The mixture was stirred at 25 °C for 2 hr. The residue was purified by pre-HPLC (NH4HCO3 conditions) to afford a brown solid (190 mg, 63.50% yield). LC-MS (m / z): Calculated: 785.32; Found: 786.2 [M+H]+

[0246]

[0177] 1H NMR (400 MHz, DMSO) δ 7.90 (d, J= 8.6 Hz, 1H), 7.72 (br t, J= 5.4 Hz, 1H), 7.65 (br d, J= 8.5 Hz, 2H), 7.34 (br d, J= 9.3 Hz, 1H), 7.25 - 7.11 (m, 3H), 7.06 (d, J= 5.9 Hz, 1H), 7.00 (dd, J= 1.9, 8.8 Hz, 1H), 6.61 (br d, J= 8.5 Hz, 2H), 6.24 (d, J= 5.9 Hz, 1H), 6.17 (br t, J= 5.3 Hz, 1H), 4.31 (s, 1H), 4.04 (br d, J= 9.1 Hz, 1H), 3.63 - 3.40 (m, 22H), 3.24 (br d, J= 5.5 Hz, 2H), 1.21 (s, 6H), 1.11 (s, 6H)

[0247] 2-[ ( 2 -chloroacetyl) amino ]-N-[ 2-[2-[ 2-[ 2-[ 2-[ 2-[ 4-[[ 3-(3-chloro-4-cyano-phenoxy)-2, 2, 4, 4- tetramethyl-cyclobutyl ] carbamoyl ]n cilino ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethyl ] thiophene -

[0248] 3-carboxamide :

[0249]

[0178] To a solution of 2-amino-N-[2-[2 -[2 -[2 -[2 -[2 -[4 -[[3 -(3- chloro-4-cyano-phenoxy)- 2, 2, 4, 4-tetramethyl cyclobutyl]carbamoyl]anilino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]thiophene-3- carboxamide (20 mg, 1 eq), 2-chloroacetyl chloride (2.87 mg, 2.03 , 1 eq) in DCMμL (2 mL) was added TEA (7.72 mg, 10.62 , 3 eμqL). The mixture was stirred at 0 °C for 1 hr. The residue was purified by pre-HPLC (TFA conditions) to a white solid (4.7 mg, 20.69% yield). LC-MS (m / z): Calculated: 861.29; Found: 862.1 [M+H]+.

[0250]

[0179] 1H NMR (400 MHz, DMSO) δ 12.93 (s, 1H), 7.62 (d, J= 8.5 Hz, 2H), 7.57 (d, J= 8.6 Hz, 1H), 7.54 - 7.46 (m, 1H), 7.29 (d, J= 5.9 Hz, 1H), 6.97 (d, J= 2.4 Hz, 1H), 6.83 - 6.79 (m, 2H), 6.67 (br d, J= 8.1 Hz, 2H), 6.13 (br d, J= 8.3 Hz, 1H), 4.25 (s, 2H), 4.15 (d, J= 8.3 Hz, 1H), 4.05 (s, 1H), 3.69 - 3.62 (m, 22H), 3.32 (t, J= 5.0 Hz, 2H), 1.27 (s, 6H), 1.22 (s, 6H).

[0251] EXAMPLE 4: Synthesis of SUTACs of Formula IA-4 (BRD4-targeting SUTAC)

[0252]

[0253]

[0180] Scheme 4: synthetic scheme: (a) HOBt / EDCI / DIEA / DMF / RT (b) TEA / ETOH / 68 °C (c) LiOH / 25 °C (d) HATU / DIEA / DMF / 25 °C (e) DCM / TEA / 0 °C tert-butyl 2-[2-[2-[(2-cyanoacetyl) amino] ethoxy] ethoxy] acetate:

[0254] 2

[0255]

[0181] To a solution of tert-butyl 2-[2-(2-aminoethoxy)ethoxy]acetate (1 g, 1 eg),2-cyanoacetic acid (504.29 mg, 1.3 eq) in DMF (10 mL) was added EDCI (2.19 g, 2.5 eq) and HOBt (924.33 mg, 1.5 eq), DIEA (1.77 g, 2.38 mL, 3 eq). The mixture was stirred at 20 °C for 16 hr. The residue was purified by pre-HPLC (NH4HCO3 conditions) to afford a yellow oil (500 mg, 38.29% yield). LC-MS (m / z): Calculated: 286.15; Found: 287.1 [M+H]+. tert-butyl 2-[2-[ 2-[ (2-aminothiophene-3-carbonyl) amino ] ethoxy ] ethoxy ] acetate:

[0256]

[0182] To a solution of tert-butyl 2-[2-[2-[(2-cyanoacetyl)amino]ethoxy]ethoxy]acetate (500 mg, 1 eq), l,4-dithiane-2,5-diol (265.84 mg, 1 eq) in EtOH (3 mL) was added TEA (353.41 mg, 486.12 μL, 2 eq). The mixture was stirred at 68 °C for 10 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by pre-HPLC (NH4HCO3 conditions) to afford a yellow oil (300 mg, 49.88% yield). LC-MS (m / z): Calculated: 342.16; Found: 341.1 [M-H]+.

[0257] 2-[ 2-[ 2-[ ( 2-aminothiophene-3-carbonyl) amino ] ethoxy ] ethoxy ]aceticacid:

[0258]

[0183] To a solution of tert-butyl 2- [2- [2- [(2 - aminothiophene- 3- carbonyl) amino] ethoxy] ethoxy] acetate (100 mg, 1 eq) in THF (1 mL), MeOH (1 mL), H2O (1 mL) was added LiOH.EEO (36.55 mg, 3 eq). The mixture was stirred at 25 °C for 3 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by pre- HPLC (NH4HCO3 conditions) to afford a yellow oil (60 mg, 71.68% yield). LC-MS (m / z): Calculated: 288.08; Found: 289.0 [M+H]+.

[0259]

[0184] 1H NMR (400 MHz, DMSO) δ 7.88 (br t, J = 5.6 Hz, 1H), 7.17 (br s, 2H), 7.12 (d, J =

[0260] 5.9 Hz, 1H), 6.23 (d, J= 5.9 Hz, 1H), 3.67 (s, 3H), 3.36 - 3.28 (m, 7H) -amino-N-[ 2-[ 2-[ 2-[ 2-[ 4-[ (Z)-l-( 4-hydr oxyphenyl) -2 -phenyl-but-l-enyl Jphenoxy Jethyl-methyl- amino ] -2 -oxo-ethoxy ] ethoxy ] ethyl ]thiophene-3-carboxamide :

[0261]

[0185] To a solution of 2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy]ethoxy]acetic acid (50 mg, 1 eq), 4-[(Z)-l-[4-[2-(methylamino)ethoxy]phenyl]-2-phenyl-but-l-enyl]phenol (64.77 mg, 1 eq) in DMF (1 mL) was added HATU (98.91 mg, 1.5 eq) and DIEA (67.24 mg, 90.62 μL, 3 eq). The mixture was stirred at 25 °C for 2 hr. The residue was purified by pre-HPLC (NH4HCO3 conditions) to afford a yellow solid (13 mg, 11.64% yield). LC-MS (m / z): Calculated: 643.27; Found: 644.1 [M+H]+. 2-[ ( 2 -chloroacetyl) amino ]-N-[ 2-[2-[ 2-[ 2-[ 4-[ (Z)-l-(4-hydroxyphenyl)-2-phenyl-but-l- enyl]phenoxy]ethyl-methyl-amino]-2-oxo-ethoxy] ethoxy] ethyl] thiophene-3 -carboxamide:

[0262]

[0186] To a solution of 2-amino-N-[2-[2-[2-[2-[4-[(Z)-l-(4-hydroxyphenyl)-2-phenyl-but-l- enyl]phenoxy]ethyl-methyl-amino]-2-oxo-ethoxy]ethoxy]ethyl]thiophene-3-carboxamide (13 mg, 1 eq), 2-chloroacetyl chloride (2.28 mg, 1.61 , 1 eq)μ iLn DCM (1 mL) was added TEA (6.13 mg, 8.43 μ,L 3 eq). The mixture was stirred at 0 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by pre-HPLC (FA conditions) to afford an off-white solid (4.47 mg, 30.73% yield). LC-MS (m / z): Calculated: 719.24; Found: 720.2 [M+H]+.

[0263]

[0187] 1H NMR (400 MHz, DMSO) δ 7.34 (dd,J = 3.3, 5.8 Hz, 1H), 7.17 - 7.05 (m, 5H), 7.01 (d, J= 8.4 Hz, 2H), 6.89 (d, J= 5.6 Hz, 1H), 6.80 - 6.70 (m, 4H), 6.53 (d, J= 8.5 Hz, 2H), 4.34 (d, J= 19.3 Hz, 2H), 4.29 - 4.17 (m, 2H), 3.98 (br d, J= 5.4 Hz, 2H), 3.68 - 3.51 (m, 10H), 3.04 - 2.84 (m, 3H), 2.48 (q, J= 7.3 Hz, 2H), 0.90 (t, J= 7.4 Hz, 3H).

[0264] EXAMPLE 5: Synthesis of SUTACs of Formula IA-5 (BRD4-targeting SUTAC) Scheme 5. presents the synthesis of & SUTAC having the structure of conjugate IA-5.

[0265]

[0188] Scheme 5: synthetic scheme: (a) EDCI / HOBt / DIEA / DMF, RT (b) TEA / EtOH, 70 °C

[0266] (c)LiOH / MeOH / THF / H2O (d) EDCI / HOBt / DIEA / DMF, RT (e) NaHCO3,THF / H2O, 0 °C tert-butyl 8-[ (2-cyanoacetyl)amino] octanoate:

[0267]

[0189] To a solution of 2-cyanoacetic acid (513.53 mg, 1.3 eq) in DMF (10 mL) was added EDCI (2.23 g, 2.5 eq) and HOBt (941.27 mg, 1.5 eq), DIEA (1.80 g, 2.43 mL, 3 eq) then added tertbutyl 8-aminooctanoate (1 g, 1 e< / ).The mixture was stirred at 25 °C for 18 hr.. The residue was purified by pre-HPLC (FA conditions) to afford a yellow oil (500 mg, 38.13% yield). LC-MS (m / z): Calculated: 282.19; Found: 283.0 [M+H]+. tert-butyl 8-[ (2-aminothiophene-3-carbonyl)amino]octanoate:

[0268]

[0190] To a solution of tert-butyl 8-[(2-cyanoacetyl) amino] octanoate (500 mg, 1 eq), 1,4- dithiane-2,5-diol (269.56 mg, 1 eq) in EtOH (5 mL) was added TEA (358.35 mg, 492.91 pL, 2 eq). The mixture was stirred at 68 °C for 10 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by pre-HPLC (NH4HCO3 conditions) to afford a yellow oil (300 mg, 49.76% yield). LC-MS (m / z): Calculated: 340.18; Found: 341.0 [M+H]+.

[0269] 8-[ ( 2-aminothiophene-3-carbonyl)amino ]octanoic acid:

[0270]

[0191] To a solution of tert-butyl 8-[(2-aminothiophene-3-carbonyl)amino] octanoate (200 mg, 1 eq) in THF (1.5 mL), MeOH (1.5 mL), H2O (1.5 mL) was added LiOH.H2O (73.95 mg, 3 eq). The mixture was stirred at 25 °C for 3hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by pre-HPLC (NH4HCO3 conditions) to afford a yellow oil (95 mg, 56.87% yield). LC-MS (m / z): Calculated: 284.12; Found: 307.1 [M+Na]+.

[0271] 2-amino-N-[8-[2-[4-[(Z)-l-(4-hydroxyphenyl)-2-phenyl-but-l-enyl]phenoxy]ethyl-methyl- amino ]-8-oxo-octyl ]thiophene-3-carboxamide :

[0272]

[0192] To a solution of 8-[(2-aminothiophene-3-carbonyl)amino]octanoic acid (95 mg, 1 eq), in DMF (1 mL) was added HATU (190.53 mg, 1.5 eq) and DIEA (129.53 mg, 174.57 , 3 eq), μL then added 4-[(Z)-l-[4-[2-(methylamino)ethoxy]phenyl]-2-phenyl-but-l-enyl]phenol (124.77 mg, 1 eq). The mixture was stirred at 25 °C for 2hr. The residue was purified by pre-HPLC (NH4HCO3 conditions) to afford a yellow solid (80 mg, 37.43% yield). LC-MS (m / z): Calculated: 639.31; Found: 638.3 [M-H]+.

[0273] 2-[(2-chloroacetyl)amino]-N-[8-[2-[4-[(Z)-l-(4-hydroxyphenyl)-2-phenyl-but-l- enyl]phenoxy]ethyl-methyl-amino]-8-oxo-octyl]thiophene-3-carboxamide:

[0274]

[0193] To a solution of 2-amino-N-[8-[2-[4-[(Z)-l-(4-hydroxyphenyl)-2-phenyl-but-l- enyl]phenoxy]ethyl-methyl-amino]-8-oxo-octyl]thiophene-3-carboxamide (11 mg, 1 eq in DCM (1 mL) was added TEA (5.22 mg, 7.18 , 3 eq μ, L then added 2-chloroacetyl chloride (1.94 mg, 1.37 μL, 1 eq at 0 °C. The mixture was stirred at 0 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by pre-HPLC (FA conditions) to afford the product as an off-white solid (5.16 mg, 41.06% yield). LC-MS (m / z): Calculated: 715.28; Found: 716.2 [M+H]+

[0275]

[0194] 1H NMR (400 MHz, DMSO-d6) δ 7.33 (dd, J= 2.5, 5.9 Hz, 1H), 7.17 - 6.97 (m, 7H), 6.94 (dd, J= 3.5, 5.8 Hz, 1H), 6.81 - 6.68 (m, 4H), 6.53 (br d, J= 7.4 Hz, 2H), 4.36 (d, J= 4.5 Hz, 2H), 4.00 (td, J= 5.1, 10.2 Hz, 2H), 3.73 - 3.59 (m, 2H), 3.37 - 3.32 (m, 2H), 3.10 - 2.88 (m, 3H), 2.52 - 2.28 (m, 4H), 1.66 - 1.52 (m, 4H), 1.38 - 1.29 (m, 6H), 0.89 (t, J= 7.4 Hz, 3H).

[0276] EXAMPLE 6: Synthesis of SUTACs of Formula IA-6 (BRD4-targeting SUTAC)

[0277]

[0195] Scheme 6: synthetic scheme: (a) DIEA / HOBt / EDCI / DMF, 25°C (b) TEA, ETOH, 68 °C (c) LiOH / THF / H2O / MeOH, 25 °C (d) HATU / DIEA / DMF / 25 °C (e) TEA, DCM, 0 °C. tert-butyl3-[2-[2-[2-[2-[2-[ (2- cyanoacetyl)amino] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]propanoate:

[0278] 3

[0279]

[0196] To a solution of tert-butyl 3-[2-[2-[2-[2-(2- aminoethoxy)ethoxy] ethoxy] ethoxy] ethoxy ]propanoate (500 mg, 1 eq), 2-cyanoacetic acid (116.38 mg, 1 eq) in DMF (5 mL) was added EDCI (655.68 mg, 2.5 eq) and HOBt (277.30 mg, 1.5 eq), DIEA (353.64 mg, 476.61 , 2μ eLq). The mixture was stirred at 25 °C for 18 hr. The residue was purified by pre-HPLC (FA conditions) to give as yellow oil (300 mg, 50.70% yield). LC-MS (m / z): Calculated 432.25; Found: 431.0 [M-H]+. tert-butyl 3- [ 2-[ 2-[ 2-[ 2-[ 2-[ (2-aminothiophene-3- carbonyl)amino ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]propanoate:

[0280]

[0197] To a solution of tert-butyl 3-[2-[2-[2-[2-[2-[(2- cyanoacetyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (231 mg, 1 eq), 1,4- dithiane-2,5-diol (81.31 mg, 1 eq) in EtOH (2.5 mL) was added TEA (108.09 mg, 148.68 pL, 2 eq). The mixture was stirred at 68 °C for 10 hr. The reaction mixture was poured into water (20 mL), extracted with Ethyl acetate (20 mL x 2). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by pre-HPLC (NH4HCO3 conditions) to give as yellow oil (150 mg, 57.24% yield). LC-MS (m / z): Calculated 490.23; Found: 491.0 [M+H]+.

[0281] 3-[ 2-[ 2-[ 2-[ 2-[ 2-[ (2-aminothiophene-3- carbonyl)amino ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy propanoicacid:

[0282]

[0198] To a solution of tert-butyl 3-[2-[2-[2-[2-[2-[(2-aminothiophene-3- carbonyl)amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (120 mg, 1 eq) in THF (1.5 mL), H2O (1.5 mL), MeOH (1.5 mL) was added LiOH.H2O (30.79 mg, 3 eq). The mixture was stirred at 25 °C for 1 hr. The residue was purified by pre-HPLC (NH4HCO3 conditions) to give as yellow oil (60 mg, 56.46% yield). LC-MS (m / z): Calculated 434.17; Found: 435.0 [M+H]+.

[0283]

[0199] 1H NMR (400 MHz, MeOH) δ = 7.00 (d, = 5.9 Hz, 1H), 6.27 (d, J= 5.9 Hz, 1H), 3.74 (t, J= 6.8 Hz, 3H), 3.69 - 3.60 (m, 21H), 3.52 - 3.48 (m, 2H), 2.50 (t, J= 6.7 Hz, 2H.

[0284] 2-amino-N-[ 2-[ 2-[ 2-[ 2-[ 2-[ 3-[ 2-[ 4-[ (Z)-l-(4-hydroxyphenyl)-2-phenyl-but-l- enyl J phenoxy Jethyl-methyl-amino J-3-oxo- propoxy ] ethoxy ] ethoxy ] ethoxy] ethoxy J ethyl ]thiophene-3-carboxami:

[0285]

[0200] To a solution of 3-[2-[2-[2-[2-[2-[(2-aminothiophene-3-carbonyl)amino]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (50 mg, 1 eq), in DMF (1 mL) was added HATU (65.63 mg, 1.5 eq) and DIEA (44.62 mg, 60.13 , 3 eq),μ 4L-[(Z)-l-[4-[2- (methylamino)ethoxy]phenyl]-2-phenyl-but-l-enyl]phenol (42.98 mg, 1 eq). The mixture was stirred at 25 °C for 2 hr. The residue was purified by pre-HPLC (FA conditions) to give (40 mg, 44.00% yield) as brown solid. LC-MS (m / z): Calculated 789.37; Found: 790.4 [M+H]+.

[0286] 2-[ ( 2 -chloroacetyl) amino ]-N-[ 2-[2-[ 2-[ 2-[ 2-[ 3-[2-[4-[ (Z)-l-(4-hydr oxyphenyl) -2 -phenyl-but-1- enyl ] phenoxy Jethyl-methyl-amino J-3-oxo- propoxy J ethoxy J ethoxy J ethoxy] ethoxy ] ethyl Jthiophene-3-carboxamide :

[0287]

[0201] To a solution of 2-amino-N-[2-[2-[2-[2-[2-[3-[2-[4-[(Z)-l-(4-hydroxyphenyl)-2-phenyl- but-l-enyl]phenoxy]ethyl-methyl-amino]-3-oxo- propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]thiophene-3-carboxamide (20 mg, 1 eq in DCM (1 mL) was added TEA (7.69 mg, 10.57 , 3 eqμL. Then was added 2-chloroacetyl chloride (2.86 mg, 2.02 ,μL 1 eq at 0 °C. The mixture was stirred at 0 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by pre- HPLC (FA conditions) to afford a brown solid (13.66 mg, 62.27% yield) as brown solid. LC- MS (m / z): Calculated 789.37; Found: 790.4 [M+H]+.

[0288]

[0202] 1H NMR (400 MHz, CDCh) δ12.93 (br s, 1H), 7.44 (br s, 1H), 7.27 - 7.25 (m, 1H), 7.21 - 7.01 (m, 7H), 6.94 - 6.62 (m, 6H), 6.50 (br d, J= 8.5 Hz, 2H), 4.30 - 4.20 (m, 2H), 4.04 - 3.90 (m, 2H), 3.78 - 3.51 (m, 24H), 3.15 - 2.89 (m, 3H), 2.67 - 2.44 (m, 4H), 0.92 (br t, J= 7.4 Hz, 3H).

[0289] EXAMPLE 7: Synthesis of SUTACs of Formula IA-7 (BRD4-targeting SUTAC)

[0290] Scheme 7. presents the synthesis of a SUTAC having the structure of conjugate IA-

[0291]

[0203] 3 pmol of 1-5 (30 μfl a o 100 mM stock in DMSO) were used in the reaction. 2 mg of JQ1 acid (5 pmol) were weighed and dissolved in a 10 pl DMF. EDC and HOBT were dissolved in DMF to a concentration of 0.4 M, and 12.5 f this solμulti oon + 3.5 pl DIPEA (20 pmol) were added to the solution of JQ1 acid. After 5 minutes this was added to the sample of 1-5 with gentle vortex. LCMS indicated complete reaction after 1 hour. The sample was dilute with 15% acetonitrile in water + 0.1% TFA and purified using reverse phase HPLC on 12-65% acetonitrile gradient. The product was lyophilized from 30% acetic acid to remove the TFA. Total yield obtained was 0.94 mg (43%). The mass was confirmed by LCMS.

[0292] EXAMPLE 8: Synthesis of SUTACs of Formula ID-1 (BRD4 targeting SUTAC 1-26)

[0293] Scheme 8, presents the synthesis of a SUTAC having the structure of conjugate ID-1.

[0294]

[0204] 6.2 pmol of 1-30 and 10.7 pmol of JQ1 acid were weighed and dissolved in 400 pl of di chloromethane. Then 10.7 pmol of EDC were added to the mixture while stirring on ice. LCMS indicated 20% conversion after 3 hours on ice, and the reaction was transferred to room temperature. After two hours, 10 f TFμAl o were added to stop the reaction. The sample was evaporated, dissolve in 80% acetonitrile:water followed by dilution with 0.1% TFA in water to about 30% acetonitrile. The products were separated using reverse phase HPLC using a 20%- 70% gradient. The products were lyophilized from 30% acetic acid to remove the TFA. Yield was 3.4 mg (4.3 pmol, 70%). The mass was confirmed by LCMS.

[0295] EXAMPLE 9: Synthesis of SUTACs of Formula IC-1 (BRD4-targeting SUTAC)

[0296]

[0205] Scheme 9, presents the synthesis of a SUTAC having the structure of conjugate IC-1.

[0297]

[0206] 6.2 pmol of 5-1 and 10.7 pmol of JQ1 acid were weighed and dissolved in 400 pl of di chloromethane. Then 10.7 pmol of EDC were added to the mixture while stirring on ice. LCMS indicated 20% conversion after 3 hours on ice, and the reaction was transferred to room temperature. After two hours, 10 f TμFlA o were added to stop the reaction. The sample was evaporated, dissolve in 80% acetonitrile: water followed by dilution with 0.1% TFA in water to about 30% acetonitrile. The products were separated using reverse phase HPLC using a 20%- 70% gradient. The products were lyophilized from 30% acetic acid to remove the TFA. Yield was 3.88 mg (4.9 pmol, 79%). The mass was confirmed by LCMS. EXAMPLE 10: Synthesis of compound of Formula 1-34 (PIAS4 binder)

[0298] Scheme 10, presents the synthesis of a PIAS4 binder having the structure of 1-34 (Figure 4A).

[0299]

[0207] 7.7 pmol of 1-30 were weighed and dissolved in methanol. They were mixed with 0.8 equivalent of 4-pentynal and 10 μl of 3 M sodium acetate pH = 5.2 (4 equivalents). After 5 minutes, 2 equivalents of sodium cyanoborohydride were added. LCMS indicated 10-20% conversion after 2 hours without any progress after additional 4 hours. Another 1.6 equivalents of aldehyde and another 16 equivalents of sodium cyanoborohydride were added. The product was purified by LCMS and its mass was confirmed by LCMS (0.56 mg, 16% yield).

[0300] EXAMPLE 11: Materials and Methods for Examples 12-25.

[0301]

[0208] Protein Expression and Purification

[0302]

[0209] PIAS1(123-419)

[0303]

[0210] The sequence of PIAS1 (123-419, SEQ ID NO: 2) was cloned into a modified pET41 plasmid encoding an N-terminal GST tag, a His tag followed by TEV cleavage site. The plasmid was transformed into BL21(DE3) bacteria. 5 liters of 2YT + 1 mM MgSO4 + 1% glucose + NPS (25 mM ammonium sulfate, 50 mM disodium hydrogen phosphate, 50 mM potassium dihydrogen phosphate) + 100 pg / ml kanamycin were inoculated with the bacterial and grown at 37°C to an optical density of 0.5 and induction took place overnight at 15°C using 0.2 mM IPTG.

[0304]

[0211] The cells were lysed in 200 ml of Tris 50 mM pH = 7.5, 0.5 M NaCl with added lysozyme and protease inhibitor mixture, followed by probe sonication (amplitude 55%, 5 second on, 10 seconds off, total sonication time 2 minutes, on ice). After sonication, the sample was centrifuged at 20000 rpm for 20 minutes, and the supernatant was filtered. Imidazole was added to 20 mM and the sample was loaded on a 5 ml Ni-NTA column. The column was washed with 30 ml of Tris 50 mM pH = 7.5, 0.5 M NaCl, 20 mM imidazole, followed by elution with Tris 50 mM pH = 7.5, 0.5 M NaCl, 0.5 M imidazole. The sample was cleaved with TEV protease (molar ratio 1:50) with dialysis into Tris 25 mm pH = 7.5., 50 mM NaCl. The sample was supplemented with 25 mM imidazole and passed again through an Ni-NTA column. PIAS1(123-419) retained weak binding to the column and did not elute in the flowthrough. The column was washed with buffer containing 25 mM imidazole, and then PIAS1(123-419) was eluted using 50 mM imidazole. The fractions containing PIAS1(123-419) were pooled, concentrated and injected into a Superdex 75 120 ml column equilibrated with Tris 25 mM pH = 7.5, 50 mM NaCl, 2 mM DTT. Fractions containing PIAS 1(123- 419) were combined and concentrated to 36 μM, followed by aliquoting, flash freezing in liquid nitrogen, and freezing at -80°C.

[0305]

[0212] PIAS4(126-411)

[0306]

[0213] A synthetic gene encoding for PIAS4 (126-411, SEQ ID NO: 4) was cloned into pET28 plasmid with a His-tagged yeast SUMO fusion protein (Integrated DNA Technologies) preceding the PIAS4 construct. BL21(DE3) bacteria transformed with the plasmid were grown in 2YT + 1 mM MgSO4 + 1% glucose + NPS + 100 pg / ml kanamycin, with induction at 16°C overnight with 0.1 mM IPTG. The bacteria were lysed in buffer A (HEPES 25 mM pH = 7.5, 20 mm imidazole, 0.5 M NaCl) supplemented with lysozyme and protease inhibitors, using sonication as described for PIAS1. The sample was centrifuged, and the supernatant was sonicated again and filtered 0.45 pm, followed by loading on an NiNTA column. The column was washed with buffer A, followed by a wash with buffer A + 48 mM imidazole. The protein was eluted using 300 mM imidazole. The tag was cleaved with His6-Ulpl overnight at 4°C while dialyzing against buffer A. The sample was loaded again on a Ni-NTA column. The flowthrough was collected, concentrated and loaded on a Superdex 75 120 ml column equilibrated with HEPES 25 mM pH = 7.5, 0.5 M NaCl. Fractions containing pure PIAS4( 126-411) were pooled (final concentration 38 μM), flash frozen in liquid nitrogen and stored at -80°C.

[0307]

[0214] Electrophile Screening

[0308]

[0215] The LC-MS runs for were performed on a Waters ACQUITY UPLC class H instrument, in positive ion mode using electrospray ionization. UPLC separation used a C4-BEH column (300 A, 1.7 pm, 21 mm x 100 mm). The column was held at 40 °C and the autosampler at 10 °C. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The run flow was 0.4 mL / min. The gradient used was 1% B for 2 min, increasing linearly to 80% B for 2.5 min, holding at 80% B for 0.5 min, changing to 20% B in 0.2 min, and holding at 1% for 0.8 min. The MS data were collected on a Waters SQD2 detector with an m / z range of 2-3071.98 at a range of 600-1900 m / z. The desolvation temperature was 500 °C with a flow rate of 800 L / h. The voltages used were 1.00 kV for the capillary and 24 V for the cone. MassLynx version 4.2 was used to operate the LC-MS and analyze the data. Raw data were processed using openLYNX and deconvoluted using MaxEnt with a range of 30000-36000 Da and a resolution of 1 Da / channel.

[0309]

[0216] 384-well polypropylene plates were prepared using Echo, design to contain pools of 5 compounds in each well with masses differing by at least 20 Da. Compounds were added to the plates as DMSO stocks. To initiate the screen, proteins were diluted to 1 μM in HEPES 25 mM pH = 7.5, 50 mM NaCl, and protein solution was added to the plates while the plate was on ice. The plates were mixed (1400 rpm for 6 seconds), centrifuged briefly, and kept at 4°C overnight for incubation. Reactions were quenched by addition of formic acid to 0.4%, and samples were analyzed by LC-MS. PIAS4 was incubated with the 15 μM compounds, and PIAS1 was incubated with 20 μM compounds.

[0310]

[0217] Dose response and further analysis of new compounds

[0311]

[0218] The identified hits were further characterized in a dose response experiment in which each compound was tested in a range of concentrations without being mixed with other compounds. Using Echo dispensing, different amounts of compounds were added to the plate, with DMSO added to maintain uniform DMSO concentrations in all samples. PIAS1 was added to these samples as in the screen, and labeling was tested using LCMS. Further derivatives of confirmed hits were synthesized and tested using similar protocol.

[0219] Measurement of compounds reactivity using DTNB assay

[0312]

[0220] Measurement of the intrinsic reactivity of the compounds was performed using a thiol- reactivity assay described in Resnick et al. (pubmed 31060360). The assay is performed in NaPi 25 mM pH = 7.4, 150 mM NaCl. 0.5 f 2μ0l o mM compound are added to each well in a black 384 well plate with transparent bottom. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) is added to a concentration of 50 μM and TCEP is added to a concentration of 200 μM, resulting of reduction of DTNB to the yellow-colored TNB anion. After several minutes, 50 f the solutiμoln o is added to each well. Compounds were measured in triplicates, and for each compound, in addition to the triplicate measured in the presence of TCEP and DTNB, another triplicate was measured in the presence of TCEP only to correct for intrinsic absorbance by the compounds if there is any. The plate was covered with transparent tape and incubated with shaking at 37°C inside a plate reader, in which absorbance at 492 nm was measured every 15 minutes over a period of 14 hours.

[0313]

[0221] DTNB assay data was analyzed as follows:

[0314] 1. Absorbance for each triplicate is averaged and the 95% confidence interval is calculated. The same is done for the blank (non-DTNB containing) samples.

[0315] 2. The blank is subtracted from the averaged signal.

[0316] 3. The data is normalized by dividing every data point by the data at time 0. The error from the confidence interval is propagated using standard formulas.

[0317]

[0222] When fiting the data, the actual concentration of the two species (compound and TNB) is calculated assuming the TNB is initially 100 μM and the compound is 200 μM and they react only with each other. The signal comes from TNB absorbance, so [TNB]=0.0001M*(normalized ab sorbance) [Compound]=[TNB]+0.0001 M

[0318]

[0223] The fiting is done based on the following analytical solution to the second order rate equation, which enables to perform a simple linear fit to obtain the rate constant.

[0319]

[0224] The first few data points are discarded when performing the fit due to the time it takes for the plate to equilibrate to 37 DC, and for high reactivity compound linearity is frequently lost when TNB concentration is low, so only the data from the first 4 hours is used for the fit. In cases in which compounds have low solubility in the aqueous buffer, addition of 20% acetonitrile to the buffer was performed, which a slight reduction in observed reactivity while the relative observed rates remain consistent with measurements performed in buffer.

[0225] Time course binding experiment for medicinal chemistry optimization

[0320]

[0226] Compounds were diluted 50-fold from a 100 μM stock in DMSO with 1 μM protein dissolved in HEPES 25 mM pH = 7.5, 50 mM NaCl. Incubation was performed at room temperature, and reactions were stopped at designated times by mixing the sample with an equal volume of 40% acetonitrile in water + 0.5% trifluoroacetic acid.

[0321]

[0227] Assessment of intrinsic reactivity of the electrophiles using N-acetylcysteine methyl ester

[0322]

[0228] For each compound, a 1 pl sample of 20 mM compound in DMSO was diluted to 50 pl with reaction buffer (NaPi 25 mM pH = 7.5, 50% acetonitrile). In parallel, NAC was dissolved to 100 mM in water freshly and diluted to 2 mM in reaction buffer. The solutions of compound and NAC were mixed in equal volumes to initiate the reaction, after which it proceeded at room temperature. At defined time points, 10 pl samples were mixed with 35 μl of 0.1% trifluoroacetic acid in water and injected to LC-MS.

[0323]

[0229] The LC-MS runs for the NAC assay were performed using the same instrument with a C 18- CSH column (300 A, 1.7 pm, 21 mm x 100 mm) using a gradient starting from 1% B for 1 minute, rising to 95% B in 4.5 minutes, holding at 95% B for 0.75 minutes, then decreasing to 1% B in 0.75 minutes and holding at 1% B for 1 minute. MS data were collected at a range of 80-2500 m / z, using identical conditions for ionization as with the protein.

[0324]

[0230] LC-MS / MS analysis of compound-bound PIAS4 complexes

[0325]

[0231] PIAS4 (38 μM) was incubated with either DMSO or 1.5 equivalents of compound 1-41 for 30 minutes at room temperature in HEPES 25 mM pH = 7.5, 0.5 M NaCl. After confirming full labelling of the protein by the compound by LC-MS, 40μl o f each sample was buffer exchanged using BioSpin columns into 100 mM tri ethylammonium bicarbonate. After buffer exchange, 40 pl of each sample was incubated with 1.25 pg of sequencing grade trypsin (Promega) over night at 37°C. Following trypsinization, 1 μl o f 200 mM DTT was added to each sample, followed by 30 minutes incubation at 37°C. At this point 1 μl of freshly dissolved 0.8 M iodoacetamide was added to each sample with 30-minute incubation at room temperature in the dark. At this point 40 μl of 0.2% TFA in water was added, and the sample were desalted using Oasis columns (Waters) and dried using speedvac. The samples were dissolved in 50 μl of 3% acetonitrile + 0.1% formic acid and 0.5 pl were injected to LC-MSMS.

[0326]

[0232] Samples were analyzed using EASY-nLC 1200 nano-flow UPLC system, using PeμMap RSLC C18 column (2 pm particle size, 100 A pore size, 75 pm diameter x 50 cm length), mounted using an EASY-Spray source onto an Exploris 240 mass spectrometer operated using Xcalibur version 4.4.16.14. uLC / MS-grade solvents were used for all chromatographic steps at 300 nL / min. The mobile phase was: (A) H2O + 0.1% formic acid and (B) δ0% acetonitrile + 0.1% formic acid. Peptides were eluted from the column into the mass spectrometer using the following gradient: 1- 40% B in 60 min, 40-100% B in 5 min, maintained at 100% for 20 min, 100 to 1% in 10 min, and finally 1% for 5 min. Ionization was achieved using a 2100 V spray voltage with an ion transfer tube temperature of 275 °C. Initially, data were acquired in data-dependent acquisition (DDA) mode. MSI resolution was set to 120,000 (at 200 m / z), a mass range of 375-1650 m / z, normalized AGC of 300%, and the maximum injection time was set to 20 ms. MS2 resolution was set to 15,000, quadrupole isolation 1.4 m / z, normalized AGC of 100%, and maximum injection time of 22 ms, and HCD collision energy at 30%. 3 injections of 0.5 pl were performed for each sample. The DDA data was analyzed using Fragpipe version 22.0. The database contained the sequence of the PIAS4 construct used in the study, and contaminants were included. Methionine oxidation and N terminal acetylation were variable modifications, and carbamidomethyl and the modification by compound 1-41 were set as variable modifications in the analysis, with up to 3 modifications per peptide. Digestion was defined as trypsin / P with up to 2 missed cleavages. The data was imported into skyline (version 22.2.0.351) and precursors from 16 peptides were selected for parallel reaction monitoring (PRM). In every acquisition cycle, one full MS spectrum was taken at a range of 350- 1400 Da, 300% AGC target, maximum injection time 20 ms at a resolution of 120,000. Data for each precursor was measured during a 4-5 min window around the retention time measured in the DDA run, with QI resolution of 2 Da, orbitrap resolution of 15,000, 300% AGC target and maximum injection time of 160 ms. The acquired data was then analyzed in skyline using a spectral library generated from the DDA runs. The 5 most intense product ions were used for quantitation relative to the DMSO control.

[0327]

[0233] DSF of PIAS4

[0328]

[0234] PIAS4, either WT or mutant was incubated at 20 μM with or without compound 1-41 for 10 minutes in HEPES 25 mM pH = 7.5, 150 mM NaCl, containing 1% DMSO. At this point, 19 pl of sample were mixed with 1 f SYμPl R o O Orange (Sigma) X100 in HEPES 25 mM pH = 7.5, 50 mM NaCl (final dilution of SYPRO Orange X5). Samples were measured in triplicates in StepOne Plus instrument, using FAM as the target and ROX as passive reference.

[0329]

[0235] Crystallography of PIAS4(126-411) PROSS mutant with compound 1-41

[0330]

[0236] The PROSS mutant of PIAS4( 126-411) (SEQ ID NO: 5) was expressed and purified using the same protocol as the wild type protein and concentrated to 724 μM prior to freezing. Crystallographic screens were performed on the free protein first. The protein was desalted using Biospin (Bio-Rad) into HEPES 25 mM pH = 7.5, 100 mM NaCl. Crystallization was performed via the sitting drop method using 96-well iQ plates, and Salt-Rx, PEG-Rx and PEG-Ion screens from Hampton. 150 nL protein and 100 nL buffer were placed in each spot and incubated at 20°C. After a week, small crystals appeared in several conditions, of which 1.3 M Ammonium tartrate dibasic, 0.1 M BIS-TRIS propane pH 7.0 was selected for preparation of seeds. The crystals were crushed, and vortex with stainless steel beads, and serially diluted in 1.4 M Ammonium tartrate dibasic, 0.1 M Tris pH 8.5.

[0331]

[0237] The crystallize the complex with compound 1-41, 90 pl protein was mixed with 2 μl of 50 mM compound in DMSO (1.5 equilvalents). LCMS confirmed full labelling after 20 minutes. The sample was centrifuged and the supernatant was desalted into HEPES 25 mM pH = 7.5, 100 mM NaCl. The sample was screened using the Salt-Rx screen by mixing 150 nl complex with 50 nl seed stock and 100 nl buffer. After 3 days a large crystal appeared in 4.0 M Sodium nitrate, Tris 0.1 M pH = 8.5. The crystal was picked up, dipped in cryo-oil, and measured.

[0332]

[0238] Gel-based characterization of proteomic selectivity of molecule 1-34

[0333]

[0239] Daudi cells were treated with 1-34 (6 hours) with various preincubations (2 hours). The cells were lysed in 100μl o f RIP A + protease inhibitors and centrifuged, and the concentration of the protein was measured by BCA. After this, the lysates were diluted to 2 mg / ml with RIPA and click reaction was performed for 50 pl samples with the following additions:

[0334] - 1.8μl o f 0.1 M CuSO4 / THPTA

[0335] - 1 μl o f 5 mM TAMRA-azide

[0336] - 1.5μl o f 150 mM sodium ascorbate, dissolved fresh in water

[0337]

[0240] All samples were precipitated with methanol chloroform (150 pl water, 200 pl methanol and 50 pl chloroform, spin, remove top, repeat with 200 pl methanol twice), then dissolved in 120 μl of IX LDS buffer with 5 mM DTT, heated to 70°C for 10 minutes, and 50 pl were loaded on the gel. The gels were run, fixed with 45% methanol, 45% water, 10% acetic acid, and imaged on Typhoon.

[0338]

[0241] Cell lines

[0339]

[0242] Daudi, OCI-AML2, Ramos, RPMI-8226 cells were grown in RPMI medium supplemented with 10% FBS (GIBCO), 2 mM glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen) and 1% Sodium Pyruvate (Invitrogen). LNCaP cells were grown in RPMI medium supplemented with 10% FBS (GIBCO), 2 mM glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen), 1% Sodium Pyruvate (Invitrogen). WT, PIAS1 KO and PIAS1 / 4 DKO TK6-cells (Mohiuddin, M. et al. SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines. Proc Natl Acad Sci U S A 115, 2018) were grown in RPMI medium supplemented with 5% Horse Serum (Hl 138 Sigma-Aldrich), 2 mM glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen) and 1% Sodium Pyruvate (Invitrogen). 293T, A549, cells were grown in DMEM medium supplemented with 10% FBS (GIBCO), 2 mM glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen) and 1% Sodium Pyruvate (Invitrogen). All cell lines were grown at 37°C with 5% CO2 and were routinely tested for mycoplasma contamination.

[0243] Plasmids overexpression

[0340]

[0244] 293T cells were transfected with either pCMV-mCherry or pCMV-FLAG-hAR (Plasmid addgene #89080) using JetPEI (PolyPlus) according to the manufacturer’s instructions.

[0341]

[0245] Characterization of the proteomic selectivity of molecule 1-34

[0342]

[0246] The following cell samples were prepared in triplicates (40 million cells in each sample): -3 replicates: DMSO (2 hours) pre-incub ati on and then DMSO (6 hours), labelled (1,2,3).

[0343] -3 replicates: DMSO (2 hours) pre-incub ati on and then IμM 1-34 (6 hours), labelled (4,5,6).

[0344] -3 replicates: 10 μM 1-12 (2 hours) pre-incubation and then IμM 1-34 (6 hours), labelled (7,8,9).

[0345]

[0247] The cells were lysed in RIPA and tested for BCA, giving 8-10 mg / ml in the lysate concentrations. The lysates were reacted with a biotin-containing peptide that is trypsin cleavable (Biotin-GGGGGGRK(az)-NH2). Samples were diluted to 2 mg / ml in RIPA in 15 ml polypropylene tube, in a total volume of 250 pl. To each sample was added 1 f 40 mM peptiμdle o azide, 2.5 pl of CuSO4:THPTA 100 mM, and 2.5 fμ sol d oium ascorbate 200 mM freshly dissolved. The reaction proceeded for 1 hour in the dark at room temperature, followed by methanol chloroform precipitation as follows: 750 pl water, 1 ml methanol and 250 f chloroform wμerle o added, followed by vigorous mixing and centrifugation for 10 minutes at 3200xg. The top layer was aspirated and 1 ml of methanol was added and the sample was mixed and centrifuged. The supernatant was removed, and the samples were left to dry and stored at -80°C.

[0346]

[0248] Samples were dispersed in 180 f2.5%μ SlD oS inPBS with sonication (16 seconds, 2 second pulses). The samples were diluted X20 with PBS and incubated with PBS-washed streptavidin beads (from cytiva - 20 pl beads per samples) at room temperature with tumbling for 3 hours. The beads were then transferred to spin columns and washed as follows:

[0347] * 3 times with 300 fμ 1l% o SDS / PBS. Before draining the third wash, 3 f 1 M DTT weμrel o added and the beads were incubated in this solution for 40 minutes room temperature with occasional mixing, then 15 fμ flre osh 0.8M iodoacetamide were added followed by 30 minutes room temperature incubation with occasional mixing. At this point the solution was drained and washing was continued.

[0348] * 3 times with 350 fμ frle oshly dissolved 6 M urea in PBS.

[0349] * 3 times with 400 fμ 2l0 o% methanol in PBS.

[0350] * 1 time with 400 pl PBS.

[0351] * 2 times with 400 pl water.

[0352]

[0249] At this point the beads were transferred to eppendorfs with two 50 pl portions of 50 mM TEAB, and 2 μlf o 0.5 pg / pl trypsin were added to each sample, followed by incubation at 37°C for 6 hours with 1150 rpm shaking. The beads were centrifuged, the supernatant was aspirated (90 pl), and the beads were further washed with 100 pl 2M NaCl in 50 mM TEAB, which was combined with the first 90 pl supernatant. The combined supernatant was mixed with 190 f 0.2% TFA in μl o water, followed by desalting using Oasis columns (Waters) according to the manufacturer’s protocols. The eluted samples were evaporated under vacuum.

[0353]

[0250] The dry peptides were dissolved in 3% acetonitrile + 0.1% formic acid (25 pl) and 2 pl were injected. Samples were analyzed using EASY-nLC 1200 nano-flow UPLC system, using PeμMap RSLC Cl 8 column (2 pm particle size, 100 A pore size, 75 pm diameter x 50 cm length), mounted using an EASY-Spray source onto an Exploris 240 mass spectrometer. uLC / MS-grade solvents were used for all chromatographic steps at 300 nL / min. The mobile phase was: (A) H2O + 0.1% formic acid and (B) δ0% acetonitrile + 0.1% formic acid. Peptides were eluted from the column into the mass spectrometer using the following gradient: 1-40% B in 160 min, 40-100% B in 5 min, maintained at 100% for 20 min, 100 to 1% in 10 min, and finally 1% for 5 min. Ionization was achieved using a 2100 V spray voltage with an ion transfer tube temperature of 275 °C. Initially, data were acquired in data-dependent acquisition (DDA) mode. MSI resolution was set to 120,000 (at 200 m / z), a mass range of 375-1650 m / z, normalized AGC of 300%, and the maximum injection time was set to 20 ms. MS2 resolution was set to 15,000, quadrupole isolation 1.4 m / z, normalized AGC of 50%, automatic maximum injection time, and HCD collision energy at 30%. 3 samples were analyzed per condition.

[0354]

[0251] Data analysis was performed using Fragpipe (version 19.1) using Msfragger search engine (version 3.8), lonQuant 1.8.10 and Philosopher 4.8.1. Analysis was performed using a human proteome database from December 2022 (Uniprot) with contaminants added and with Streptavidin added manually as a contaminant. Msfragger analysis was performed using Trypsin as the enzyme that cuts after Arg and Lys, with up to 2 missed cleavages, peptide length 7-50 and the N terminal methionine removed. N terminal acetylation and methionine oxidation were defined as variable modifications and carbamidomethyl was defined as a fixed modification. False discovery rate of 0.01 was used both at the peptide and the protein level. Label -Free Quantification was performed using lonQuant with 1 minimum peptide, with Match Between Runs was enabled with a tolerance of 1 minute. After analysis, the combined protein file was analyzed using Perseus.

[0355]

[0252] Intensities were converted to Log2 values. One sample from the dataset with preincubation of 1-12 was contaminated with SDS and removed from the analysis. The triplicates of the first two sets (DMSO treated; 1-34 treated) and the duplicate of the last set (1-34 treated with 1-12 preincubation) were grouped, and all proteins for which there were at least 2 valid values in one of the groups were kept in the analysis. Missing values were replaced by imputation from a normal distribution (downshift 2.7, width 0.3), and differences and P-values were calculated using student’s t-test for two pairs of groups: 1-34-treated cells compared to DMSO-treated cells; and 1-34-treated cells compared to 1-34-treated cells with 1-12 preincubation. The results were filtered based on both tests and selected proteins that gave significant differences for both of them (proteins that were effectively pulled down by 1-34 but competed effectively by 1-12).

[0356]

[0253] Characterization of the proteomic selectivity of compound 1-43

[0357]

[0254] Daudi cells (20 million per sample) were incubated with 1 μM of compound 1-43 (or DMSO) for 2 hours, followed by incubation with 0.1 μM of compound 1-42 (or DMSO) for 1 hour. Four replicates were measured for each sample. After incubation, the cells were washed with PBS, harvested, and lysed in 200 pl RIPA buffer (Sigma) supplemented with protease inhibitors, by dispersing the cells in buffer and incubating the samples on ice for 15 minutes with occasional vortexing. The samples were centrifuged at 21,000 x g for 10 minutes at 4°C, and the protein concentration in the supernatant was estimated using BCA assay. At this point, a sample of 200 pl / 2.5 mg / ml protein was prepared from each replicate by dilution with RIPA buffer.

[0358]

[0255] For each sample, a preclick mix containing 0.5 f 50 mMμ blio otin azide in DMSO, 4.5 pl DMSO, 22.5 pl RIPA buffer and 12.5 f aμqlu oeous 100 mM THPTA: 20 mM CuSO4 was added (total volume 40 pl). Then, 10 f freμslhl oy dissolved 200 mM sodium ascorbate was added and the sample was vortexed and incubated 1.5 hours at room temperature in the dark. Following the reaction, each sample was precipitated by adding 750 pl water, 1 ml methanol and 250 pl chloroform, followed by strong vortexing and centrifugation (3200 x g, 10 minutes, 4°C). The upper layer was aspirated, and the pellet was washed twice with cold 1 ml methanol and air dried.

[0359]

[0256] The pellets were dispersed in 250 f 2.5%μl S oDS in PBS, followed by sonication (20%, 8 pulses of 2 seconds with 2 second interval). The samples were then diluted 20-fold with PBS, and 10 μl o f streptavidin beads (cytiva) were added. The samples were tumbled for 3 hours at room temperature and processed using small spin columns in a vacuum manifold. The beads were washed 3 times with 1% SDS in PBS (400 pl), and then 400 pl 1% SDS in PBS was added, and 4 pl of 1 M DTT was added with intermittent mixing. After 30 minutes, 25 f freshlyμl d oissolved 0.8M iodoacetamide was added, with 30 minutes further incubation in the dark. Then the buffer was pumped out, and the samples were washed as follows: 3 times with fresh 6 M urea in PBS, 4 times with 20% methanol in PBS, 2 times with PBS and 2 times with water.

[0360]

[0257] Beads were then transferred to eppendorf tubes using 2 portions of 100 f 100 mM TEAB, μl o and 0.5 pg trypsin was added, followed by incubation at 37°C overnight with shaking (1300 rpm). The samples were then centrifuged, the supernatant was removed, and the beads were washed once with TEAB, and once with TEAB + 2 M NaCl. Washes were combined with the supernatant, and then TFA was added to 0.1%, and the samples were desalted using Oasis desalting columns (Waters). They were dissolved in 3% ACN + 0.1% formic acid and injected (5 pl) to the LCMSMS instrument.

[0258] Samples were analyzed using EASY-nLC 1200 nano-flow UPLC system, using PeμMap RSLC Cl 8 column (2 pm particle size, 100 A pore size, 75 pm diameter x 50 cm length), mounted using an EASY-Spray source onto an Exploris 240 mass spectrometer. C / MS-gradeμ sLolvents were used for all chromatographic steps at 300 nL / min. The mobile phase was: (A) 1420 + 0.1% formic acid and (B) δ0% acetonitrile + 0.1% formic acid. Peptides were eluted from the column into the mass spectrometer using the following gradient: 1 40% B in 160 min, 40-100% B in 5 min, maintained at 100% for 20 min, 100 to 1% in 10 min, and finally 1% for 5 min. Ionization was achieved using a 2100 V spray voltage with an ion transfer tube temperature of 275 °C. Initially, data were acquired in data-independent acquisition (DIA) mode. MSI resolution was set to 60,000 (at 200 m / z), a mass range of 370-1450 m / z, normalized AGC of 300%, and the maximum injection time was set to 20 ms. MS2 resolution was set to 30,000, with 31 isolation windows of 19 Da with a 1 Da overlap, maximum injection time of 50 ms, and HCD collision energy at 27%. Four samples were analyzed per condition.

[0361]

[0259] Data analysis was performed using Fragpipe (version 22.0) using Msfragger search engine (version 4.1, PMID 28394336), and DIANN 1.9 (PMID 31768060). Analysis was performed using a human proteome database from December 2022 (Uniprot) with contaminants added and with Streptavidin added manually as a contaminant. Msfragger analysis was performed using Trypsin as the enzyme that cuts after Arg and Lys, with up to 2 missed cleavages, peptide length 7-50 and the N terminal methionine removed. N terminal acetylation and methionine oxidation were defined as variable modifications and carbamidomethyl was defined as a fixed modification. False discovery rate of 0.01 was used both at the peptide and the protein level. Label-Free Quantification was performed using DIANN. After analysis, the quantified protein file was analyzed using Perseus (PMID 27348712). Intensities were converted to Log2 values, the quadruplicates of each type were grouped, and missing values were replaced by 13. Differences and P-values were calculated using double-sided student’s t-test. -LogP value of 2 and a difference value of 1 (2 - fold) were used as limits for significance.

[0362]

[0260] In vitro SUMOylation assay of BRD4 by 1-26 SUTAC

[0363]

[0261] Recombinant full-length MYC-DDK-PIAS4 (TP306748, Origene) was pre-incubated with 3.5 μM 1-26 SUTAC or DMSO for 4 hours at 25°C. After which, they were incubated with the following recombinant proteins: 10 μM SUMO1 (UL-712, Boston Biochem) , 10 μM SUMO2 (UL- 752, Boston Biochem), 1 μM SAE1 / SAE2 (E315 Boston Biochem), ± 1 μMUBC9 (E2-645 Boston Biochem), ± 350 nM BRD4 (SP-600, R&D systems), in a reaction buffer composed of 50 mM HEPES, 4 mM ATP, 15 mM creatine phosphate, 0.7 p / ml creatine phosphate kinase, 10 mMMgC12, 10 mM MgOAC, 50 mM KOAc, and 0.5 mM DTT. This mixture was then allowed to incubate for 60 minutes at 30°C, after which they were subsequently subjected to western blot analysis.

[0364]

[0262] In vitro SUMOylation assay of BRD4 by ID-2

[0365]

[0263] Recombinant full-length MYC-DDK-PIAS4 (TP306748, Origene) at 200 nM was preincubated with 200 nM ID-2 or DMSO for 90 minutes at 25°C. After which, they were incubated with the following recombinant proteins: 4 μM SUMO1 (UL-712, Boston Biochem) , 4 μM SUMO2 (UL-752, Boston Biochem), 0.5 μM SAE1 / SAE2 (E315 Boston Biochem), ± 0.2 μM UBC9 (E2- 645 Boston Biochem), ± 200 nM BRD4 (SP-600, R&D systems), in a reaction buffer composed of 50 mM HEPES, 4 mM ATP, 15 mM creatine phosphate, 0.7 p / ml creatine phosphate kinase, 10 mM MgC12, 10 mM MgOAC, 50 mM KOAc, and 0.5 mM DTT. This mixture was then allowed to incubate for 60 minutes at 30°C, after which they were subsequently subjected to western blot analysis.

[0366]

[0264] Denaturating lysis buffer for whole cell lysates

[0367]

[0265] Cells were harvested, washed and counted. Equal numbers of cells were taken for lysis. 5x lysis buffer (2% SDS, 50 mM Tris pH7.5, 10 mM NaCl, Protease Inhibitor Cocktail 1 :250 (Merck), 20 mM NEM(Merck)) was used to lyse the cells. Lysates were then incubated on ice for 10 minutes, followed by the addition of 4 volumes of PBS+ / + supplemented with Protease Inhibitor Cocktail, 20 mM NEM, and 1 pl in 1000 ml of Benzonase (71205-3, Merck). Lysates were then incubated on ice for 5 minutes, followed by an incubation at 30°C for 10 minutes. Finally, lysates were centrifuged (15000rcf, 15 minutes, 4°C) and the supernatant was collected for further analysis.

[0368]

[0266] Chromatin fractionation

[0369]

[0267] Cells were harvested, washed, and counted. Equal numbers of cells were resuspended in lysis buffer (10 mM HEPES pH 7.4, 10 mM KC1, 0.05% NP-40) and incubated on ice for 20 minutes. The lysates were then centrifuged at 14,000 RPM for 10 minutes at 4°C, separating the cytoplasmic and nucleoplasmic fractions. The pellet was resuspended in Low-Salt Buffer (10 mM Tris-HCl pH 7.4, 0.2 mM MgCL, 1% Triton X-100) and incubated on ice for 15 minutes. After centrifugation under the same conditions, the chromatin and nucleoplasmic fractions were separated. The chromatin-containing pellet was resuspended in 0.2 N HC1, incubated on ice for 30 minutes, and centrifuged. The resulting supernatant was collected and neutralized by mixing 1 : 1 with 1 M Tris-HCl pH 8.0. All buffers were supplemented with 25 mM NEM, a protease inhibitor cocktail (Sigma-Aldrich), and 1 mM PMSF.

[0370]

[0268] Active extract

[0371]

[0269] Cells were collected, rinsed, and then subjected to lysis using a swelling buffer containing 25 mM HEPES (pH 7.5), 1.5 mM MgC12, 5 mM KC1, 1 mM DTT, and protease inhibitors cocktail. The lysates were further homogenized through a freeze-thaw process and passed through a needle. To remove impurities, the extracts underwent consecutive centrifugation steps, with the first at 5,000 rpm for 5 minutes and the second at 14,000 rpm for 60 minutes. A portion of the resulting extract (20 μL, 25 mg / mL) was combined with an energy mix comprising 150 mM creatine phosphate, 20 mM ATP, 2 mM EGTA, and 20 mM MgC12 (pH 7.6). 1-5 μM SUMO 1 -aldehyde and SUMO2- aldehyde (R&D Systems) were included in some downstream assays.

[0372]

[0270] Immunoprecipitation

[0373]

[0271] Flag magnetic beads (M2, Sigma) were utilized for immunoprecipitation. Lysates were subjected to a 2 hour incubation at 4°C while rotating with these magnetic beads. Following this incubation, the beads were used to remove unbound proteins, and subsequently, they were incubated with an elution buffer consisting of 150 mM Tris-HCl (pH 7.4), 1.5 mM MgC12, 150 mM NaCl, and 0.5 mg / ml X3flag peptide (Sigma). All buffers were supplemented with 25 mM of NEM, 1 :250 protease inhibitor cocktail, and 1 mM PMSF. The samples were subsequently subjected to analysis by western blot, as described in further detail below.

[0374]

[0272] Western blotting

[0375]

[0273] The protein concentration was assessed using the BCA Kit (ThermoScientific). Proteins were separated by SDS-PAGE on a 4-15% Criterion™ TGX Stain-Free gels and transferred onto nitrocellulose membranes using an iBlot 2 Gel Transfer Device (Thermo Fisher Scientific). The membranes were blocked in 5% milk prepared in TBS-0.1% Tween and incubated with primary antibodies overnight at 4C: anti-SUMOl Cell Signaling Technology Cat# 4930S (1: 1000), anti- UBC9 Cell Signaling Technology Cat#4918S (1: 1000), anti-Ubiquitinated proteins (FK2) Sigma- Aldrich #04-263 (1:1000), anti-Ubiquitin (P4D1) Cell Signaling Technology #3936 (1:1000), anti- BRD4 Cell Signaling Technology #13440 (1: 1000), anti-BRD4 abeam abl28874 (1: 1000), anti- H3K9me3 abeam ab8898 (1 :1000), anti -PI AS I abeam ab32219 ( 1 : 1000), anti-PIAS2 abeam ab 155556 ( 1 : 1000), anti-PIAS3 Cell Signaling Technology Cat#4164, anti-PIAS4 Cell Signaling Technology Cat#4392, anti-P300 anti-cMYC Cell Signaling Technology #9402 (1 :1000), anti-AR Cell Signaling Technology #5153 (1: 1000), anti-P300 C20 Santa Cruz SC-585 (1000), anti-Actin Cell Signaling Technology Cat#4970, anti-HSP90 abeam abl3495(l:20000), anti-Vinculin (E1E9V) Cell Signaling Technology #13901 (1:1000), anti-gamma Tubulin (GTU-88) abeam abl l316 (1:10000), anti antiH2B abeam abl790. For the secondary antibody detection, peroxideconjugated AffiniPure goat anti-rabbit (at a 1:5,000 dilution, product number 111-035-003 from Jackson ImmunoResearch), goat anti-mouse (1:5,000, Jackson ImmunoResearch), and donkey antisheep (1 : 5,000, Jackson ImmunoResearch) were utilized. Finally, the blots were examined using the SuperSignal West Pico Chemiluminescent substrate from ThermoScientific and the ChemiDoc XRS+ system from BIO-RAD.

[0274] Proliferation assay

[0376]

[0275] To evaluate the impact of the compounds on cell proliferation, the Cell Titer Gio (Promega, #G7572) was used according to the manufacturer instructions. Briefly, cells were plated at a density of -5,000 cells with the compounds in 100 pl in 96-well tissue culture plates. After a 72-hour incubation period, 100 fμ relc oonstituted Cell-Titer-Glo reagent was added to each well, and the luminescence was measured using a plate reader. The relative cell growth was determined by comparing the assay readings of the treated cells with those of control cells treated with DMSO.

[0377] EXAMPLE 12: Electrophile screen and initial dose response against PIAS1

[0378]

[0276] Goal: identify candidate molecules that bind to a SUMO-specific E3 ligase.

[0379]

[0277] An in-house electrophile-fragment screen (Resnick, E. et al. Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening. J Am Chem Soc 141, 2019) was utilized to screen a recombinant domain of the human protein PIAS1 (sequence 123-419) (Figure IB). The screen performed at 20 μM pooled molecules was analyzed using OpenLynx and the output was parsed to reveal the initial hits. Some of the molecules revealed by the screen are frequent hitters in the screens of other proteins as well and were filtered out. Following this step, six candidate hits remained. As secondary screen, a dose-response measurement of the protein with the candidates (overnight at 4°C) was performed. Three of the molecules did not label the protein at all, leaving three confirmed hits from two distinct scaffolds (Figure 1C) - molecule 1 was based on a thiophene scaffold with a proximal amide group, and molecules 2 and 3 are based on a phenyl group with a para-positioned electron-rich group relative to the chloroacetamide. Inspection of data from the original screen revealed some structure-activity relationship (SAR), although the quantitative value of this data is limited due to the pooling of compounds.

[0380]

[0278] To optimize the binding potency, modified versions of the three hits were created. In parallel to performing dose-response experiments on the derivatives, for a subset of molecules the intrinsic thiol reactivity of the compounds was also measured to reveal how intrinsic reactivity contributes to their binding to the protein. The derivatives of molecules 2 and 3 were prepared with modifications to the bulky substituent on the para position and other modifications on the phenyl ring (Figure 2). Several substitutions on the ring enhanced the binding, especially for molecules 3- 3, 3-5 and 3-7, but this was accompanied by enhanced intrinsic reactivity, particularly for the meta position, indicating these alterations mainly improved binding by enhancing the reactivity of the electrophile and not through better recognition. The only change made on the para substituent resulting in slightly better potency was the addition of a methyl group to the difluoro group on the sulfur, but this is also probably due to slightly enhanced reactivity.

[0279] The thiophene scaffold of molecule 1 was tested with several derivatizations. First was the amide substituent on the ring. Several amides were tested and found that the molecule tolerates a relatively large range of amide substituents, but does not tolerate secondary amides (Figure 3A). This result is also in agreement with the observation from the initial screen, that molecules similar to molecule 1 but with an ester group instead of an amide showed poor binding to PIAS1. Various modifications to the ring structure on molecule 1 were also tested (Figure 3B). The most potent derivatives contain a fused cyclohexane or phenyl ring to the thiophene, with relatively restricted directions for expansion as seen by the differential effects of chlorine substitutions on the rings (molecules 1-12 through 1-19). Replacing the chloroacetamide warhead with sulfamate acetamide warheads (Lu, J. et al. Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4. Chem Biol 22, 2015) was also tested.

[0381] EXAMPLE 13: Characterization of cellular targets of molecule 1-12

[0382]

[0280] Goal: verify PIAS 1 as a bona fide cellular target of the molecules.

[0383]

[0281] The most potent binder, compound 1-12, was selected and an alkyne-modified derivative, compound 1-34 (Figure 4A) was prepared. The molecule displayed potent binding of PIAS 1 in vitro but also exhibited high intrinsic reactivity, a property observed for several other 1-12 derivatives with positively charged chains. When cells were incubated with 1-34, and labeled the lysates with TAMRA azide using copper-catalyzed azide-alkyne coupling (CuAAC), an extensive labelling of many proteins was observed (Figure 4B), almost all of which were not outcompeted by the less promiscuous 1-12. For the proteomics experiments, cells were incubated with 1-34 and CuAAC was used to attach biotin to the labeled proteins, followed by pull-down, trypsinization and LC-MS / MS. Targets labeled by 1-34 were identified relative to DMSO-treated control, and targets labeled by 1- 12 were identified by preincubating the lysates prior to incubation with 1-34 and comparing the obtained proteins. As expected, 1-12 was far more selective than 1-34. PIAS1 was identified only through a single peptide and was possibly pulled down and competed but not with high statistical significance, possibly due to its low abundance. Yet, two other homologs, PIAS2 and PIAS4, were identified more robustly and were clearly bound by 1-34 and competed effectively by 1-12 (Figure 4C). Importantly, preliminary LC-MS / MS uncovered that the 1-5 PIAS binder covalently binds Cys315 of PIAS 1 (data not shown), which is conserved across the PIAS family (PIAS1 / 2 / 3 / 4) and may underlie the non-selective labelling of the PIAS family.

[0384] EXAMPLE 14: BRD4-targeting SUTAC reduces the levels of BRD4 and its downstream target c-MYC

[0385]

[0282] Goal: test the feasibility of the SUTAC modality in modifying and altering a POI.

[0283] The transcriptional activator BRD4 was chosen as a first target. This protein is critical for the expression of c-MYC-driven oncogenic programs and the proliferation of various cancer types including acute myeloid leukemia (AML) and Burkitt’s lymphoma (BL). Further, BRD4 is widely used as a model substrate for PROTAC development as it has potent small molecule binders (e.g., the BET inhibitor JQ1) and it was hypothesized that SUMOylation of BRD4 by SUTAC may disrupt its binding to the chromatin and inhibit its activity. Conjugates of the PIAS binder 1 with +JQ1, connected by linkers with either 2 or 5 polyethylene glycol (PEG) groups, were prepared, thus forming 1-24 (IA-7) and 1-25 BRD4-targeting SUTACs, respectively (Figure 5A). Invariably, the conjugation reduced the binding potency of the molecules to PIAS1. Nevertheless, 20 hours of treatment of the B cell lymphoma cell line Daudi, with 1-24 but not 1-25 BRD4-targeting SUTACs, resulted in a significant reduction in the levels of BRD4 (Figure 5B).

[0386]

[0284] This effect was achieved at 100 nM and coupled with a reduction in c-MYC levels. Importantly, the conjugation between the building blocks of SUTAC, namely JQ1 and the PEG- modified PIAS binder 1.5 (Figure 5C), was essential for mediating its effects and the 100-fold improvement in lowering c-MYC levels compared to free JQ1 (Figure 5D). Collectively, these results support the proximity -inducing mechanism of action of SUTAC.

[0387]

[0285] To examine whether the effects of BRD4-targeting SUTAC are indeed facilitated by SUMOylation machinery, the same reaction was performed with pre-incubation of the SUMO E2 conjugating enzyme inhibitor, 2D08. It was found that SUMOylation inhibition partly cancels the reduction in BRD4 levels induced by SUTAC (Figure 6A). Furthermore, in cells where both PIAS1 and PIAS4 were double knocked out (DKO), but not in cells where only PIAS1 was knocked out, BRD4 levels were less effectively reduced (Figure 6B). This observation aligns with the fact that the identified PIAS1 binders interact not only with PIAS1 but also with PIAS2 and PIAS4 (Figures 4A-4C)

[0388] EXAMPLE 15: SUTAC modifies recombinant BRD4 only under SUMOylation-promoting conditions

[0389]

[0286] Goal: test the ability of 1-24 (IA-7) BRD4-targeting SUTAC to induce SUMOylation on a recombinant BRD4 (short form: (Glu49-Glu460)-FLAG-BRD4) under controlled cell-free conditions.

[0390]

[0287] Remarkably, only under SUMOylation-preserving conditions (supplementation of SUMO- aldehyde), the SUTAC treatment induced a high molecular weight BRD4 band, indicating that BRD4 was SUMOylated (Figure 7). Notably, the anti-SUMOl antibody used failed to detect it, most likely due to the inefficiency of anti-SUMO antibodies to detect all SUMOylated proteins (Garvin, A. J., Lanz, A. J. & Morris, J. R. SUMO monoclonal antibodies vary in sensitivity, specificity, and ability to detect types of SUMO conjugate. Sci Rep 12, 2022).

[0391] EXAMPLE 16: Optimization of the PIAS binder enhances the effects of SUTACs against

[0392] BRD4

[0393]

[0288] Goal: test whether the enhanced binders 1-9 and 1-12 (Figures 3A-3C) can result in enhancing the effects of BRD4-targeting SUTAC.

[0394]

[0289] The conjugation of JQ1 to binders 1-9 and 1-12, to form 1-26 and 1-27 SUTACs, respectively, significantly reduced the binding potency of the SUTACs to a recombinant PIAS1 (Figure 8A). The reductions in the levels of BRD4 and cMYC following a 20-hours treatment of Daudi cells with the 1-26 and 1-27 SUTACs were stronger than the original 1-24 SUTAC (Figure 8B) Of note, the reduction in BRD4 levels was readily seen with a 20 nM concentration of 1-26.

[0395] EXAMPLE 17: BRD4-targeting SUTACs suppress the growth of hematological cancer cells in vitro

[0396]

[0290] The significant decrease in c-MYC levels resulting from treatment with SUTACs compared to JQ1, suggests that SUTACs may elicit a high degree of killing of lymphoma cells. SUTAC variants demonstrated a more substantial inhibition (up to 120-fold) of the proliferation of Daudi cells compared to JQ1, upon 72 hours of treatment (Figure 9A). Consistently, these effects were also more pronounced than those observed with JQ1 in the OCI-AML2 AML cell line (Figure 9B) with IC50 values of 3.8-10 nM. Importantly, treatment with the binder alone was considerably less toxic than SUTAC (Figures 9B-9C).

[0397]

[0291] The BRD4-targeting SUTAC was able to alter the ability of BRD4 to activate a c-MYC- dependent pro-malignant program in hematological cancer cells, leading to cell death.

[0398] EXAMPLE 18: AR-targeting SUTACs lowered AR protein levels in prostate cancer cells

[0399]

[0292] In order to establish SUTAC as a modality that is broadly applicable against other protein targets, Androgen receptor (AR)-targeting SUTACs were generated. SUTACs were utilized to induce the SUMOylation of AR, aiming to mitigate its hyperactivity that is associated with promoting cancer progression. To that end, AR-targeting SUTAC molecules based on 1 PIAS binder were generated (Figure 10A). The conjugation to AR binder significantly inhibited the binding to PIAS1, as no binding to PIAS1 was observed at 20 μM and could not detect a reaction with the DTNB compound, even at 200 μM. The activity of this SUTAC was assessed as it may still operate by other PIAS proteins. The AR-targeting SUTAC treatment was able to lower the protein levels of AR in the LNCaP prostate cancer cell line (Figure 10B) as well as the levels of overexpressed flag- tagged AR (Figure IOC).

[0400]

[0293] The AR-targeting SUTAC promoted AR degradation in prostate cancer cells, which demonstrates the broad utility and versatility of the SUTAC approach.

[0401] EXAMPLE 19: SUMOylation of recombinant BRD4 is enhanced by ID-1 (1-26) SUTAC and drives chromatin eviction of BRD4

[0402]

[0294] Goal: further validatation that ID-1 SUTAC could enhance the SUMOylation of a recombinant BRD4 by PIAS4 in a purified reaction.

[0403]

[0295] PIAS4 or vehicle were pre-incubated with ID-1 SUTAC or DMSO control. After which, they were incubated with SUM01,2 and SUM0-E1 (SAE1 / 2), with or without SUM0-E2 enzyme (UBC9), with or without recombinant BRD4. The samples were then subjected to western blot. It was found that BRD4 was SUMOylated by ID-1 SUTAC only when all required SUMOylation enzymes were present (Figure 11 A).

[0404]

[0296] When examined how ID-1 SUTAC alters BRD4 and cMYC in 0CI-AML2 cells, which exhibited a pronounced growth attenuation when treated with ID-1 SUTAC for 72 hours, it was found that while cMYC levels were reduced, BRD4 levels were not significantly altered. This suggested that cMYC downregulation may be driven by the dissociation of BRD4 from the chromatin. Indeed, the ID-ltreatment resulted in increased cytoplasmic levels of BRD4, coupled with a reduction in its chromatin levels (Figure 11B and Figure 11C). This was different than how a canonical PROTAC against BRD4 worked (e.g., MZ1), altogether highlighting a distinct mechanism of action from PROTACs.

[0405] EXAMPLE 20: Electrophile screens to discover binders for PIAS4 and PIAS1 and Medicinal chemistry campaign for binder optimization

[0406]

[0297] Goal: identify general binders for the PIAS family proteins.

[0407]

[0298] Two PIAS isoforms, PIAS1 and PIAS4, were screened against a library of -1500 chloroacetamides. Liquid chromatography-mass spectrometry (LC-MS) was used to estimate binding (Figure 12). In general, PIAS4 was considerably more reactive than PIAS1 and displayed a much higher hit rate. Nevertheless, a small group of compounds showed extensive labeling of both proteins. The vast majority of these hits were aminothiazole-based chloroacetamides, which are known from previous screens to be intrinsically reactive compounds that label most proteins. However, a large group of thiophene-based chloroacetamide fragments emerged as hits. The thiophene compounds were divided into three classes based on the substituents adjacent to the chloroacetamide: amides, esters and nitriles. While nitriles and esters labeled primarily PIAS4, amide-containing thiophenes showed significant binding to both isoforms. Therefore the amide-containing thiophene scaffold was selected, starting from compound 1.

[0408]

[0299] Due to the higher reactivity of PIAS4, an optimization campaign was performed using this protein. The optimization campaign proceeded in 2 stages:

[0409]

[0300] A. Expansion of the aromatic system around the thiophene: the ring system was expanded and substituents added at various positions (Figure 13A). Attempting several substituents led to compound 1-12, which bound PIAS4 10-fold more potently than compound 1. Then chlorine substituents were systematically added on the fused phenyl of compound 1-12 (compounds 1- 16- 1-19) showing only one permissible position (compound 1-18). Replacement of the chloring with a methyl group (compound 1-40) resulted in further improvement.

[0410]

[0301] B. Expansion from the amide - first, it was tested whether the amide position can be modified. The corresponding acid was almost unreactive, while methylation, but no dimethylation of the amide, was permissible. Therefore, it was reasoned that a large variety of primary amines can be coupled to compound 1-36 to yield active binders (Figure 13A). A high throughput experiment was set up, in which 1-36 was coupled to a library of primary amines at pl-scale volumes and tested the crude reaction for binding to PIAS4. Since the acid 1-36 is virtually inactive, any labeling was expected to arise from the formed amide and was normalized to the measured yield of the coupling reaction. From the top scoring amines, a benzyl glycine derivative was selected with the reasoning that it would serve as a scaffold easily modified with a linker and recruiters for target proteins. This yielded compound 1-39, which was 6 times more potent than compound 1.

[0411]

[0302] Finally, the features from compounds 1-40 and 1-39 were combined to obtain compound 1-41 (Figure 13A), which was 100-fold more potent than compound 1. Introduction of an alkyne into the para position of the benzyl group (compound 1-42) resulted in a slight improvement in potency. Finally, introduction of a PEG handle (1-44) and the BRD4 binder JQ1 (ID-2) were tested for their effect on binding to PIAS4 (Figure 13B). The conjugates displayed diminished binding towards PIAS4 compared to compound 1-41, but still labeled PIAS4 in a time scale of minutes, making them potential candidates for targeted-PIAS4 recruiters.

[0412]

[0303] The effect of changes in the intrinsic reactivity of the electrophile on the binding of PIAS4 by the compounds was determined. To this end a reactivity assay against N-acetylcysteine methyl ester (NAC) was performed. It was found that compound 1-41 was 6-7 times more reactive towards NAC than compound 1, compared to > 100-fold difference in binding rate to PIAS4. These results indicate that enhanced recognition to PIAS4 plays a significant role in the increase in potency from compound 1 to compound 1-41. On the other hand, ID-2 was only 33% more reactive than compound 1, indicating that enhanced reactivity only slightly contributes to the improved binding observed along the series.

[0413] EXAMPLE 21: Identification of PIAS4 binding site

[0414]

[0304] Goal: identify the binding site of the compounds in PIAS4.

[0415]

[0305] To identify the binding site of the compounds in PIAS4, the protein was incubated with compound 1-41 and digested with trypsin, and LC-MS / MS was used to identify the binding site. Among the non-modified peptides, the peptide comprising residues 322-330 was specifically depleted in the compound-treated sample, pointing to Cys326 as the site of modification. Furthermore, the peptide modified by compound 1-41 was clearly identified, thus confirming Cys326 as the target site (Figure 14A).

[0416]

[0306] PIAS4 was crystallized with compound 1-41 to elucidate the structure of the complex and to confirm the binding site. Wild type PIAS4 was not sufficiently soluble to enable crystallography. Therefore PROSS (1) was used to design stabilized mutants for PIAS4(126- 411). After inputting the protein sequence and the AlphaFold-derived structure, mutant number 7 that contained 5 mutations: N140D / C405R / M141V / S388N / I291C focused on. The mutations were inspected visually on the AlphaFold structure, and were found to be reasonable except I291C, which replaces a surface isoleucine with a nonpolar cysteine. To consider alternatives, the sequence of PIAS4 was compared with the sequences of the other PIAS isoforms. All of them were found to contain a lysine in that position. The N140D / C405R / M141V / S388N / I291K mutant was therefore opted. After cloning the gene for the mutant, it was expressed in bacteria and about 10-fold high yields were obtained compared to the wild type. Differential scanning fluorimetry (DSF) experiments revealed that the mutant was stabilized by 10°C relative to the wild type, and that for both proteins binding of compound 1-41 destabilized the protein by 3- 4°C.

[0417]

[0307] The structure of mutated PIAS4 with compound 1-41 was determined at a resolution of 2.2A (Figure 14B). As predicted by proteomics data, the compound binds to Cys326. In order to bind to the cysteine, the compound displaces a loop comprising residues 324-333 (Figure 14B, left). The displacement of the loop exposes a hydrophobic pocket (Figure 14B, right) to which the chloroacetamide and the thiophene scaffold insert and form hydrophobic interactions with the loop and with the alpha helix comprising residues 350-359. This perturbation to the fold of the protein may explain the destabilization observed in DSF. Even though the hydrogen in the amide group is essential for binding, the crystal structure does not indicate hydrogen bonding in the complex. Furthermore, the benzylglycine that dramatically increases the potency does not form any direct interactions except crystal contacts. It is possible that both features contribute to binding by enabling the compound to adopt the necessary conformation for binding, or by participating in the 324-333 loop dynamics prior to covalent bond formation.

[0418] EXAMPLE 22: A derivative of compound 1-41 (compound 1-43) binds PIAS proteins in cells

[0419]

[0308] Goal: verify that PIAS proteins are indeed cellular targets of the optimized binder, compound 1-41.

[0420]

[0309] To verify that PIAS proteins are indeed cellular targets of the optimized binder, compound 1-41, an alkyne-modified derivative was prepared, compound 1-42 (Figure 13A) that allows the identification of its interacting proteins via proteomics and used compound 1-43 as a competitor. Specifically, Daudi cells were incubated with compound 1-42 and used CuAAC to attach biotin to the labeled proteins, followed by pull-down, trypsinization and LC-MS / MS. Targets labeled by 1-42 were identified relative to DMSO-treated control, and targets labeled by 1-43 (chloro competitor, Figure 13A) were identified by preincubating the lysates prior to incubation with 1-42 and comparing the obtained proteins (Figure 15). Indeed, PIAS4 and PIAS2 (PIAS1 also identified, but difference was not significant) were identified among a large number of proteins pulled down (1032 proteins). As expected, competition by preincubation with 1-43 is an indicator of how much of these proteins are labeled, and in the competition data only 17 proteins were identified, one of which was PIAS4. Many of the off targets are common reactive proteins that come up in many experiments, such as GSTO1 and selenoproteins.

[0421] EXAMPLE 23: BRD4-targeting SUTAC (ID-2) enhances the SUMOylation of recombinant BRD4 and its downstream target c-MYC

[0422]

[0310] Goal: test the ability of compound 1-41 in functioning in a SUTAC settings.

[0423]

[0311] Compound 1-41 was conjugated to JQ1 that targets BRD4 to obtain ID-2 (Figure 13B). To assess its ability to facilitate BRD4 SUMOylation, an in vitro SUMOylation assay was performed. Using a purified reaction containing recombinant BRD4, SUMOs, and the essential enzymes of the SUMOylation pathway, it was observed that ID-2 (SUTAC) significantly enhanced BRD4 SUMOylation, only in the presence of all required SUMOylation enzymes, including PIAS4 (Figure 16A), which validates the compound 1-41 is a binder of PIAS4 that could be used to induce its proximity with a POI.

[0424]

[0312] To examine the capacity of ID-2 to impact the cellular levels and functionality of BRD4, different cancer cell lines (Daudi, OCI-AML2, RAMOS and RPMI-8226) were treated with increasing concentrations of ID-2 for an overnight treatment. Indeed, ID-2 treatment resulted in a significant reduction of cMYC levels across all test lines (Figure 16B). Notably, the reduction in cMYC was much more pronounced than that induced by JQ 1-1 alone (Figure 16C).

[0313] Importantly, the reduction in the levels of BRD4 was not observed in all cell lines suggesting that the underlying mechanism of cMYC downregulation, is not solely driven by degradation, at least for OCI-AML2 cells. Notably, in Daudi cells, where the reduction in cMYC levels was coupled to a reduction in BRD4 levels, proteasomal inhibition could completely abrogate the reduction in BRD4 levels induced by ID-2 (Figure 16D).

[0425]

[0314] Furthermore, the reduction in BRD4 levels by ID-2 in cells that are double knock outs (DKO) for PIAS1 and PIAS4, was partly yet consistently revoked. This validates the mechanism of action of SUTACs and underscores the importance of PIAS in mediating the effects of PIAS in cells (Figure 16E).

[0426] EXAMPLE 24: BRD4-targeting SUTAC (ID-3) enhances the SUMOylation of recombinant BRD4

[0427]

[0315] Although the hydrogen in the amide group of the binder is critical for the covalent binding to the recombinant PIAS4( 126-411) (Figures 13A-13B), the crystal structure did not show evidence of hydrogen bonding in the complex (Figure 14B). Therefore, it was hypothesized that a binder with a methyl group could still bind full-length PIAS4, either covalently or non-covalently. The results revealed that a JQl-based SUTAC incorporating such a binder (ID-3, Figure 17A) enhanced the SUMOylation of BRD4 by full-length PIAS4 even more effectively than ID-2 (Figure 17B). Remarkably, ID-3 also induced poly-SUMOylation of BRD4 (Figure 17B). This demonstrates the utility of methyl-derivate of compound 1-41 (i.e 1-46) in the SUTAC approach.

[0428] EXAMPLE 25: P300-targeting SUTACs reduce cMYC levels and proliferation of cancer cells

[0429]

[0316] Goal: test the generalizability of compound 1-41 and its methyl-derivate in altering other targets.

[0430]

[0317] To further test the generalizability of compound 1-41 and its methyl-derivate 1-46 in altering other targets, the target of P300 / CBP was selected. P300 is a histone acetyltransferase that drives cancer progression, and its activity is known to be negatively regulated by SUMOylation. A SUTAC based on compound 1-41 and P300 / CBP inhibitor GNE-207 was generated (ID-4, Figure 18A). Indeed, it was found that P300 was altered in two cancer cell lines, Daudi and OCI-AML2 by the P300-targeting SUTAC (Figure 18B). Importantly, this also resulted in lowering the levels of cMYC, a downstream gene that is activated by P300, suggesting that P300 activity is altered. Importantly, 72 hours treatment of OCI-AML2 cells with ID-4 (Figure 18C) resulted in their growth attenuation, while 1-41 had no significant effect on their growth. Finally, when comparing the effects of ID-4 with a methylamine derivative of the P300 inhibitor GNE-207 (compound 10, Figure 18D), and a P300-targeting SUTAC based on the methyl derivative of compound 1-41 (ID-5, Figure 18D), we observed that ID-5 was the most effective in reducing cMYC levels (Figure 18E).

[0431] EXAMPLE 26: Synthesis of SUTACs of ID-2 (BRD4-targeting SUTAC)

[0432]

[0318] Scheme 11, presents the synthesis of a SUTAC having the structure of conjugate ID-2.

[0433]

[0319] 10 mg of molecule 1-44 was dissolved in 600 pl dichloromethane and 140 pl trifluoroacetic acid. After tumbling for 40 minutes at room temperature, LC-MS confirmed full deprotection. The solution was evaporated under argon flow, and dichloromethane was added and evaporated three times to fully remove trace TFA. From the product, 2.5 mg (4.12 pmol) were used for coupling.

[0434]

[0320] For coupling, 6 mg of JQ1 acid (14.9 pmol) wee dissolve in dichloromethane and mixed with the amine. To the mixture, 50 f 0.2 Mμl E oDC / 0.6M DIPEA in DCM were added (10 pmol EDC, 30 pmol DIPEA). Reaction proceeded at room temperature for 24 hours, DCM was evaporated and the product was purified by HPLC. Obtained 1.32 mg of pure RG104 (yield 32%).

[0435] I l l The Synthesis of 1-44:

[0436] Synthetic scheme: (a) KOH, p-xylene, 25 °C, 12 h (b) PtO2, MeOH, 25 °C, 16 h (c) EDCI, Py, 30 °C, 2 h. tert-butyl N-[ 2-[ 2-[ 2-[ 2-( 4-cyanophenyl) ethoxy ] ethoxy ] ethoxy ] ethyl ] carbamate:

[0437] 3

[0438]

[0321] A mixture of tert-butyl N-[2-[2-(2-bromoethoxy)ethoxy]ethyl]carbamate (1.59 g, 1.5 eq), 4-(2-hydroxyethyl)benzonitrile (500 mg, 1 eq), KOH (953.05 mg, 5 eq) ,tetrabutylammonium;bromide (547.60 mg, 0.5 eq) in p-xylene (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 25 °C for 12 hr under N2 atmosphere. The reaction mixture was quenched by NH4CI (100 mL ), extracted with Ethyl acetate (100 mL- 2). The combined organic layers were washed with brine (50 mL *2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC to afford a yellow oil (430 mg, 33.44% yield).

[0439]

[0322] LC-MS (m / z): Calculated: 378.22; Found: 379.1 [M+H ]+. tert-butyl N-[ 2-[ 2-[ 2-[ 2-[ 4-(aminomethyl)phenyl ] ethoxy ] ethoxy ] ethoxy ] ethyl ] carbamate:

[0440]

[0323] To a solution oftert-butyl N-[2-[2-[2-[2-(4-cyanophenyl)ethoxy]ethoxy]ethoxy]ethyl] carbamate (200 mg, 1 eq) in MeOH (5 mL) was added PtCF (144.00 mg, 1.20 eq). The mixture was degassed and purged with H2 for 3 times, and then the mixture was stirred at 25 °C for 16 hr under H2 (30 Psi) atmosphere. The reaction mixture was filtered and the mother solution was concentrated. The residue was purified by pre-HPLC to afford a white solid (150 mg, 74.21% yield).

[0441]

[0324] LC-MS (m / z): Calculated: 382.25; Found: 383.1 [M+H]+.

[0442]

[0325] 1H NMR (400 MHz, DMSO) δ 7.36 - 7.31 (m, 2H), 7.28 - 7.22 (m, 2H), 6.78 (br t, J = 5.2 Hz, 1H), 3.88 (s, 2H),3.52 - 3.49 (m, 10H), 3.38 (br t, J = 6.0 Hz, 3H), 3.07 (q, J = 6.0 Hz, 2H), 2.81 (t, J = 7.0 Hz, 2H), 1.38 (s, 9H) tert-butyl N-[ 2-[ 2-[ 2-[ 2-[ 4-[[[2-[ [ 2-[ ( 2-chloroacetyl)amino ]-5-methyl-benzothiophene-3- carbonyl amino ] acetyl amino ] methyl phenyl ethoxy ] ethoxy ] ethoxy ] ethyl carbamate:

[0443]

[0326] To a solution of tert-butyl N-[2-[2-[2-[2-[4-(aminomethyl) phenyl] ethoxy] ethoxy] ethoxy] ethyl] carbamate (100 mg, 1 eq), 2-[[2-[(2-chloroacetyl) amino] -5-methyl- benzothiophene-3 -carbonyl] amino] acetic acid (89.09 mg, 1 eq) in Py (2.5 mL) was added EDCI (75.18 mg, 1.5 eq). The mixture was stirred at 30 °C for 2hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by prep- HPLC to afford a brown solid (41.69 mg, 22.61% yield).

[0444]

[0327] LC-MS (m / z): Calculated: 704.26; Found: 705.1 [M+H]+

[0445]

[0328] 1H NMR (400 MHz, CDCh) δ 12.91 (s, 1H), 7.81 (s, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.27 - 7.17 (m, 6H), 6.50 (br d, J = 4.2 Hz, 1H), 5.05 (br s, 1H), 4.51 (d, J = 5.6 Hz, 2H), 4.30 - 4.25 (m, 4H), 3.69 (t, J = 7.0 Hz, 2H), 3.65 - 3.59 (m, 8H), 3.50 (t, J = 5.1 Hz, 2H), 3.23 (q, J = 5.4 Hz, 2H), 2.90 (t, J = 7.0 Hz, 2H), 2.54 (s, 3H), 1.44 (s, 9H).

[0446] EXAMPLE 27: Synthesis of SUTACs of ID-3 (BRD4-targeting SUTAC)

[0447]

[0329] Scheme 12, presents the synthesis of a SUTAC having the structure of conjugate ID-3.

[0448]

[0449]

[0330] The amine derivative (a) of scheme 12 and JQ1 acid were dissolved in DMSO to 200 mM. Oxyma pure and was dissolved to 0.4 M in DMS and EDC was dissolved to 0.4 M in DMSO. 18 μl of EDC solution and 36 fμ Olx oyma pure solution were premixed and added to 30 μl of the JQ1 acid (6 pmol). The mixture was incubated at room temperature for 5 minutes, followed by the addition of 57 pl DMF, 3 pl DIPEA and 20 f thμel a omine derivative (a) (4 pmol). Reaction proceeded for 3 hours at room temperature, after which the product was purified by HPLC. Yield 2.65 mg (66%).

[0450] EXAMPLE 28: Synthesis of SUTACs of ID-4 (P300 / CBP-targeting SUTAC)

[0451] Scheme 13, presents the synthesis of a SUTAC having the structure of conjugate ID-4.

[0452]

[0453]

[0331] 2.3 mg of compound (b) of scheme 13 (4.62 pmol) were dissolved in 50 pl DMF. A mixture containing 13.9 f 0μ.l4 o M EDC (DMSO) and 27.8 f 0.4 M μ oxl y oma pure (DMF) was added, followed by 5 minutes incubation at room temperature. The sample was diluted to 5 ml with 10 mM DIPEA in DMF, and 3.6 mg of deprotected amine (a) was added. Reaction proceeded for 48 hours at 37°C. At this point HC1 was added to 20 mM and the sample was evaporated and purified by HPLC. Obtained 1.39 mg of pure product (yield 28%).

[0454] EXAMPLE 29: Synthesis of SUTACs of ID-5 (P300 / CBP-targeting SUTAC)

[0455] Scheme 14, presents the synthesis of a SUTAC having the structure of conjugate ID-5.

[0456]

[0457]

[0332] The acid (b) of scheme 14 was dissolved to 200 mM in DMSO. 30 μl o f 0.2 M EDC / DMSO and 30 μl of 0.4 M Oxyma / DMF were premixed and added to 25 μl of the acid (b) of scheme 14 solution (5 pmol). After 5 minutes at room temperature, 4.3μl o f DIPEA and 30 μl o f the amine (a) of Scheme 14, 200 mM in DMSO were added. Reaction proceeded at room temperature for 24 hours, after which the sample was evaporated and purified by HPLC. Yield 1.02 mg (19%).

[0458] EXAMPLE 30: Synthesis of Compound 10 (P300 / CBP-inhibitor)

[0459] Scheme 15A and 15B, present the synthesis of compound 10.

[0460] Scheme 15 A)

[0461]

[0462] Synthetic scheme: (a) Py, 0~25 °C, 2 h (b) Pd(dppf)C12, K2CO3, THF, H2O, 80 °C, 3 h (c) Pd2(dba)3, KO Ac, dioxane, 120 °C, 8 h (d) XPhos Pd G 2 , XPhos, CSCO3, dioxane, H2O, 90 °C, 1 h

[0463] (8-chloro-3-isoquinolyl) trifluoromethanesulfonate:

[0464]

[0333] 8-chloroisoquinolin-3-ol (2 g, 1 eq) in Py (80 mL) was cooled to 0 °C and carefully treated with trifluoromethyl sulfonyl trifluoromethanesulfonate (5.03 g, 1.6 eq) at 0 °C. The mixture was allowed to warm to 25 °C and stirred 2 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column to afford a yellow solid (2.91 g, 83.85% yield).

[0465]

[0334] LC-MS (m / z): Calculated: 310.96; Found: 312.1 [M+H]+. methyl 5-(8-chloro-3-isoquinolyl)pyridine-2-carboxylate:

[0466]

[0335] A mixture of (8-chloro-3-isoquinolyl) trifluoromethanesulfonate (2.91 g, 1 eq), methyl 5-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)pyridine-2-carboxylate (3.68 g, 1.5 eq), Pd(dppf)Cl2(683.19 mg, 0.1 eq) and K2CO3 (3.87 g, 3 eq) in THF (35 mL) and H2O (7 mL) was stirred at 80 °C for 3 h. The reaction mixture is poured into 10ml H2O, filtered and collected, then washed with 15mL THF collected solids, and dried in high vacuum. The residue was purified by filtered to afford a gray solid (2.1 g, 75.29% yield).

[0467]

[0336] LC-MS (m / z): Calculated: 298.72; Found: 299.1 [M+H]+. methyl 5-[ 8-( 4, 4, 5, 5-tetramethyl-l, 3, 2-dioxaborolan-2-yl)-3-isoquinolyl ] pyridine-2- carboxylate:

[0468]

[0337] A mixture of methyl 5-(8-chloro-3-isoquinolyl)pyridine-2-carboxylate (1 g, 1 eq), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l,3,2-dioxaborolane (1.28 g, 1.5 eq) in dioxane (30 mL) was added tricyclohexylphosphane (375.50 mg, 0.4 eq), KO Ac (985.62 mg, 10.04 mmol, 3 eq) and Pd2(dba)3 (306.54 mg, 0.1 eq) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 120 °C for 8 hr under N2 atmosphere. The reaction mixture was poured into 20 mL of dioxane and collected by filtration and dried under high vacuum The residue was purified by filtration to afford a yellow solid (600 mg, 45.93% yield).

[0469]

[0338] LC-MS (m / z): Calculated: 390.18; Found: 391.1 [M+H]+

[0470] 5-[8-(5-acetyl-l-tetrahydropyran-4-yl-6, 7-dihydro-4H-pyrazolo [4,3-c] pyridin- 3-yl)-3-isoquinolyl] pyridine -2 -carboxylic acid:

[0471]

[0472]

[0339] To a mixture of dicyclohexyl-[2-(2,4,6-triisopropylphenyl) phenyl] phosphane (61.08 mg, 0.1 eq), [2-(2- aminophenyl) phenyl]-chloro-palladium;dicyclohexyl-[3-(2,4,6- triisopropylphenyl) phenyl] phosphane (100.81 mg, 0.1 eq) , and CS2CO3 (1.25 g, 3 eq) were added to a solution of methyl 5-[8-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)-3-isoquinolyl] pyridine-2-carboxylate (500 mg, 1 eq) and l-(3-iodo- l-tetrahydropyran-4-yl-6,7-dihydro-4H- pyrazolo[4,3-c] pyridin-5-yl) ethanone (480.74 mg, 1 eq) in dioxane (20 mL) and H2O (10 mL). The reaction mixture was heated to 90 °C for 2 h. The reaction mixture was quenched by H2O (20 mL ), extracted with Ethyl acetate (20 mL / 2), the aqueous solution was adjust to pH=6 with saturated FA, extracted with Ethyl acetate (20 mL / 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC to afford a off-white solid (36.77 mg, 5.25% yield).

[0473]

[0340] LC-MS (m / z): Calculated: 497.21; Found: 498.1 [M+H]+

[0474]

[0341] 1H NMR (400 MHz, DMSO) δ 10.01 (s, 1H), 9.55 (br s, 1H), 8.83 - 8.66 (m, 2H), 8.19 (br d, J = 8.0 Hz, 1H), 8.09 (br d, J = 8.0 Hz, 1H), 7.93 (br s, 1H), 7.80 - 7.67 (m, 1H), 4.59(br s, 2H), 4.55 - 4.43 (m, 1H), 4.08 - 4.00 (m, 2H), 3.90 - 3.80 (m, 2H), 3.58 - 3.51 (m, 2H), 2.99 (br t, J = 5.2 Hz, 1H), 2.87 (br d, J = 4.8 Hz, 1H), 2.21 - 2.10 (m, 4H), 2.05 - 1.94 (m, 3H). Scheme 15B)

[0475]

[0342] The product of scheme 15A (structure (a) in Scheme 15B) was dissolved to 200 mM in DMSO. 60 μl of 0.2 M EDC / DMSO and 60 f 0μ.l4 o M Oxyma / DMF were premixed and added to 50 μfl o Compound (a) of scheme 15A solution (10 pmol). After 5 minutes at room temperature, 17.2 fμ Dl I oPEA and 50 f 1 Mμl m o ethylamine hydrochloride in DMSO were added. Reaction proceeded at room temperature for 24 hours, after which the sample was evaporated and purified by HPLC. Yield 1.21 mg (24%).

[0476] EXAMPLE 31 : Synthesis of SUMOylation compounds which are used as SUMOylation enzyme binder group in the Chimeras of this invention.

[0477] The synthesis of 1-2

[0478] Synthetic scheme: (a) TEA / EtOH (b) NaHCO3 / THF / H2O / 0 °C

[0479] 2-amino-N-methylthiophene-3-carboxamide:

[0343] To a solution of l,4-dithiane-2,5-diol (500 mg, 3.28 mmol) in EtOH (15 mL) was added 2-cyano-N,N-dimethylacetamide (368.28 mg, 3.28 mmol) and TEA (664.69 mg, 6.57 mmol, 914.29 uL). The mixture was stirred at 70 °C for 16 hr. After cooling to room temperature, the mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate = 3 / 1) to afford a yellow solid (320 mg).

[0480]

[0344] LC-MS (m / z): Calculated: 156.04; Found: 157.01 [M+H]+

[0481]

[0345] 1H NMR (400 MHz, DMSO) δ 6.75 (d, J= 5.6 Hz, 1H), 6.48 (s, 2H), 6.32 (d, J= 5.6 Hz, 1H), 2.96 (s, 6H).

[0482] 2-(2-chloroacetamido)-N,N-dimethylthiophene-3-carboxamide :

[0483]

[0023] To a solution of 2-amino-N-methylthiophene-3-carboxamide (100 mg, 587.43 umol), NaHCOs (98.70 mg, 1.17 mmol, 45.69 uL) in THF (1 mL), H2O (1 mL) was added 2-cyano- N,N-dimethylacetamide (79.62 mg, 704.92 umol, 56.07 uL) at 0 °C. The mixture was stirred at 20 °C for 0.5 h. The mixture was concentrated under vacuum to give a residue. The residue was purified by prep-HPLC (FA conditions) to afford a white solid (62.49 mg).

[0484]

[0024] LC-MS (m / z): Calculated: 246.02; Found: 247.1 [M+H]+

[0485]

[0025] 1H NMR (400 MHz, DMSO) δ 11.36 (s, 1H), 7.15-7.03 (m, 2H), 4.49 (s, 2H), 3.01 (s, 6H).

[0486] The synthesis of 1-18.

[0487] Synthetic scheme: (a) NaH / DMSO / 15~100 °C (b) NaOH / DMSO / lOO °C (c) H2SO4 / 6O °C (d) / NaHCO3 / THF / H2O / 0 °C ethyl N-(5-chloro-3-cyano-benzothiophen-2-yl)carbamate:

[0488]

[0026] A mixture of 2-(4-chloro-2-fluoro-phenyl)acetonitrile (5 g, 29.48 mmol, 1 eq), NaH (2.83 g, 35.38 mmol, 30% purity, 1.2 eq) in DMSO (50 mL) was degassed and purged with N2, and then the mixture was stirred at 15 °C for 0.5 hr under N2 atmosphere. Add ethyl N- (thi oxomethylene) carbamate (3.87 g, 29.48 mmol, 1 eq), and then the mixture was stirred at 100 °C for 2.5 hr under N2 atmosphere. The reaction is not post-processed and is directly used for the next step.

[0489]

[0027] LC-MS (m / z): Calculated: 280.01; Found: 278.9 [M-H]

[0490] 2-amino-5-chloro-benzothiophene-3-carbonitrile:

[0491]

[0028] A mixture of ethyl N-(5-chloro-3-cyano-benzothiophen-2-yl)carbamate (5 g, 17.81 mmol, 1 eq , NaOH (2.14 g, 53.43 mmol, 3 eq) in DMSO (50 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100 °C for 2 h under N2 atmosphere. Filtrate the reaction liquid, and the solid is the crude product. The product was obtained as a yellow solid (3.7 g, 17.73 mmol, 99.56% yield).

[0492]

[0029] LC-MS (m / z): Calculated: 207.99; Found: 206.81 [M-H]

[0493]

[0030] 1H NMR (400 MHz, DMSO) δ 7.95 (br s, 2H), 7.64 (d, J= 7.9 Hz, 1H), 7.29 (d, J= 7.9 Hz, 1H), 7.08 (t, J= 7.9 Hz, 1H). -amino-5-chloro-benzothiophene-3-carboxamide:

[0494]

[0031] To a solution of 2-amino-5-chloro-benzothiophene-3-carbonitrile (500 mg, 2.40 mmol, 1 eq in H2SO4 (3 mL). The mixture was stirred at 60 °C for 1 h. Pour the reaction solution into the NaHCO, solution (100 mL), then extracted with Ethyl acetate (20 mL x 2). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (NH4HCO3)-ACN]; gradient: 6%-36% B over 15 min). The product was obtained as a white solid (180 mg, 794.06 pmol, 33.14% yield).

[0495]

[0032] LC-MS (m / z): Calculated: 226; Found: 226.9 [M+H]+

[0496]

[0033] 1H NMR (400 MHz, DMSO) δ 7.59 (dd, J= 0.8, 7.8 Hz, 1H), 7.43 - 7.27 (m, 2H), 7.22 (dd, J= 0.8, 7.8 Hz, 1H), 7.00 (t, J= 7.8 Hz, 1H), 6.58 (s, 2H) -chlor o-2 -[ ( 2 -chloroace tyl)amino ]benzothiophene-3-carboxamide :

[0497]

[0034] A solution of 2-amino-5-chloro-benzothiophene-3-carboxamide (100 mg, 441.15 pmol, 1 eq), 2-chloroacetyl chloride (59.79 mg, 529.38 pmol, 42.16 , 1.2 eq) in TμLHF (1 mL), H2O (1 mL) was added NaHCOs (148.24 mg, 1.76 mmol, 68.66 , 4 eq). ThμeL mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove solvent. The product was obtained as a white solid (12.46 mg, 39.46 pmol, 8.94% yield).

[0498]

[0035] LC-MS (m / z): Calculated: 301.97; Found: 300.9 [M-H]+

[0499]

[0036] 1H NMR (400 MHz, DMSO) δ 11.13 (br s, 1H), 8.03 (s, 1H), 7.92 - 7.89 (m, 1H), 7.70 (br s, 1H), 7.42 (dd, J= 0.9, 7.8 Hz, 1H), 7.31 - 7.26 (m, 1H), 4.54 (s, 2H). The synthesis of 1-19.

[0500] Synthetic scheme: (a) NaH / DMSO / 15~100 °C (b) NaOH / DMSO / lOO °C (c) H2SO4 / 6O °C (d) / NaHCO3 / THF / H2O / 0 °C ethyl N-(4-chloro-3-cyano-benzothiophen-2-yl)carbamate:

[0501]

[0037] A mixture of 2-(2-chloro-6-fluoro-phenyl)acetonitrile (1 g, 5.90 mmol, 1 eq), ethyl N- (thioxomethylene)carbamate (773.38 mg, 5.90 mmol, 1 eq , NaH (283.02 mg, 7.08 mmol, 60% purity, 1.2 eq) in DMSO (10 mL) was degassed and purged with N2, and then the mixture was stirred at 15 °C for 0.5 hr under N2 atmosphere. Add ethyl N-(thi oxomethylene) carbamate (773.38 mg, 5.90 mmol, 1 eq), and then the mixture was stirred at 100 C for 2.5 h under N2 atmosphere. The reaction is not post-processed and is directly used for the next step.

[0502]

[0038] LC-MS (m / z): Calculated: 280.01; Found: 279.0 [M-H]+ -amino-4-chloro-benzothiophene-3-carbonitrile:

[0503] 3 4

[0504]

[0039] A mixture of ethyl N-(4-chloro-3-cyano-benzothiophen-2-yl)carbamate (1 g, 3.56 mmol, 1 eq), NaOH (5 M, 15 mL, 21.05 eq) in DMSO (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100 °C for 3hr under N2 atmosphere. Add 50 mL of water to the reaction solution, cool to 15 degrees Celsius, filter the residue is coarse product. The product was obtained as a yellow solid (720 mg, 3.45 mmol, 96.86% yield).

[0505]

[0040] LC-MS (m / z): Calculated: 207.99; Found: 206.81 [M-H]+.

[0506]

[0041] 1H NMR (400 MHz, DMSO) δ 7.95 (br s, 2H), 7.65 (br d, J= 7.9 Hz, 1H), 7.29 (br d, J = 7.9 Hz, 1H), 7.08 (br t, JM= 7.8 Hz, 1H) -amino-4-chloro-benzothiophene-3-carboxamide:

[0507] 4 5

[0508]

[0042] To a solution of 2-amino-4-chloro-benzothiophene-3-carbonitrile (300 mg, 1.44 mmol, 1 eq) in H2SO4 (3 mL). The mixture was stirred at 60 °C for 0.5 hr. Pour the reaction solution into the NaHCOi solution (30 mL) with EA (10*3) The residue was purified by prep-HPLC (column: waters Xbridge Prep OBD Cl 8 150*40mm*10um; mobile phase: [water ( NH4HCO3)-ACN];gradient:5%-35% B over 15 min). The product was obtained as a white solid (25 mg, 110.29 pmol, 7.67% yield).

[0509]

[0043] LC-MS (m / z): Calculated: 226; Found: 225.1 [M-H]+

[0510]

[0044] 1H NMR (400 MHz, DMSO) δ 7.58 (dd, J= 0.8, 7.8 Hz, 1H), 7.42 - 7.28 (m, 2H), 7.22 (dd, J= 0.9, 7.8 Hz, 1H), 6.99 (t, J= 7.8 Hz, 1H), 6.58 (s, 2H) 4-chloro-2-[(2-chloroacetyl)amino]benzothiophene-3-carboxamide:

[0511]

[0045] To a solution of 2-amino-4-chloro-benzothiophene-3-carboxamide (25 mg, 110.29 pmol, 1 eq), 2-chloroacetyl chloride (14.95 mg, 132.34 pmol, 10.54 , 1.2 eq) in TμHLF (1 mL), H2O (1 mL) was added NaHCOs (37.06 mg, 441.15 pmol, 17.16 , 4 eq). TheμL mixture was stirred at 0 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The product was obtained as a white solid (8.67 mg, 27.74 pmol, 25.15% yield).

[0512]

[0046] LC-MS (m / z): Calculated: 301.97; Found: 300.9 [M-H]+

[0513]

[0047] 1H NMR (400 MHz, DMSO) δ 11.14 (br s, 1H), 8.04 (s, 1H), 7.91 (d, J= 7.9 Hz, 1H), 7.71 (br s, 1H), 7.43 (d, J= 7.8 Hz, 1H), 7.32 - 7.27 (m, 1H), 4.55 (s, 2H)

[0514] The synthesis of 1-36.

[0515] 1-36

[0516]

[0048] 143 mmol of chloroacetic acid (1.5 mmol) were dissolved in dichloromethane and mixed with 173 mg EDC (0.9 mmol), 138 mg HOBT hydrate (0.9 mmol) and 695 f DIPEμAl o (4 mmol). After 30 minutes, 80 mg of 2-aminothiophene-3 -carboxylic acid (0.56 mmol) were added, and the reaction proceeded for 1 hour at room temperature. The reaction was washed twice with IN HC1, followed by wash with brine and evaporation. The crude product was purified by HPLC to obtain 1-36 as a bright brown solid, total weight 11.71 mg (yield 9.5%). The synthesis of 1-39

[0517] Synthetic scheme: (a) NaOH, MeOH, H2O, 80 °C, 2 h (b) HOBt, EDCI, TEA, DMF, 25 °C, 12 h (c) TEA, DCM, 0 °C, 1 h.

[0518] 2-aminothiophene-3-carboxylic acid:

[0519] 1 2

[0520]

[0049] A mixture of methyl 2-aminothiophene-3 -carboxylate (1 g, 1 eq , NaOH (763.35 mg, 3 eq in MeOH (10 mL), H2O (2 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80 °C for 2 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The reaction mixture was filtered and the mother solution was concentrated. The residue was purified by prep-HPLC (NH4HCO3 conditions) to afford a yellow solid (800 mg, 87.84% yield).

[0521]

[0050] LC-MS (m / z): Calculated: 143; Found: 141.9[M-H]+

[0522]

[0051] 1H NMR (400 MHz, DMSO) δ 7.31 - 6.33 (m, 3H), 6.04 (d, J = 5.6 Hz, 1H) 2-amino-N-[2-(benzylamino)-2-oxo-ethyl]thiophene-3-carboxamide:

[0523] 2 3 4

[0524]

[0052] To a solution of 2-aminothiophene-3 -carboxylic acid (300 mg, 1 eq in DMF (5 mL) was added HOBt (424.73 mg, 1.5 eq) and EDCI (602.57 mg, 1.5 eq), TEA (636.13 mg, 875.00 pL, 3 eq), 2-amino-N-benzyl-acetamide (344.09 mg, 1 eq . The mixture was stirred at 25 °C for 12 hr. The reaction mixture was poured into water (50 mL), extracted with Ethyl acetate (50 mL x 2). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column to afford a pink oil (120 mg, 19.79% yield).

[0525]

[0053] LC-MS (m / z): Calculated: 289.09; Found: 290.1 [M+H]+.

[0526] N-[2-(benzylamino)-2-oxo-ethyl]-2-[(2-chloroacetyl)amino]thiophene-3-carboxamide:

[0527]

[0054] To a solution of 2-amino-N-[2-(benzylamino)-2-oxo-ethyl] thiophene-3 -carboxamide (60 mg, 1 eq) in DCM (1.5 mL) was added TEA (62.95 mg, 3 eq) was added 2-chloroacetyl chloride (46.84 mg, 2 eq) at 0 °C. The mixture was stirred at 0 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by prep-HPLC (NH4HCO3 conditions) to afford a yellow solid (27.06 mg, 35.67% yield).

[0528]

[0055] LC-MS (m / z): Calculated: 365.06; Found: 366.1 [M+H]+.

[0529]

[0056] 1H NMR (400 MHz, DMSO) δ 12.73 (s, 1H), 8.82 (t, J= 6.0 Hz, 1H), 8.51 (t, J = 6.0 Hz, 1H), 7.51 (d, J = 6.0 Hz, 1H), 7.34 - 7.26 (m, 4H), 7.25 - 7.19 (m, 1H), 7.09 (d, J = 5.7 Hz, 1H), 4.58 (s, 2H), 4.30 (d, J = 6.0 Hz, 2H), 3.91 (d, J = 6.0 Hz, 2H). The synthesis of 1-40.

[0530] Synthetic scheme: (a) NH4Ac, AcOH, Tol, 60 °C, 12 h (b) S, NaHCCh, EtOH, 80 °C, 2 h (c) NaHCO3, THF / H2O, 0 °C, 1 h.

[0531] (2Z)-2-cyano-2-(3-methylcyclohex-2-en-l-ylidene) acetamide:

[0532]

[0057] To a solution of 3-methylcyclohex-2-en-l-one (5 g, 1 eq), 2-cyanoacetamide (10.88 g, 2.85 eq) in Tol. (70 mL) was added CH3COONH4 (8.92 g, 2.55 eq) and CH3COOH (8.15 g, 2.99 eq). The mixture was stirred at 60 °C for 12 hr. The reaction mixture was poured into water (200 mL), extracted with Ethyl acetate (200 mL x 2). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiCh, Petroleum ether / Ethyl acetate=l / l, Rf=0.2) to afford a yellow solid (3.5 g, 47.36% yield).

[0533]

[0058] LC-MS (m / z): Calculated: 176.09; Found: 175.1 [M-H]+. -amino-5-methyl-benzothiophene-3-carboxamide:

[0534] 3 4

[0535]

[0059] To a solution of (2Z)-2-cyano-2-(3-methylcyclohex-2-en-l-ylidene) acetamide (500 mg, 1 eq in EtOH (16 mL) was added S (0.72 g, 7.91 eq) and NaHCOs (2.4 g, 10.07 eq . The mixture was stirred at 80 °C for 2 hr. The reaction mixture was filtered and the mother solution was concentrated. The residue was purified by pre-HPLC (FA conditions) to afford a yellow solid (250 mg, 42.72% yield).

[0536]

[0060] LC-MS (m / z): Calculated: 206.05; Found: 207.1 [M+H]+.

[0537]

[0061] Tf NMR (400 MHz, DMSO) 57.75 - 7.67 (m, 2H), 7.59 - 7.49 (m, 2H), 7.02 (s, 2H), 6.90

[0538] (d, J= 8.0 Hz, 1H), 2.40 - 2.37 (m, 3H) -[ ( 2 -chloroacetyl) amino ]-5-methyl-benzothiophene-3-carboxamide:

[0539]

[0062] To a solution of 2-amino-5-methyl-benzothiophene-3-carboxamide (50 mg, 1 eq), 2- chloroacetyl chloride (27.38 mg, 19.31 , 1 eq μ iLn THF (0.5 mL), H2O (0.5 mL) was added NaHCOs (61.09 mg, 3 eq . The mixture was stirred at 0 °C for 1 hr. The residue was purified by filtered to afford a yellow solid (24.22 mg, 34.63% yield).

[0540]

[0063] LC-MS (m / z): Calculated: 282.02; Found: 283.1 [M+H] +.

[0541]

[0064] 1H NMR (400 MHz, DMSO) δ 12.55 (s, 1H), 7.89 - 7.76 (m, 4H), 7.17 (d, J= 8.0 Hz, 1H), 4.58 (s, 2H), 2.43 (s, 3H). The synthesis of 1-41.

[0542] 1-41

[0543] Synthetic scheme: (a) EDCI, HOBt, TEA, DMF, 25 °C, 1 h (b) NaHCCh, THF, H2O, 0 C, 1 h.

[0544] 2-amino-N-[2-(benzylamino)-2-oxo-ethyl]-5-methyl-benzothiophene-3-carboxamide:

[0545] 1 2 3

[0546]

[0065] To a solution of 2-amino-5-methyl-benzothiophene-3-carboxylic acid (220 mg, 1 eq) in DMF (2 mL) was added HOBt (215.16 mg, 1.5 eq) and EDCI (305.24 mg, 1.5 eq), TEA (322.24 mg, 3 eq), 2-amino-N-benzyl-acetamide (174.31 mg, 1 eq). The mixture was stirred at 25 °C for 1 hr. The reaction mixture was filtered and the mother solution was concentrated. The residue was purified by prep-HPLC to afford a yellow solid (50 mg, 13.33% yield).

[0547]

[0066] LC-MS (m / z): Calculated: 353.12; Found: 354.1 [M+H]+.

[0548]

[0067] 1H NMR (400 MHz, DMSO) δ 8.43 (br t, J = 6.0 Hz, 1H), 7.60 - 7.52 (m, 4H), 7.48 (d, J = 8.0 Hz, 1H), 7.38 - 7.15 (m, 5H), 6.87 (d, J = 8.2 Hz, 1H), 4.32 (d, J = 6.0 Hz, 2H), 3.95 (d, J = 5.6 Hz, 2H), 2.35 (s, 3H). N-[2-(benzylamino)-2-oxo-ethyl]-2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3- carboxamide:

[0549]

[0068] To a solution of 2-amino -N-[2-(benzylamino) -2-oxo-ethyl] -5-methyl- benzothiophene-3 -carboxamide (25 mg, 1 eq) in THF (2 mL), H2O (1 mL) was added NaHCO, (17.83 mg, 3 eq) was added 2- chloroacetyl chloride (15.98 mg, 2 eq) at 0 °C. The mixture was stirred at 0 °C for 1 hr. The residue was purified by filtered to afford a off-white solid (14.88 mg, 48.93% yield).

[0550]

[0069] LC-MS (m / z): Calculated: 429.09; Found: 430.1 [M+H]+.

[0551]

[0070] 1H NMR (400 MHz, DMSO) δ 12.30 (s, 1H), 8.67 (t, J = 6.0 Hz, 1H), 8.49 (t, J = 6.0 Hz, 1H), 7.89 - 7.76 (m, 2H), 7.37 - 7.28 (m, 4H), 7.28 - 7.22 (m, 1H), 7.21 - 7.14 (m, 1H), 4.56 (s, 2H), 4.36 (d, J = 6.0 Hz, 2H), 4.07 (d, J = 6.0 Hz, 2H), 2.43 (s, 3H).

[0552] Synthesis of 1-43

[0553] Synthetic scheme: (a) EDCI, Py, 30 °C, 2 h

[0554] 2-[ ( 2 -chloroacetyl) amino ]-N-[ 2-[ ( 4-chlorophenyl)methylamino ] -2 -oxo-ethyl ]-5-methyl- benzothiophene- 3-carboxamide:

[0555] 1-43

[0071] To a solution of 2-[[2-[(2-chloroacetyl)amino]-5-methyl-benzothiophene-3-carbonyl] amino] acetic acid (40 mg, 1 eq, (4-chlorophenyl)methanamine (33.24 mg, 2 eq) in Py (1 mL) was added EDCI (33.75 mg, 1.5 eq). The mixture was stirred at 40 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by prep-HPLC to afford a white solid (8.68 mg, 15.92% yield).

[0556]

[0072] LC-MS (m / z): Calculated: 463.05; Found: 464.1 [M+H]+.

[0557]

[0073] 1H NMR (400 MHz, DMSO) δ 12.28 (s, 1H), 8.68 (br t, J = 6.0 Hz, 1H), 8.48 (br t, J = 6.0 Hz, 1H), 7.86 - 7.79 (m, 2H), 7.41 - 7.36 (m, 2H), 7.36 - 7.31 (m, 2H), 7.18 (d, J = 7.6 Hz, 1H), 4.55 (s, 2H), 4.34 (d, J = 6.0 Hz, 2H), 4.06 (d, J = 5.8 Hz, 2H), 2.43 (s, 3H).

[0558] Synthesis of 1-45

[0559]

[0560] Synthetic scheme: (a) CH3COOH, CH3COONH4, Tol., 60 °C, 12 h (b) S, NaHCO3, EtOH, 80 °C, 2 h (c) NaOH, MeOH, THF, H2O, 80 °C, 12 h (d) HOBt, EDCI, TEA, DMF, 25 °C, 12 h (e) DCM, TEA, 0 °C, 1 h (f) TFA, DCM, 25 °C, 2 h (g) EDCI, Py, 30 °C, 2 h methyl (2Z)-2-cyano-2-(3-methylcyclohex-2-en-l-ylidene) acetate:

[0561]

[0074] To a solution of 3-methylcyclohex-2-en-l-one (10 g, 1 eq), methyl 2-cyanoacetate (25.64 g, 2.85 eq) in Tol. (100 mL) was added CH3COOH (16.30 g, 2.99 eq) and CH3COONH4 (17.84 g, 2.55 eq). The mixture was stirred at 60 °C for 12 hr. The reaction mixture was poured into water (200 ml), extracted with Ethyl acetate (100 mL x 3). The combined organic layers were washed with brine (100 mL ), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by pre-HPLC (FA conditions) to afford a yellow oil (12 g, 69.12% yield).

[0562]

[0075] LC-MS (m / z): Calculated: 191.09; Found: 192.0[M+H]+

[0563]

[0076] 1H NMR (400 MHz, DMSO) δ 7.65 - 6.47 (m, 1H), 3.85 - 3.72 (m, 3H), 3.06 - 2.67 (m, 2H), 2.32 (d, J= 7.0 Hz, 2H), 2.05 (d, J= 12.6 Hz, 3H), 1.89 - 1.70 (m, 2H) methyl 2-amino-5-methyl-benzothiophene-3-carboxylate:

[0077] A mixture of methyl (2Z)-2-cyano-2-(3-methylcyclohex-2-en-l-ylidene) acetate (7 g, 1 eq) in EtOH (100 mL) NaEICCh (30.75 g, 10 eq), S (7.01 g, 5.98 eq) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80 °C for 2 hr under

[0564] N2 atmosphere. The reaction mixture was filtered and the mother solution was concentrated. The residue was purified by column to afford a yellow solid (3.4 g, 41.98% yield).

[0565]

[0078] 1H NMR (400 MHz, DMSO) δ 7.94 (s, 2H), 7.79 (d, J= 0.6 Hz, 1H), 7.47 (d, J= 8.0 Hz, 1H), 6.90 (dd, J= 1.1, 8.0 Hz, 1H), 3.82 (s, 3H), 2.34 (s, 3H)

[0566] 2-amino-5-methyl-benzothiophene-3-carboxylic acid:

[0567]

[0079] To a solution of methyl 2-amino-5-methyl-benzothiophene-3-carboxylate (3.4 g, 1 eq) in THF (30 mL), MeOH (30 mL), H2O (30 mL) was added NaOH (1.54 g, 2.5 eq). The mixture was stirred at 80 °C for 12 hr. The reaction mixture was adjust to pH=7 with HC1 (Imol / L), the filtered solids are crude products. The residue was purified by prep-HPLC (NH4HCO3 conditions) to afford a red soild (6 g, crude).

[0568]

[0080] LC-MS (m / z): Calculated: 207.04; Found: 208.1 [M+H]

[0569]

[0081] 1H NMR (400 MHz, DMSO) δ 12.74 - 11.82 (m, 1H), 7.99 - 7.78 (m, 3H), 7.47 (d, J= 7.9 Hz, 1H), 6.95 - 6.85 (m, 1H), 2.35 (s, 3H) tert-butyl 2-[(2-amino-5-methyl-benzothiophene-3-carbonyl)amino]acetate:

[0570]

[0082] To a solution of 2-amino-5-methyl-benzothiophene-3-carboxylic acid (2 g, 1 eq, HC1) in DMF (20 mL) was added HOBt (1.66 g, 1.5 eq) and EDCI (2.36 g, 1.5 eq), TEA (2.49 g, 3 eq), tert-butyl 2-aminoacetate (1.08 g, 1 eq). The mixture was stirred at 25 °C for 2 hr. The residue was purified by prep-HPLC (NH4HCO3 conditions) to afford a yellow soild (491 mg, 18.67% yield).

[0571]

[0083] LC-MS (m / z): Calculated: 320.12; Found: 319.2 [M-H]+.

[0084] 1H NMR (400 MHz, DMSO) δ 7.65 (br t, J= 5.8 Hz, 1H), 7.55 (s, 3H), 7.49 (d, J= 7.9 Hz, 1H), 6.88 (d, J= 8.0 Hz, 1H), 3.91 (d, J= 6.0 Hz, 2H), 2.36 (s, 3H), 1.44 (s, 9H). tert-butyl 2-[[2-[(2-chloroacetyl) amino]-5-methyl-benzothiophene-3-carbonyl] amino] acetate:

[0572]

[0085] To a solution of tert-butyl 2-[(2-amino-5-methyl-benzothiophene-3 -carbonyl) amino]acetate (780 mg, 1 eq) in DCM (10 mL) was added TEA (739.01 mg, 3 eq), 2- chloroacetyl chloride (549.90 mg, 2 eq) at 0 °C. The mixture was stirred at 0 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The was to afford a yellow soild (700 mg, 72.45% yield).

[0573]

[0086] LC-MS (m / z): Calculated: 396.09; Found: 395.1 [M-H]+.

[0574] 2- [ [2- [(2-chloroacetyl)amino]-5-methyl-benzothiophene-3-carbonyl] amino] acetic acid:

[0575]

[0087] To a solution of tert-butyl 2-[[2-[(2-chloroacetyl) amino]-5-methyl-benzothiophene-3- carbonyl] amino] acetate (800 mg, 1 eq) in DCM (16 mL) was added TFA (6.14 g, 26.71 eq). The mixture was stirred at 25 °C for 2 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The reaction mixture was poured into 20 mL of Petroleum ether and collected by filtration and dried under high vacuum. The was to afford a yellow soild (550 mg, 80.07% yield).

[0576]

[0088] LC-MS (m / z): Calculated: 340.03; Found: 339.1 [M-H]+.

[0089] 1H NMR (400 MHz, DMSO) δ 13.32 - 12.38 (m, 1H), 12.17 (br s, 1H), 8.54 (s, 1H), 7.88 (br d, J= 8.8 Hz, 2H), 7.22 (d, J= 8.1 Hz, 1H), 4.60 (s, 2H), 4.07 (d, J= 5.8 Hz, 2H), 2.47 (s, 3H)

[0577] 2-[ ( 2 -chloroacetyl) amino ]-N-[ 2-[ ( 4-ethynylphenyl)methylamino ]-2-oxo-ethyl ]-5-methyl- benzothiophene-3-carboxamide:

[0578]

[0090] To a solution of 2-[[2-[(2-chloroacetyl) amino]-5-methyl-benzothiophene-3-carbonyl] amino] acetic acid (30 mg, 1 eq , (4-ethynylphenyl) methanamine (23.10 mg, 2 eq in Py (1 mL) was added EDCI (25.31 mg, 1.5 eq). The mixture was stirred at 30 °C for 2hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by pre-HPLC (FA conditions) to afford a white soild (3.31 mg, 8.28% yield).

[0579]

[0091] LC-MS (m / z): Calculated: 453.09; Found: 454.4 [M+H]+.

[0580]

[0092] 1H NMR (400 MHz, DMSO) δ 12.29 (s, 1H), 8.75 - 8.64 (m, 1H), 8.49 (s, 1H), 7.89 - 7.78 (m, 2H), 7.44 (d, J= 8.0 Hz, 2H), 7.32 (d, J= 8.0 Hz, 2H), 7.18 (d, J= 8.1 Hz, 1H), 4.55 (s, 2H), 4.37 (d, J= 5.8 Hz, 2H), 4.14 (s, 1H), 4.07 (d, J= 6.0 Hz, 2H), 2.43 (s, 3H).

Claims

CLAIMS1. A SUMOylation-targeting chimera (SUTAC) comprising a SUMOylation enzyme binder group attached to a target protein binder group via a linker.

2. The SUTAC of claim 1, wherein said SUMOylation enzyme binder group is capable of binding a SUMO E3 ligase.

3. The SUTAC of claim 1, wherein said SUMO E3 ligase is selected from the group consisting of PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, TOPORS, Triml9, Trim28, PML, ZNF451, and RANBP2.

4. The SUTAC of claim 1, represented by the structure of formula I or salt thereof:wherein,E is H, amide, acetyl amide, or an electrophilic group;Z is O, S and NH; each of R6, R7are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, - C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl; or R6, R7form together a 5-8 membered ring, wherein the ring is substituted or unsubstituted;R11 is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;L2is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-,substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and W is a target protein binder group.

5. The SUTAC of claim 4, represented by the structure of formula I’ or salt thereof:

6. The SUTAC of claim 4, or claim 5, wherein Z is S.

7. The SUTAC of any one of claims 4-6, represented by the structure of formula IB or salt thereof:wherein: the represents a saturated or non-saturated bond; wherein if is a saturated bond, then X is C, N, O or S; wherein if is a non-saturated bond, X is C or N; each of Rs,,,, R9, R10are independently selected from the group consisting of: H, halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, haloaryl, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, thioalkoxy, thioalkyl, thioalkenyl, thiohaloalkyl, cyano, nitro, azide, amino, -COOH, -C(O)NHR’, -NHCOR”-; wherein R’ is H, alkyl, alkenyl, aryl, heterocycloalkyl, heteroaryl; and R” is hydroxy, alkoxyl, aryloxy, alkyl, alkenyl, aryl, heterocycloalkyl or heteroaryl;R11 is H, substituted or unsubstituted alkyl, alkenyl, aryl, heterocyclic or heteroaryl; n is an integer 1, 2 or 3; L1is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linear or branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10;L2 is a bond, -(CH2CH2O)q, -(CH2CH2O)q(CH2CH2)NH(CO)-, -(CH2CH2O)q(CH2CH2)-, - (CH2CH2O)q(CH2CH2)NH-, -NHCH2(CO)NHCH2Ph-, -N(alkyl)CH2(CO)NHCH2Ph-, substituted or unsubstituted linear or branched alkylene, substituted or unsubstituted linearor branched alkenylene, substituted or unsubstituted linear or branched alkynylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an ether group, an ester, an amine group, an amide group or any combination thereof, wherein q is an integer 2-10; and W is a target protein binder group.

8. The SUTAC of any one of claims 1-7, wherein said target protein binder group comprises a Bromodomain-containing protein 4 (BRD4)-targeting binder to form a BRD4-targeting SUTAC.

9. The SUTAC of claim 8, wherein said BRD4-targeting binder comprises JQ1 or an acid or ester thereof.

10. The SUTAC of claim 8, wherein said BRD4-targeting SUTAC is represented by one of theID-3IC-1.

11. The SUTAC of any one of claims 1-7, wherein said target protein binder group comprises an Androgen receptor (AR)-targeting binder to form an AR-targeting SUTAC.

12. The SUTAC of claim 11, wherein said AR-targeting SUTAC is represented by one of the following structures:

13. The SUTAC of any one of claims 1-7, wherein said target protein binder group comprises a P300-targeting binder to form a P300-targeting SUTAC.

14. The SUTAC of claim 13, wherein said P300-targeting binder comprises GNE-207, or a derivative thereof.

15. The SUTAC of claim 13, wherein said P300-targeting SUTAC is represented by one of the following structures:ID-5.

16. A pharmaceutical composition comprising the SUTAC of any one of claims 1-15 and a suitable acceptable carrier.

17. The SUTAC of any one of claims 1-15 for use in treating, improving the condition, inhibiting the decline of a subject afflicted with cancer.

18. The SUTAC for use according to claim 17, wherein the cancer is selected from the group consisting of: kidney cancer, lung cancer, endometrial / uterine cancer, esophageal cancer, breast cancer, cervical cancer, liver cancer, gastric cancer, esophageal cancer, head and neck cancer, ovarian cancer, skin cancer, bile duct cancer, leukemia, lymphoma, rhabdoid, brain cancer, colon / colorectal cancer, pancreatic cancer, myeloma, prostate cancer, neuroblastoma, gastric cancer, sarcoma, thyroid cancer, bladder cancer, bone cancer or eye cancer.

19. The SUTAC of any one of claims 1-15 for use in treating, improving the condition, inhibiting the decline of a subject afflicted with inflammation, neuro-inflammation, allergy, aging, autoimmunity, viral infections, bacterial infections, obesity, neurodegenerative diseases, fibrosis, cardiovascular diseases, diabetes, Crohn’s disease, osteoporosis, Multiple Sclerosis (MS), SLE, or non-alcoholic fatty liver disease.

20. The SUTAC for use according to claim 19, wherein the diabetes is type II diabetes.

21. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject decreases the degradation of the target protein compared to a non -treated subject.

22. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject increases the degradation of the target protein compared to a non-treated subject.

23. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject decreases the stabilization of the target protein compared to a nontreated subject.

24. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject increases the stabilization of the target protein compared to a non-treated subject.

25. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject decreases the activation of the target protein compared to a non-treated subject.

26. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject increases the activation of the target protein compared to a non-treated subject.

27. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject decreases the repression of the target protein compared to a non-treated subject.

28. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject increases the repression of the target protein compared to a non-treated subject.

29. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject decreases the binding of the target protein to other protein(s) or DNA compared to a non-treated subject.

30. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject increases the binding of the target protein to other protein(s) or DNA compared to a non-treated subject.

31. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject decreases the solubilization of the target protein compared to a nontreated subject.

32. The SUTAC for use according to any one of claims 17-20, wherein administering said SUTAC to a subject increases the solubilization of the target protein compared to a nontreated subject.

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