WO2024010776A1 - Agents with transcription factor targeting moieties - Google Patents
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- WO2024010776A1 WO2024010776A1 PCT/US2023/026875 US2023026875W WO2024010776A1 WO 2024010776 A1 WO2024010776 A1 WO 2024010776A1 US 2023026875 W US2023026875 W US 2023026875W WO 2024010776 A1 WO2024010776 A1 WO 2024010776A1
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- RLMUBIZOFHUHBI-UHFFFAOYSA-N trifluoromethyl hypochlorite Chemical compound FC(F)(F)OCl RLMUBIZOFHUHBI-UHFFFAOYSA-N 0.000 description 1
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- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
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- ZDPHROOEEOARMN-UHFFFAOYSA-N undecanoic acid Chemical compound CCCCCCCCCCC(O)=O ZDPHROOEEOARMN-UHFFFAOYSA-N 0.000 description 1
- 125000002948 undecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
Definitions
- TFs sequence-specific transcription factors
- TF binding motifs typically locate in the enhancer and promotor regions of respective genes.
- These TFs promote or repress genetic transcription and play pivotal roles in the development of various human diseases, including cancer.
- the cancer dependency map project (DepMap) uncovered that TFs represent a major class of essential genes that maintain the proliferation and tumorigenesis of cancer cells, indicating a high potential of TFs as therapeutic targets.
- DepMap uncovered that TFs represent a major class of essential genes that maintain the proliferation and tumorigenesis of cancer cells, indicating a high potential of TFs as therapeutic targets.
- there are approximately 1600 putative TFs which could be classified into tens of families largely based on their distinct DNA binding domains, such as C2H2, bZIP, bHLH and Homeodomain 21.
- TF binding motif for each individual TF.
- PBM protein binding microarray
- SELEX protein binding microarray
- DAP-seq DAP-seq
- HiTS-FLIP HiTS-FLIP
- EMSA EMSA
- CHIP low-throughput and high-throughput methods
- the DNA binding motif has been successfully defined for more than 600 putative human TFs by experimental approaches and more than 1300 by theoretical methods, which presents valueless treasury that may aid the development of TFs-targeting therapies.
- TFs typically lack active sites or allosteric regulatory pockets that normally exist in kinases or other types of enzymes, thereby making it difficult to design and screen for small molecule inhibitors (SMIs) of TFs.
- SIs small molecule inhibitors
- the DNA binding motif in theory may define the biological and potentially biochemical specificity of different TFs. Indeed, a large number of studies have theoretically and/or experimentally defined the unique DNA binding motif for most TFs. Nevertheless, identification of compounds capable of binding to TFs to modify their transcriptional activity has proved problematic such that TFs are often considered “undruggable.”
- the Proteolysis-Targeting Chimeras (Pro-TAC) technology is an emerging novel class of bifunctional synthetic drug that engages the cell’s endogenous ubiquitin/proteasome system to target specific oncoprotein for proteasomal degradation in order to combat cancers.
- PROTACs are more robust, high potency and durability, capable of degrading protein targets in a catalytic manner.
- proteins that are considered “undruggable” by traditional small molecule inhibitors will be targeted using the PROTAC technology, a process that is functioning like CRISPR-mediated knockout, but on the protein level.
- this new technology has drawn a lot of attention in the drug development field.
- Tumorigenesis is initiated by either induction/activation of oncoproteins or repression/inactivation of tumor suppressor proteins. Genetic alternations, including amplification or mutation of oncogenes, as well as deletion or mutation of tumor suppressor genes have been wildly detected and play a driver role in tumorigenesis and development.
- Small molecule degraders such as molecule glues and the proteolysis targeting chimeras (PROTACs) have been developed for targeting these oncoproteins for proteasomal degradation.
- PROTACs proteolysis targeting chimeras
- PPAR ⁇ a covalent ligand
- AMPK A769662 and Compound 991
- PP2A a covalent ligand for OTUB1
- a recent preprint paper has identified a covalent ligand (EN523) for OTUB1, a OTU family member of deubiquitinase (DUB).
- EN523 binds the allosteric cysteine C23 rather than the catalytic C91 residue of OTUB1, thus does not interrupt its DUB enzymatic function.
- TFs transcription factors
- POI protein of interest
- a covalent ligand of OTUB1 an OTU family member of deubiquitinase (DUB), EN523, has been identified to develop a DUBTAC (Deubiquitinase-Targeting Chimera) platform for stabilization of CFTR (cystic fibrosis transmembrane conductance regulator), a protein responsible for cystic fibrosis (Henning et al., Nat. Chem. Biol.2022, 18:412-421 and WO 2022232634, which are incorporated herein by reference in its entirety).
- CFTR cystic fibrosis transmembrane conductance regulator
- PROTACs typically adopt small molecule inhibitor (SMIs) as ligand for protein of interest (POI), but inhibitor moieties cannot be used for DUBTAC, due to a high possibility of residual inhibitory effect on protein of interest from these engineered DUBTACs. Moreover, it is still unknown whether the OTUB1 ligand could be used for stabilizing tumor suppressive proteins.
- SIs small molecule inhibitor
- POI protein of interest
- OTUB1 ligand could be used for stabilizing tumor suppressive proteins.
- transcription factor based TF-PROTAC platforms Liu et al, JACS 143.23 (2021): 8902- 8910 and WO 2022251614, each of which are hereby incorporated by reference in their entirety and particularly in relation to transcription factor conjugation strategies
- TFs degrade oncogenic transcription factors
- TFs degrades oncogenic transcription factors
- a nucleotide- based TF-DUBTAC platform for targeted stabilization of TFs with tumor suppressive function is provided.
- the TF-DUBTAC strategy avoids or minimizes the degradation of TFs to afford stabilization of certain POIs such as tumor suppressive transcription factors.
- the agents e.g., DUBTACs
- the present disclosure may be able to stabilize the protein of interest such as tumor suppression propteins and result in therapeutic efficacy.
- the DUBTAC may comprise an oligonucleotide that hybridizes to a transcription factor of interest (e.g., a tumor suppressor transcription factor), wherein the oligonucleotide is directly or indirectly bound to a ligand of a deubiquitinase.
- the deubiquitinase is OTUB1.
- the oligonucleotide may be covalently bound to the ligand via a linker.
- the linker comprises an azide/alkyne cycloaddition reaction product.
- the linker may comprise a strained click chemistry reaction product.
- the oligonucleotide has a double-strand hairpin structure formed via intra- dimerization.
- the oligonucleotide is a double-stranded DNA comprising a sense chain and an anti-sense chain.
- the transcription factor is a tumor suppressor.
- the oligonucleotide may be p53, FOXO3A, or IRF3.
- the DUBTACs (or agents) of the present disclosure may have the structure of formula (I): ODN–L1–DUB (I) wherein DUB is a deubiquitinase binding moiety; L1 is absent or a linker; ODN is a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing. Typically, conjugation occurs between the DNA oligomer protein and the DUB moiety via an azide/alkynyl cycloaddition reaction to form the functioning PROTAC.
- DUB is a deubiquitinase binding moiety
- L1 is absent or a linker
- ODN is a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing.
- L 1 often comprises an optionally substituted heteroarylene group (e.g., a five membered heteroarylene, a six membered heteroarylene, a divalent triazole, a divalent imidazole, a divalent pyrrole, a multicyclic (e.g., bicyclic, tricyclic) heteroarylene such as a five or six membered membered heteroaryl including triazolyl, imidazolyl, pyrrolyl which may be optionally fused to an mono or bicyclic cycloalkyl such as C 3 -C 12 monocyclic cycloalkyl or C 4 -C 12 bicyclic cycloalkyl including bicyclo[6.1.0] nonane).
- an optionally substituted heteroarylene group e.g., a five membered heteroarylene, a six membered heteroarylene, a divalent triazole, a divalent imidazole, a divalent pyrrole
- L1 comprises a triazole fused to a C 6 -C 12 monocyclic or bicyclic cylcoalkyl (e.g., azide/alkynyl SPAAC reaction products).
- L 1 comprises an optionally substituted mono or multicyclic heteroarylene group or an optionally substituted mono or multicyclic heterocyclene group (e.g., a five membered heterocyclene, a six membered heterocycene, a mono or multicyclic heteroarylene (e.g., dihydroisoxazolyl which may be optionally fused to a mono or bicyclic cycloalkyl such as C 3 -C 12 monocyclic cycloalkyl or C 4 -C 12 bicyclic cycloalkyl including bicyclo[6.1.0] nonane).
- a mono or bicyclic cycloalkyl such as C 3 -C 12 monocyclic cycloalkyl or C 4 -C 12 bicyclic
- the DUB binds to a deubiquitinase.
- the deubiquitinase may be any deubiquitinase, e.g., in a cell, including cysteine protease deubiquitinases and metalloprotease deubiquitinases.
- the deubiquitinase is a cysteine protease, e.g., comprising a catalytic site cysteine amino acid residue.
- the compound may have the structure of Formula (IIA), (IIB), (IIC), or (IID): wherein Ring A is a cycloalkyl, heterocyclyl, aryl, or heteroaryl (and conjugation to L1 may occur through a substitution group) which may have 1-10 points of optional substitution which may be independently substituted with, for example, alkyl (e.g., C 1 -C 6 alkyl), haloalkyl (e.g., C 1 -C 6 haloalkyl), heteroalkyl (e.g., C 1 -C 6 heteroalkyl), halo (e.g., F, Cl, Br); R8 is hydrogen, alkyl (e.g., C1-C20 alkyl, C 1 -C 6 alkyl, lower alkyl, C1-C4 alkyl), or an electrophilic moiety; n may be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; R 9 is alkyl (e.g.
- Ring A is heteroaryl (e.g., monocyclic heteroaryl). In some embodiemnts, Ring A is a 5-membered heteroaryl (e.g., furanyl).
- R 8 is an electrophilic moiety which may be, for example, C 2 -C 6 alkenyl, C 2 - C6 alkynyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, halo, cyano, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl which may be optionally substituted (e.g., have from 1-10 points of substitution which may be independently substituted with, for example, alkyl (e.g., C 1 -C 6 alkyl), haloalkyl (e.g., C 1 -C 6 haloalkyl), heteroalkyl (e.g., C 1 -C 6 heteroalkyl), halo (
- the DUB may have the structure of: wherein p is 0, 1, 2, or 3; and indicates the point of attachment to L 1 .
- DUB may have the structure of EN523 and the agent may have the structure of formula (III): The ability to degrade the transcription factor may be strongly dependent on the linker between the chimera.
- the agent has the structure of formula (IV): ODN–Y 1 –Y 2 –Y 3 –Y 4 –Y 5 –Y 6 –Y 7 –Y 8 –Y 9 –DUB (IV) wherein Y1-Y9 are independently selected from absent (i.e., it is a bond), an optionally substituted heteroarylene group or optionally substituted heterocycloalkylene (e.g., a five membered heteroarylene, a six membered heteroarylene, a triazolylene, a imidazole, a divalent pyrrole) optionally fused to an mono or bicyclic cycloalkyl (e.g., C 3 -C 10 monocyclic cycloalkyl, C 4 -C 10 bicyclic cycloalkyl such as bicyclo[6.1.0] nonane) –C(O)–, –O–, –OC(O)–, –NR a –, –N(
- At least one of Y 1 -Y 9 comprises a linker moiety that is a click chemistry reaction product including click chemistry reaction products synthesized via another reaction mechanism.
- at least one of Y 1 -Y 9 may include a moiety that could be formed from Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction product (e.g., a divalent triazole), a strain-promoted azide-alkyne cycloaddition (SPAAC) product (e.g., a divalent fused triazole), a strain-promoted alkyne-nitrone cycloaddition (SPANC) product (e.g., a divalent fused dihydroisoxazole), or another product of strained alkene reactions such as alkene-azide cycloaddition.
- CuAAC Copper(I)-catalyzed azide-alkyne cycloaddition
- Click-chemistry compatible reactions may also be considered to include alkene-tetrazine inverse-demand Diers-Alder reactions, alkene-tetrazole photoclick reactions, Michael additions of thiols, nucleophilic substitution of thiols with amines, and certain Diels-Alder reactions, such as those disclosed by Becer, et al. Angew. Chem. Int. Ed.48 (2009): 4900-4908, which is hereby incorporated by reference in its entirety.
- one of Y 1 -Y 9 includes a triazole product (e.g., similar to those produced from a Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, or a Strain- promoted azide-alkyne cycloaddition (SPAAC)).
- one of Y 1 -Y 9 includes an isoxazole product such as fused dihydroisoxazole (e.g., similar to those produced from a Strain- promoted alkyne-nitrone cycloaddition (SPANC) reaction).
- SPANC Strain- promoted alkyne-nitrone cycloaddition
- the compound has the structure of formula (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), or (IVt): ODN–Y 1 –Y 2 –Y 3 –NH–(CH 2 ) 1-12 –NH–C(O)–DUB (IVa) ODN–Y1–Y2–Y3–(CH2)1-12–NH–C(O)–(CH2)1-12–DUB (IVb) ODN–Y1–Y2–Y3–(CH2)1-12–NH–(CH2)1-12–DUB (IVc) ODN–Y1–Y2–Y3–NH–(CH2)1-12–NH–C(O)–DUB (IVd) ODN–Y1–Y2–Y3–C(O)–(CH2)1-12–DUB (IVd
- the compound has the structure of formula, (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), or (IVt1): ODN–NHCO–(CH 2 ) 1-12 –Y 3 –NH–(CH 2 ) 1-12 –NH–C(O)–DUB (IVa1) ODN–NHCO–(CH2)1-12–Y 3 –(CH2)1-12–NH–C(O)–(CH2)1-12–DUB (IVb1) ODN–NHCO–(CH2)1-12–Y 3 –(CH2)1-12–NH–(CH2)1-12–DUB (IVc1) ODN–NH–(CH 2 ) 1-12 –Y 3 –NH–(CH 2 ) 1
- At least one of Y 1 -Y 7 may be a linker thaty may be formed from a strained click chemistry reaction (also referred to herein as –LSCC—) such as those having the structure:
- each indicates the point of attachment to the neighboring linker group (e.g., Y1, Y2, Y3 , Y 4 ); m is an integer from 0-12 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12); p is an integer from 0-10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); Z1 is –N– or –CR3–; Z 2 is O, C(O), or C(R 3 ) 2 ; and R3 is independently selected at each occurrence from hydrogen, –N(R a )(R a ), alkyl (e.g., C1-C7 alkyl, C 1 -C 3 alkyl, etc.), or alkoxy (e.g., C 1 -C 7 alkoxy, C 1 -C 3 alkoxy, etc.) and wherein any two vicinal R 3 groups of the C8 ring may together form a five or six membered optionally substituted optionally aromatic ring (e.g., ary
- At least one of Y 1 -Y 7 comprises the structure:
- the compound has the structure of formula (V): ODN–Y 1 –Y 2 –L SCC –Y 4 –Y 5 –Y 6 –Y 7 –Y 8 – Y 9 –DUB.
- the compound of the present disclosure may have the structure: ODN–NHCO–(CH2)1-12–LSCC—NH–(CH2)1-12–NH–C(O)–DUB ODN–NHCO–(CH 2 ) 1-12 – L SCC –(CH 2 ) 1-12 –NH–C(O)–(CH 2 ) 1-12 –DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2)1-12–NH–(CH2)1-12–DUB ODN–NH–(CH 2 ) 1-12 – L SCC –NH–(CH 2 ) 1-12 –NH–C(O)–DUB ODN–NH–(CH2)1-12– LSCC –C(O)–(CH2)1-12–DUB ODN–NH–(CH 2 ) 1-12 – L SCC –NH–(CH 2 ) 1-12 –DUB ODN–NH–(CH2)1-12– LSCC –C(O)–(CH2)1-12–DUB ODN
- the compound may have the structure of formula (Va):
- the linker constituents and ultimate length alter the activity of the DUBTAC.
- the compound has the structure of formula (Vb), (Vc), (Vd), or (Ve):
- Y7, Y8, and Y9 are independently selected from absent (i.e., it is a bond), –(C(R a )(R a ))1-12, – N(Ra)C(O)– and –(C(R a )(R a )C(R a )(R a )O)1-12–; and R a is independently selected at each occurrence from hydrogen and optionally substitued alkyl (e.g., C 1 -C 4 alkyl).
- Y 8 is absent and Y 7 is –(CH 2 ) 1-12 –.
- Y7 is –(CH2CH2O)1-12– and Y8 is –(CH2)1-12–.
- Y7 is –(CH2)1-12–
- Y8 is – NH(R a )–
- Y 9 is –(CH 2 ) 1-12 –.
- –Y 7 –Y 8 –Y 9 – may be part of a linker as shown in Table 1.
- the compound comprises linkers 19, 20, 21, 22, 23, 24, 25, 26, or 27 from Table 1.
- any one of Y 1 -Y 9 e.g., Y 7
- the compound may have the structure of formula (Vf): wherein r and s are independently 1-12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) and the wavy bond illustrates conjugation to ODN (e.g., via (e.g., directly to) the 5’ end of the oligonucleotide).
- s is 5.
- ODN targetting factor is typically guided by those DNA oligomer protein sequences (e.g., double stranded DNA oligomers) which bind to a transcription factor binding motif (e.g., tumor suppressor transcription factors).
- ODN may bind to SMAD4, RFX7, REST, FOXP3, FOXP1, p53, FOXO3A, or IRF3.
- the ODN may bind to p53, FOXO3A, or IRF3.
- ODN has a double-band hairpin structure optionally formed via intra-dimerization.
- ODN is a double-stranded DNA comprising a sense chain and an anti-sense chain.
- the transcription factor has one or more forkhead consensus binding sites.
- the DNA oligomer protein of ODN may have from 1-30 bases (e.g., 10-20 bases, 1-10 bases, 20-30 bases) and comprises transcription factor binding motif.
- the transcription factor binding motif is G/TAAAC/TA.
- Suitable binding motifs are described in Brunet, A. Cell 96.6 (1999): 857-868 and Tsai, K. L., et al, Nucleic Acids Res 35.20 (2007): 6984-6994, which are hereby incorporated by reference in their entirety and particularly in relation to suitable binding motifs.
- ODN comprises 5'- PuPuPuC(A/T)(T/A)GPyPyPy-3' (where Pu stands for purine and Py for pyrimidine) separated by 0- 13 base-pairs.
- ODN comprises two tandem repeates of 10 base-pair motif 5'- PuPuPuC(A/T)(T/A)GPyPyPy-3' (where Pu stands for purine and Py for pyrimidine) separated by 0- 13 base-pairs separated by 0-13 base pairs.
- ODN comprises AANNGAAA (N stands for any nucleotide).
- Suitable binding motifs are described in Panne D., et al, Cell 129.9 (2007): 1111-1123 and Panne, D., et al.
- ODN may comprise, be substantially identical to, or is the sequence CTATGTAAACAACTTTGTTGTTTACATAG, AGACATGCCTAGACATGCCT, TCCGTACAGATCCGTACAGA, GAAACTGAAACTTTTAGTTTCAGTTTC, 5’-CTATGTAAACAACTTTGTTGTTTACATAG-3’, 5’-AGACATGCCTAGACATGCCT-3’, 5’-TCCGTACAGATCCGTACAGA-3’, or 5’-GAAACTGAAACTTTTAGTTTCAGTTTC-3’; each of which may form a double stranded DNA hairpin structure via interdimerization.
- ODN is a double stranded DNA having a sense chain and an anti-sense chain, wherein the sense chain.
- the sense chain may be 5’-AGACATGCCTAGACATGCCT- 3’; and the anti-sense chain may be 5’-TCCGTACAGATCCGTACAGA-3’.
- the ODN oligonucleotide may be conjugated to L 1 through the 5’ end of the oligonucleotide (e.g., via the amino modifier C6).
- the compound may be, for example, any compound in Table 2 and formula (Vg) (where the ODN is conjugated through the 5’ end of the oligonucleotide).
- Y7 may be a linear or branched linker, wherein the linking portion between adjacent linking moieties has from 1-12 atoms.
- Y 7 is –(CH 2 ) 2-11 – and ODN is 5’-CTATGTAAACAACTTTGTTGTTTACATAG-3’, 5’-GAAACTGAAACTTTTAGTTTCAGTTTC-3’, or 5’-AGACATGCCTAGACATGCCT-3’ (bound to 5’-TCCGTACAGATCCGTACAGA-3’) Table 2
- compositions comprising the DUBTACs (or agents) are also provided. These compositions may comprise one or more pharmaceutically acceptable salts, carriers, or diluents, and a DUBTAC.
- the composition comprises an agent comprising an oligonucleotide that hybridizes to a transcription factor of interest (e.g., a tumor suppressor transcription factor), wherein the oligonucleotide is directly or indirectly bound to a ligand of a deubiquitinase.
- a transcription factor of interest e.g., a tumor suppressor transcription factor
- the composition may comprise an agent having the structure of formula (I): ODN–L 1 –DUB (I) wherein DUB is a deubiquitinase binding moiety; L 1 is absent or a linker; ODN is a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing.
- Methods for reducing the proliferation or survival of a neoplastic cell comprising a transcription factor of interest are also provided comprising contacting the cell with a DUBTAC. Contact may stabilize the transcription factor in the cell.
- the comprises comprises contacting the cell with an agent comprising an oligonucleotide that hybridizes to the transcription factor of interest (e.g., a tumor suppressor transcription factor), wherein the oligonucleotide is directly or indirectly bound to a ligand of a deubiquitinase.
- an agent comprising an oligonucleotide that hybridizes to the transcription factor of interest (e.g., a tumor suppressor transcription factor), wherein the oligonucleotide is directly or indirectly bound to a ligand of a deubiquitinase.
- the composition may comprise an agent having the structure of formula (I): ODN–L 1 –DUB (I) wherein DUB is a deubiquitinase binding moiety; L 1 is absent or a linker; ODN is a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing.
- a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing.
- these methods may comprise administering a DUBTAC of the present disclosure (or a pharmaceutical composition comprising a DUBTAC of the present disclosure) to the subject in need thereof.
- the method comprises administering an agent comprising an oligonucleotide that hybridizes to a transcription factor of interest (e.g., a tumor suppressor transcription factor), wherein the oligonucleotide is directly or indirectly bound to a ligand of a deubiquitinase.
- a transcription factor of interest e.g., a tumor suppressor transcription factor
- the composition may comprise an agent having the structure of formula (I): O DN–L1–DUB (I) wherein DUB is a deubiquitinase binding moiety; L 1 is absent or a linker; ODN is a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing.
- a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing.
- Compounds for synthesis of the DUBTACs as described herein are also provided.
- These synthetic intermediates may be a compound having the structure of formula (VI): Y2–Y3–Y4–Y5–Y6–Y7–Y8–Y9–DUB (VI) wherein DUB is a deubiquitinase binding moiety; and wherein Y2 is optionally substituted alkynyl (e.g., C1-C12 alkynyl), optionally substituted monocyclic or bicyclic cycloalkynyl (e.g., C 3 -C 12 monocyclic or multicyclic cycloalkynyl, C 3 -C 12 bicyclic cycloalkynyl), optionally substituted heteroalkynyl (e.g., C1-C12 heteroalkynyl), or optionally substituted monocyclic or bicyclic heterocycloalkynyl (e.g., C 3 -C 12 monocyclic heterocycloalkynyl, C3-C12 bicyclic heterocycloalkynyl);
- m is an integer from 0-12 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12); p is an integer from 0-10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10);
- Z1 is –N– or –CR3–;
- Z 2 is O, C(O), or C(R 3 ) 2 ;
- R3 is independently selected at each occurrence from hydrogen, –N(R a )(R a ), alkyl (e.g., C1-C7 alkyl, C 1 -C 3 alkyl, etc.), or alkoxy (e.g., C 1 -C 7 alkoxy, C 1 -C 3 alkoxy, etc.) and wherein any two vicinal R 3 groups of the C8 ring may together form a five or six membered optionally aromatic ring (e.g., aryl such as phenyl) fused to the C 8 ring.
- aromatic ring e.g., aryl such as phen
- the compound may have the structure of formula (VIa):
- the compound may, for example, have the structure of formula (VIb): .
- Y7 and Y9 are independently selected from absent (i.e., it is a bond), –(C(R a )(R a ))1-12, and –(C(R a )(R a )C(R a )(R a )O) 1-12 –; and R a is independently selected at each occurrence from hydrogen and optionally substitued alkyl (e.g., C 1 -C 4 alkyl).
- Y 9 is –(CH 2 ) 2 –.
- the synthetic intermediate is a compound having the structure of formula (VIb) with the Y7, Y8, and Y9 linkers in Table 1.
- the synthetic intermediates may have a linker length and be characterized with a click efficiency (e.g., as measured by reaction between a synthetic intermediate of the present disclosure with 10 fold excess of an azide linked ODN group) may be above 10%, above 20% or above 30% or above 40% or above 60% or above 70% or above 80% or above 90% (e.g., from 10%-99%, from 20%-99%, from 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, from 10%-99%, from 20%- 99%, from 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, from 20%-90%, from 30%-90%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, from 20%-90%, from 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-99%, from 20%-90%, from 30%-90%, 40%-90%, 50%-90%, 60%
- Methods for forming the DUBTACs of the present disclosure a compound ((e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (
- these methods comprise reacting (e.g. via an azide-alkyne cycloaddition reaction) a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof; where said DNA oligomer protein is conjugated to an azide with a compound having the structure of formula (VI): Y 2 –Y 3 –Y 4 –Y 5 –Y 6 –Y 7 –Y 8 –Y 9 –DUB (VI) wherein DUB is a deubiquitinase binding moiety; and wherein Y 2 is optionally substituted alkynyl (e.g., C 1 -C 12 alkynyl), optionally substituted monocyclic or bicyclic cycloalkynyl (e.g., C3-C12 monocyclic or multicyclic cycloalkynyl, C3-C12 bicyclic cycloalkynyl), optionally substituted heteroalkynyl (e.g.,
- the method may further include the step of conjugating an azide to the DNA oligomer protein.
- the azide can be introduced to an oligonucleotide by attaching an azide-NHS ester functional group to 5', 3' or through internal amino modified base or amino linkers such as amino C6 or amino C7 for the 3' end of an oligo. Synthetic steps may occur in physiological conditions such as in phosphate buffered saline (PBS) at from 30°C-40°C.
- PBS phosphate buffered saline
- These synthetic methods be characterized with a click efficiency (e.g., as measured by reaction between a synthetic intermediate of the present disclosure with 10 fold excess of an azide linked ODN group) may be above 10%, above 20% or above 30% or above 40% or above 60% or above 70% or above 80% or above 90% (e.g., from 10%-99%, from 20%-99%, from 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, from 10%-99%, from 20%-99%, from 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%- 99%, from 20%-90%, from 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%, from 20%-90%, from 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%) efficient.
- a click efficiency e.g., as measured by reaction between a synthetic intermediate of the present disclosure with 10 fold excess of an azide linked ODN group
- a click efficiency e.g., as measured by reaction between a synthetic intermediate of the present disclosure
- another means at least a second or more.
- all ranges of numeric values include the endpoints and all possible values disclosed between the disclosed values. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. For example, the exact values of all half-integral numeric values are also contemplated as specifically disclosed and as limits for all subsets of the disclosed range. For example, a range of from 0.1% to 3% specifically discloses a percentage of 0.1%, 1%, 1.5%, 2.0%, 2.5%, and 3%.
- a range of 0.1 to 3% includes subsets of the original range including from 0.5% to 2.5%, from 1% to 3%, from 0.1% to 2.5%, etc. It will be understood that sum of all weight percentages does not exceed 100% unless otherwise specified.
- OTUB1 polypeptide is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_060140.2 or a fragment thereof having deubiquitinase activity.
- An exemplary OTUB1 amino acid sequence follows: 1 maaeepqqqk qeplgsdseg vnclaydeai maqqdriqqe iavqnplvse rlelsvlyke 61 yaeddniyqq kikdlhkkys yirktrpdgn cfyrafgfsh leallddske lqrfkavsak 121 skedlvsqgf teftiedfhn tfmdlieqve kqtsvadlla sfndqstsdy lvvylrllts 181 gylqreskff ehfieggrtv kefcqqevep mckesdhihi ialaqalsvs iqveymdrge 241 ggttnphifp egsepkv
- OTUB1 polynucleotide sequence is provided at NCBI Accession No. NM_017670, which is reproduced below: 1 agtgcggcgc tgtttaaaga tggcggcgga ggaacctcag cagcagaagc aggagccgct 61 gggcagcgac tccgaaggtg ttaactgtct ggcctatgat gaagccatca tggctcagca 121 ggaccgaatt cagcaagaga ttgctgtgca gaaccctctg gtgtcagagc ggctggagct 181 ctcggtccta tacaaggagt atgctgaaga tgacaacatc tatcaacaga agatcaagga 241 cctccacaaaaagtactc
- An exemplary p53 amino acid sequence follows: 1 meepqsdpsv epplsqetfs dlwkllpenn vlsplpsqam ddlmlspddi eqwftedpgp 61 deaprmpeaa prvapapaap tpaapapaps wplsssvpsq ktyqgsygfr lgflhsgtak 121 svtctyspal nkmfcqlakt cpvqlwvdst pppgtrvram aiykqsqhmt evvrrcphhe 181 rcsdsdglap pqhlirvegn lrveylddrn tfrhsvvvpy eppevgsdct tihynymcns 241 scmggmn
- IRF3 amino acid sequence follows: 1 mgtpkprilp wlvsqldlgq legvawvnks rtrfripwkh glrqdaqqed fgifqawaea 61 tgayvpgrdk pdlptwkrnf rsalnrkegl rlaedrskdp hdphkiyefv nsgvgdfsqp 121 dtspdtnggg stsdtqedil dellgnmvla plpdpgppsl avapepcpqp lrspsldnpt 181 pfpnlgpsen plkrllvpge ewefevtafy rgrqvfqqti scpeglrlvg sevgdrtlpg 241 wpvtlpdpg
- IRF3 polynucleotide sequence is provided at NCBI Accession No. NG_ 31810.1, which is reproduced below: 1 tcaagaagtc gatcaaaag aaagccccag cgctctagag ctcagctgac gggaaagggg 61 gtgcgcagcc tcgagtttga gagctacccg gagctccaag acaggggtgg gttccagctg 121 cccgcacgccccgaccttcc atcgtaggcc ggaccatggg aaccccaaag ccacggatcc 181 tgccctggct ggtgtcgcag ctggacctgg ggcaactgga gggcgtggcc tgggtgaaca 241 agagccgcac gcgcgcgc
- An exemplary FOXO3a amino acid sequence follows: 1 maeapaspap lspleveldp efepqsrprs ctwplqrpel qaspakpsge taadsmipee 61 eddeddedgg gragsamaig ggggsgtlgs gllledsarv lapggqdpgs gpataaggls 121 ggtqallqpq qplpppqpga aggsgqprkc ssrrnawgnl syadlitrai esspdkrltl 181 sqiyewmvrc vpyfkdkgds nssagwknsi rhnlslhsrf mrvqnegtgk sswwiinpdg 241 gksgkaprrr avsmdnsnky
- An exemplary SMAD4 amino acid sequence follows: 1 mdnmsitntp tsndaclsiv hslmchrqgg esetfakrai eslvkklkek kdeldslita 61 ittngahpsk cvtiqrtldg rlqvagrkgf phviyarlwr wpdlhknelk hvkycqyafd 121 lkcdsvcvnp yhyervvspg idlsgltlqs napssmmvkd eyvhdfegqp slsteghsiq 181 tiqhppsnra stetystpal lapsesnats tanfpnipva stsqpasilg gshsegllqi 241 asgpqpgqqqqqq ngftg
- SMAD4 polynucleotide sequence is provided at NCBI Accession No. NM_001407041.1, which is reproduced below: 1 atgctcagtg gcttctcgac aagttggcag caacaacacg gccctggtcg tcgtcgcgc 61 tgcggatcaa aattgcttca gaaattggag acatatttga tttaaagga aaacttgaa 121 caaatggaca atatgtctat tacgaataca ccaacaagta atgatgcctg tctgagcatt 181 gtgcatagtt tgatgtgcca tagacaaggt ggagagagtg aaacatttgc aaaaagagca 241 attgaaagtt tggggagagag
- An exemplary RFX7 amino acid sequence follows: 1 msssraqqmh afswirntle ehpetslpkq evydeyksyc dnlgyhplsa adfgkimknv 61 fpnmkarrlg trgkskycys glrkkafvhm ptlpnldfhk tgdglegaep sgqlqnidee 121 vissacrlvc ewaqkvlsqp fdtvlelarf lvkshyigtk smaaltvmaa apagmkgitq 181 psafiptaes nsfqpqvktl pspidakqql qrkiqkkqqe qklqsplpge saakksesat 241 sngvtnl
- An exemplary REST amino acid sequence follows: 1 matqvmgqss gggglftssg nigmalpndm ydlhdlskae laapqlimla nvaltgevng 61 sccdylvgee rqmaelmpvg dnnfsdseeg egleesadik gephglenme lrslelsvve 121 pqpvfeasga pdiyssnkdl ppetpgaedk gkssktkpfr ckpcqyeaes eeqfvhhirv 181 hsakkffvee saekqakare sgsstaeegd fskgpircdr cgyntnrydh ytahlkhhtr 241 agdnervykc iictyttvse yhwr
- An exemplary FOXP3 amino acid sequence follows: 1 mpnprpgkps apslalgpsp gaspswraap kasdllgarg pggtfqgrdl rggahassss 61 lnpmppsqlq lstvdahart pvlqvhples pamisltppt tatgvfslka rpglppginv 121 aslewvsrep allctfpnps aprkdstlsa vpqssyplla ngvckwpgce kvfeepedfl 181 khcqadhlld ekgraqcllq remvqsleqqq lvlekeklsa mqahlagkma ltkassvass 241 dkgsccivaa gsqgpvvpaw sgpreap
- An exemplary FOXP3 polynucleotide sequence is provided at NCBI Accession No. NM_001114377.2, which is reproduced below: 1 agtttcccac aagccaggct gatccttttc tgtcagtcca cttcaccaag cctgccctg 61 gacaaggacc cgatgcccaa ccccaggcct ggcaagccct cggcct cggcctc cttt 121 ggcccatccc caggagcctc gcccagctgg agggctgcac ccaaagcctc agacctgctg 181 ggggcccggg gcccaggggg aaccttccag ggccgagatc ttcgaggcgg ggcccatgcc 241 tcctcttt
- FOXP1 amino acid sequence follows: 1 mmqesgtetk sngsaiqngs ggsnhllecg glregrsnge tpavdigaad lahaqqqqqq 61 whlinhqpsr spsswlkrli sspwelevlq vplwgavaet kmsgpvcqpn pspf
- FOXP1 polynucleotide is meant a polynucleotide encoding a FOXP1 polypeptide.
- An exemplary FOXP1 polynucleotide sequence is provided at NCBI Accession No.
- ameliorate is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
- alteration is meant a change (increase or decrease) in the expression levels, structure, or activity of a gene or polypeptide as detected by standard art known methods such as those described herein.
- an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
- analog is meant a molecule that is not identical but has analogous functional or structural features.
- a polypeptide analog retains the biological activity of a corresponding naturally occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding.
- An analog may include an unnatural amino acid.
- the term “antisense strand” refers to a polynucleotide that is substantially or 100% complementary to a target nucleic acid of interest.
- an antisense strand may be complementary, in whole or in part, to a molecule of mRNA (messenger RNA), an RNA sequence that is not mRNA (e.g., microRNA, piwiRNA, tRNA, rRNA and hnRNA) or a sequence of DNA that is either coding or non-coding.
- mRNA messenger RNA
- RNA sequence that is not mRNA
- guide strand are used interchangeably herein.
- “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S.
- an antisense molecule is in large part complementary to a target sequence.
- the complementarity need not be perfect, but may include mismatches at 1, 2, 3, or more nucleotides.
- Detect refers to identifying the presence, absence or amount of the analyte to be detected.
- detectable label is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
- useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
- disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
- expression or “expressed” as used herein in reference to a gene means the transcriptional and/or translational product of that gene.
- the level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88).
- Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell.
- Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.
- effective amount is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient.
- the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
- the term “effective amount” or “therapeutically effective amount” of an agent, as used herein, may be that amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied.
- an effective amount is the amount of an irradiated compound described herein sufficient to affect the degradation of a protein of interest.
- an effective amount of an agent in the context of administering an agent that is an anticancer agent, is, for example, an amount sufficient to achieve alleviation or amelioration or prevention or prophylaxis of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition (e.g., cancer, etc.); and remission (whether partial or total), whether detectable or undetectable, as compared to the response obtained without administration of the agent.
- the effective amount assumes that more than 50% of the compounds administered release the photolabile group under irradiation conditions (e.g., more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%, 100%, etc.) to achieve the active compound capable of degrading a protein of interest.
- the invention provides a number of targets that are useful for the development of highly specific drugs to treat, or a disorder characterized by the methods delineated herein.
- the methods of the invention provide a facile means to identify therapies that are safe for use in subjects.
- the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
- fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
- a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
- “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
- a “host cell” or “cell” is any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
- isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation.
- a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography (HPLC). The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
- isolated polynucleotide is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
- the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
- the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
- an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it.
- the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated.
- the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
- An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
- a “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
- reference is meant a standard or control condition.
- a “reference sequence” is a defined sequence used as a basis for sequence comparison.
- a reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
- the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids.
- the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
- specifically binds is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
- Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof.
- nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity.
- Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
- Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity.
- Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
- hybridize pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
- complementary polynucleotide sequences e.g., a gene described herein
- stringency See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol.152:507).
- stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
- Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
- Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
- Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
- concentration of detergent e.g., sodium dodecyl sulfate (SDS)
- SDS sodium dodecyl sulfate
- Various levels of stringency are accomplished by combining these various conditions as needed.
- hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
- hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
- hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
- washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
- Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C.
- wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
- wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
- wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
- Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
- substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
- a reference amino acid sequence for example, any one of the amino acid sequences described herein
- nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
- such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
- Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications.
- Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
- a BLAST program may be used, with a probability score between e -3 and e -100 indicating a closely related sequence.
- subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. Ranges provided herein are understood to be shorthand for all of the values within the range.
- a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
- the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
- the present disclosure features a method of treating or preventing a disease, disorder or condition that can be suppressed by a transcription factor, e.g., a tumor suppressing transcription factor and, unstable tumor suppressing transcription factors, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
- a transcription factor e.g., a tumor suppressing transcription factor and, unstable tumor suppressing transcription factors
- the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.
- the disease, disorder, or condition is selected from the group consisting of a respiratory disorder, a proliferative disorder, an autoimmune disorder, an autoinflammatory disorder, an inflammatory disorder, a metabolic disorder, a neurological disorder, and an infectious disease.
- the disease, disorder, or condition is selected from the group consisting of a respiratory disorder, a proliferative disorder, an autoimmune disorder, an autoinflammatory disorder, an inflammatory disorder, a neurological disorder, and an infectious disease.
- the disease, disorder, or condition comprises a respiratory disorder.
- the disease, disorder, or condition comprises a proliferative disorder.
- the disease, disorder, or condition comprises an autoinflammatory disorder.
- the disease, disorder, or condition comprises an inflammatory disorder.
- the disease, disorder, or condition comprises a metabolic disorder.
- the disease, disorder, or condition comprises a neurological disorder.
- the disease, disorder, or condition comprises an infectious disease.
- the disease, disorder, or condition is cancer. In some embodiments, the disease, disorder, or condition is cystic fibrosis. In some embodiments, the disease, disorder, or condition is diabetes.
- the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural. By “consist essentially” it is meant that the ingredients include only the listed components along with the normal impurities present in commercial materials and with any other additives present at levels which do not affect operation, for instance at levels less than 5% by weight or less than 1% or even 0.5% by weight.
- alkyl groups described herein refer to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of 1-30 carbon atoms (e.g., 1-16 carbon atoms, 6-20 carbon atoms, 8-16 carbon atoms, or 4-18 carbon atoms, 4-12 carbon atoms, 1-4 carbon atoms, 1-8 carbon atoms) which are optionally substituted.
- the alkyl group may be substituted with 1, 2, 3, or 4 substituent groups as defined herein.
- Alkyl groups may have from 1-26 carbon atoms.
- alkyl groups will have from 6-18 or from 1-8 or from 1-6 or from 1-4 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. Any alkyl group may be substituted or unsubstituted. Examples of alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl groups.
- Heteroalkyl groups may refer to branched or straight-chain monovalent optionally saturated aliphatic hydrocarbon radicals with one or more heteroatoms (e.g., N, O, S, etc.) in the carbon chain. Heteroalkyl groups may have 1-30 carbon atoms (e.g., 1-16 carbon atoms, 6-20 carbon atoms, 8-16 carbon atoms, or 4-18 carbon atoms, 4-12 carbon atoms, etc.) with one or more heteroatoms (e.g., N, O, S, etc.) replacing a carbon in the chain. In some embodiments, the heteroalkyl group may be substituted with 1, 2, 3, or 4 substituent groups as defined herein.
- Heteroalkyl groups may have from 1-26 carbon atoms. In other embodiments, heteroalkyl groups will have from 6-18 or from 1-8 or from 1-6 or from 1-4 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. In some embodiments, the heteroalkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups. Examples of heteroalkyl groups are an “alkoxy” which, as used herein, refers alkyl-O-; and “alkoyl” which, as used herein, refers to alkyl-CO-.
- Alkoxy substituent groups or alkoxy-containing substituent groups may be substituted by, for example, one or more alkyl groups.
- Alkenyl groups include alkyl groups having a double bond between any two carbon atoms.
- Alkynyl groups include alkyl groups having a triple bond between carbon atoms.
- Aryl groups may be aromatic mono- or polycyclic radicals of 6 to 12 carbon atoms having at least one aromatic ring. Aryl groups may be optionally substituted. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalyl, 1,2- dihydronaphthalyl, indanyl, and 1H-indenyl.
- Aryl groups as used herein may be substituted or unsubstitued.
- heteroaryls include mono- or polycyclic radical of 5 to 12 atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, and S, with the remaining ring atoms being C.
- One or more ring carbon atoms of the heteroaryl group may be replaced with a carbonyl group.
- heteroaryl groups are pyridyl, triazolyl, benzooxazolyl, benzoimidazolyl, and benzothiazolyl.
- Linking groups when present, may be referred to as divalent hydrocarbon groups (e.g., alkylene, heteroalkylene, alkynylene, alkeneylene, heteroalkynylene, heteroalkeneylene, arylene, heteroarylene), where each radical position is the point of attachment to the remaining portions of the compound.
- divalent hydrocarbon groups e.g., alkylene, heteroalkylene, alkynylene, alkeneylene, heteroalkynylene, heteroalkeneylene, arylene, heteroarylene
- Alkylene groups may refer to a straight or branched chain divalent hydrocarbon radical having from one to ten carbon atoms, optionally substituted with substituents, for example, selected from the group which includes lower alkyl (e.g., C 1 -C 4 , etc.), lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen and lower perfluoroalkyl, multiple degrees of substitution being allowed.
- substituents for example, selected from the group which includes lower alkyl (e.g., C 1 -C 4 , etc.), lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alky
- alkylene examples include, but are not limited to, methylene, ethylene, n- propylene, n-butylene, and the like.
- Alkylene groups may be saturated or unsaturated.
- Heteroalkylene groups may be alkylene groups comprising one or more heteroatoms (e.g., N, S, O) in the carbon chain.
- Cycloalkylene groups may be divalent hydrocarbons comprising one or more saturated or unsaturated cycloalkyl groups. The two points of attachment on cycloalkylene groups may be at two points in the ring, for example, at vicinal positions or at geminal positions.
- Cycloalkylene groups may be divalent saturated mono- or multicyclic ring system, in certain embodiments of 3 to 10 carbon atoms, in other embodiments 3 to 6 carbon atoms; cycloalkenylene and cycloalkynylene refer to divalent mono- or multicyclic unsaturated ring systems that respectively include at least one double bond and at least one triple bond. Cycloalkylene, Cycloalkenylene and cycloalkynylene groups may, in certain embodiments, contain 3 to 10 carbon atoms, with cycloalkenylene groups in certain embodiments containing 4 to 7 carbon atoms and cycloalkynylene groups in certain embodiments containing 8 to 10 carbon atoms.
- any divalent linking moiety with multiple points of attachment may be attached to the specified moieties in either direction to the extent permitted by valency, unless otherwise indicated (e.g., by indicating the attachments on each side).
- a linking moiety having the structure –Y 1 –Y 2 – may be used to link two portions of a compound in the –Y 1 –Y 2 – orientation or in the –Y 2 –Y 1 – orientation.
- the ring systems of the saturated, partially saturated, or unsaturated cycloalkyl, aryl, heterocylcoalkyl, heteroaryl, cycloalkylene, cycloalkenylene cycloalkynylene, heterocycloalkylene, heterocycloalkenylene and heterocycloalkynylene groups may be composed of one ring, two rings, or three or more rings (e.g., from 1 to 5 rings) which may be joined together in a fused, bridged or spiro-connected fashion, each of which may be optionally substituted.
- Heteocyclo and Heterocyclene groups may be monocyclic or multicyclic non-aromatic ring system, in certain embodiments of 3 to 10 members, in one embodiment 4 to 7 members, in another embodiment 5 to 6 members, where one or more, including 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
- Aryl and Arylene groups may be monocyclic or polycyclic, in certain embodiments monocyclic, aromatic group, in some embodiments having from 5 to 20 carbon atoms and at least one aromatic ring, in another embodiment 5 to 12 carbons.
- Arylene groups include, but are not limited to, 1,2-, 1,3- and 1,4-phenylene.
- Heteroarylene groups are typically divalent monocyclic or multicyclic aromatic ring systems, in one embodiment of 5 to 15 atoms in the ring(s), where one or more, in certain embodiments 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
- heteroarylene or heterocyclene groups are divalent optionally substituted multicyclic heteroaryl or heterocyclyl fused with a monocyclic or multicyclic (e.g., bicyclic, tricyclic) optionally saturated optionally substituted cycloalkyl, wherein one point of attachment to the rest of the compound is on the heteroaryl moiety and the other point of attachment to the rest of the compound is on the cycloalkyl moiety.
- a monocyclic or multicyclic e.g., bicyclic, tricyclic
- the multicyclic heteroarylene or heterocyclene groups may have have the structure: wherein wherein each indicates a point of attachment to the compound; ring “A” is optionally aromatic five or six membered ring; r and s are independently 0, 1, 2, 3, 4, or 5; t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; X1-X4 are independently selected at each occurrence from absent, CR, C(R)2, N, NR, O, S or SR; one of X 1 -X 4 comprises a (e.g., in place of an R group); and at least one of X 1 -X 4 is not CR or C(R)2; X 5 is CR or N; X6 is absent, C(R)2, NR, or O; R is independently selected at each occurrence from hydrogen, halogen, –OR a
- substituted refers to a group “substituted” on, e.g., an alkyl, at any atom of that group, replacing one or more hydrogen atoms therein.
- the substituent(s) on a group are independently any one single, or any combination of two or more of the permissible atoms or groups of atoms delineated for that substituent.
- a substituent may itself be substituted with any one of the substituents described herein.
- a substituted hydrocarbon group may have as a substituent one or more hydrocarbon radicals, substituted hydrocarbon radicals, or may comprise one or more heteroatoms. Examples of substituted hydrocarbon radicals include, without limitation, heterocycles, such as heteroaryls.
- a hydrocarbon substituted with one or more heteroatoms will comprise from 1-20 heteroatoms. In other embodiments, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-12 or from 1-8 or from 1-6 or from 1-4 or from 1-3 or from 1-2 heteroatoms.
- heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorous, halogen (e.g., F, Cl, Br, I, etc.), boron, silicon, etc.
- heteroatoms will be selected from the group consisting of oxygen, nitrogen, sulfur, phosphorous, and halogen (e.g., F, Cl, Br, I, etc.).
- a heteroatom or group may substitute a carbon.
- a heteroatom or group may substitute a hydrogen.
- a substituted hydrocarbon may comprise one or more heteroatoms in the backbone or chain of the molecule (e.g., interposed between two carbon atoms, as in “oxa”).
- a substituted hydrocarbon may comprise one or more heteroatoms pendant from the backbone or chain of the molecule (e.g., covalently bound to a carbon atom in the chain or backbone, as in “oxo”).
- the phrase “substituted with a[n],” as used herein, means the specified group may be substituted with one or more of any or all of the named substituents.
- a group such as an alkyl or heteroaryl group
- the group may contain one or more unsubstituted C 1 -C20 alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.
- R substituent e.g., a hydrocarbon
- the group may be referred to as “R- substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different.
- all groups described herein may optionally contain one or more common substituents, to the extent permitted by valency.
- halogen e.g., F, Cl, etc.
- C 1-12 straight chain or branched chain alkyl C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C6-12 aryl, C3-12 heteroaryl, C3-12 heterocyclyl, C 1-12 alkylsulfonyl, nitro, cyano, –COOR, –C(O)NRR’, –OR, –SR, –NRR’, and oxo, such as mono- or di- or tri-substitutions with moieties such as halogen, fluoroalkyl, perfluoroalkyl, perfluroalkoxy, trifluoromethoxy, chlorine, bromine, fluorine, methyl, methoxy, pyridyl, furyl, triazyl, piperazinyl, pyrazoyl, imidazoyl, and the like, each optionally containing one or more heteroatom
- R and R’ are independently hydrogen, C 1-12 alkyl, C 1-12 haloalkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C4-24 cycloalkylalkyl, C6-12 aryl, C7-24 aralkyl, C 3-12 heterocyclyl, C 3-24 heterocyclylalkyl, C 3-12 heteroaryl, or C 4-24 heteroarylalkyl.
- the phrase optionally substituted indicates the designated hydrocarbon group may be unsubstituted (e.g., substituted with H) or substituted with, for example one or more independently chosen common substituents.
- substituted hydrocarbons are hydrocarbons with a hydrogen atom removed and replaced by a substituent (e.g., a common substituent). It is understood by one of ordinary skill in the chemistry art that substitution at a given atom is limited by valency.
- a substituent (radical) prefix names such as alkyl without the modifier optionally substituted or substituted is understood to mean that the particular substituent is optionally substittued.
- haloalkyl without the modifier optionally substituted or substituted is still understood to mean an alkyl group, in which at least one hydrogen atom is replaced by halo.
- Agents 1-30 can have one or more of: (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agents 1-30) can have one or
- optically active forms can be obtained for example by resolution of the racemates, by asymmetric synthesis or asymmetric chromatography (chromatography with a chiral adsorbent or eluant). That is, certain of the disclosed compounds may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. “Enantiomer” means one of a pair of molecules that are mirror images of each other and are not superimposable.
- Diastereomers are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent the conFIGuration of substituents around one or more chiral carbon atoms.
- Enantiomers of a compound can be prepared, for example, by separating an enantiomer from a racemate using one or more well-known techniques and methods, such as chiral chromatography and separation methods based thereon. The appropriate technique and/or method for separating an enantiomer of a compound described herein from a racemic mixture can be readily determined by those of skill in the art.
- Racemate or “racemic mixture” means a mixture containing two enantiomers, wherein such mixtures exhibit no optical activity; i.e., they do not rotate the plane of polarized light.
- “Geometric isomer” means isomers that differ in the orientation of substituent atoms (e.g., to a carbon-carbon double bond, to a cycloalkyl ring, to a bridged bicyclic system, etc.). Atoms (other than H) on each side of a carbon- carbon double bond may be in an E (substituents are on opposite sides of the carbon- carbon double bond) or Z (substituents are oriented on the same side) conFIGuration.
- R,” “S,” “S*,” “R*,” “E,” “Z,” “cis,” and “trans,” indicate configurations relative to the core molecule.
- Certain of the disclosed compounds may exist in atropisomeric forms. Atropisomers are stereoisomers resulting from hindered rotation about single bonds where the steric strain barrier to rotation is high enough to allow for the isolation of the conformers.
- the compounds disclosed herein may be prepared as individual isomers by either isomer-specific synthesis or resolved from an isomeric mixture.
- Conventional resolution techniques include forming the salt of a free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming the salt of the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each of the isomers of an isomeric pair using an optically pure acid, amine or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of either a starting material or a final product using various well known chromatographic methods.
- the stereochemistry of a disclosed compound is named or depicted by structure
- the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9%) by weight relative to the other stereoisomers.
- the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight optically pure.
- the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight pure.
- Percent optical purity is the ratio of the weight of the enantiomer or over the weight of the enantiomer plus the weight of its optical isomer. Diastereomeric purity by weight is the ratio of the weight of one diastereomer or over the weight of all the diastereomers.
- the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction pure relative to the other stereoisomers.
- the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction pure.
- the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction pure.
- Percent purity by mole fraction is the ratio of the moles of the enantiomer or over the moles of the enantiomer plus the moles of its optical isomer.
- percent purity by moles fraction is the ratio of the moles of the diastereomer or over the moles of the diastereomer plus the moles of its isomer.
- composition represents a composition containing a compound described herein (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve
- the pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal.
- Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gel cap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other formulation described herein (see below).
- the pharmaceutical composition may comprise, for example, from 0.1% to 25% of the compounds of the present disclosure by weight of the composition.
- compositions hereof can be solids, liquids, or gases.
- the compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g., binding on ion-exchange resins or packaging in lipid-protein vesicles), sustained release formulations, solutions, suspensions, elixirs, and aerosols.
- the carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, and sesame oil.
- formulations for intravenous administration comprise sterile aqueous solutions of the active ingredient(s) which are prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution and rendering the solution sterile.
- Suitable pharmaceutical excipients include starch, cellulose, chitosan, talc, glucose, lactose, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol.
- the compositions may be subjected to conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, and buffers.
- Suitable pharmaceutical carriers and their formulation are described in Remington’s Pharmaceutical Sciences by E. W. Martin, which is hereby incorporated by reference in its entirety.
- compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for administration to the recipient.
- pharmaceutically acceptable salt refers to salts of any of the compounds described herein that within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio.
- Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G.
- Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases.
- Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, dichloroacetate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glutamate, glycerophosphate, hemisulfate, heptonate, hexanoate, hippurate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, isethionate, lactobionate, lactate, laur
- Representative basic salts include alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, aluminum salts, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, caffeine, and ethylamine.
- Transcription factors typically refer to a DNA-binding protein that regulates transcription of DNA into RNA, for example, by activation or repression of transcription. Some transcription factors effect regulation of transcription alone, while others act in concert with other proteins. Some transcription factors can both activate and repress transcription under certain conditions.
- transcription factors bind a specific target sequence or sequences highly similar to a specific consensus sequence in a regulatory region of a target gene. Transcription factors may regulate transcription of a target gene alone or in a complex with other molecules. Examples of transcription factors include, but are not limited to, Sp1, NF1, CCAAT, GATA, HNF, PIT-1, MyoD, Myf5, Hox, Winged Helix, SREBP, p53, CREB, AP-1, Mef2, STAT, R-SMAD, NF- ⁇ B, Notch, TUBBY, and NFAT. Transcription factors generally bind DNA in a sequence-specific manner and either activate or repress transcription initiation. In some embodiments, the transcription factor is a tumor suppressor.
- the transcription factor may be SMAD4, RFX7, REST, FOXP3, FOXP1, p53, FOXO3A, or IRF3.
- At least three types of separate domains have been identified within transcription factors.
- the term “subject” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. In most embodiments, the subject is a human. Other subjects may include mammals such as mice, rats, rabbits, cats, dogs, non-human primates.
- the subject may be domesticated animals (e.g., cows, calves, sheep, goat, lambs, horses, poultry, foals, pigs, piglets, etc.), or animals in the family Muridae (e.g., rats, mice, etc.), or animals in the family Felidae.
- a subject may seek or be in need of treatment, require treatment, be receiving treatment, may be receiving treatment in the future, or a human or animal that is under care by a trained professional for a particular disease or condition (e.g., cancer, etc.).
- Physiological conditions typically refer to a set of conditions including temperature, salt concentration, pH that mimic those conditions of a living subject. A host of physiologically relevant conditions for use in in vitro assays and chemical syntheses have been established.
- a physiological buffer contains a physiological concentration of salt and at adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5.
- a variety of physiological buffers may be used to produce physiological conditions in, for example, reaction medium, including phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- Physiologically relevant temperature ranges from 25° C. to 40° C or from 30° C. to 38° C.
- a proliferative disease refers to the physiological condition in a subject characterized by unregulated cell growth such as cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
- cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer (“NSCLC”), vulval cancer, thyroid cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
- squamous cell cancer e.g., epithelial squamous cell cancer
- lung cancer including small cell lung cancer, non-small cell lung cancer (“NSCLC”), vulval cancer, thyroid cancer,
- the cancer is at least one selected from the group consisting of ALL, T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma, B- cell ALL, Philadelphia chromosome positive ALL, Philadelphia chromosome positive CML, lymphoma, leukemia, multiple myeloma, myeloproliferative diseases, large B cell lymphoma, and B cell Lymphoma.
- unit dosage form refers to a physically discrete unit suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active material (e.g., a compound of the present disclosure) calculated to produce the desired therapeutic effect, in association with any suitable pharmaceutical excipient or excipients.
- active material e.g., a compound of the present disclosure
- exemplary, non- limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, gel cap, and syrup.
- FIG.1 FIG.1 (FIGs.1A-1B) provide information regarding the TF-DUBTAC platform.
- FIG.1A is a schematic diagram of the TF-DUBTAC platform.
- FIG.1B illustrates a general synthetic scheme for the TF-DUBTACs of the present disclosure.
- FIG.2 is an NMR of the DUBL-X-BCN #1 compound which may be used to form DUBTACs.
- FIG.3 is an NMR of the DUBL-X-BCN #2 compound which may be used to form DUBTACs.
- FIG.4 is an NMR of the DUBL-X-BCN #3 compound which may be used to form DUBTACs.
- FIG.5 is an NMR of the DUBL-X-BCN #4 compound which may be used to form DUBTACs.
- FIG.6 (FIGs.6A-6C) provide measurements of DUBL-X-BCN #5 compound which may be used to form DUBTACs including NMR (FIGs.6A-6B) and mass spectrometry data (FIG.6C).
- FIG.7 (FIGs.7A-7C) provide measurements of DUBL-X-BCN #6 compound which may be used to form DUBTACs including NMR (FIGs.7A-6B) and mass spectrometry data (FIG.7C).
- FIG.8 (FIGs.8A-8C) provide measurements of DUBL-X-BCN #7 compound which may be used to form DUBTACs including NMR (FIGs.8A-8B) and mass spectrometry data (FIG.6C).
- FIG.9 is an NMR of the DUBL-X-BCN #8 compound which may be used to form DUBTACs.
- FIG.10 is an NMR of the DUBL-X-BCN #9 compound which may be used to form DUBTACs.
- FIG.11 is an NMR of the DUBL-X-BCN #10 compound which may be used to form DUBTACs.
- FIG.12 (FIGs 12A-12V) illustrate how FOXO-DUBTACs, and particularly FOXO- DUBTAC#6, stabilize FOXO3A in an OTUB1-dependent manner.
- FIG.12A is a schematic diagram for the DNA binding motifs of FOXO family members (top) and a diagram for the FOXO3A motif and FOXO-ODN (bottom).
- FIG.12B illustrates that FOXO-ODN binds both FOXO3A and its close family member FOXO1 and the binding can be antagonized by free FOXO-ODN.
- Cell lysates with Flag-FOXO3A or HA-FOXO1 protein were extracted from HEK293T cells that expressed indicated constructs for 48 hours.
- FIGs.12C-D show that Biotin-FOXO-ODN binds FOXO3A (C) and FOXO1 (D) in a streptavidin-biotin pulldown assay.
- FIG.12E illustrates how FOXO-ODN binds both FOXO3A and FOXO1, which can be antagonized by free FOXO-ODN. Repeat assay for FIG.12B.
- FIG.12F shows that linkers with 6-8 methylene groups are optimal for the in vitro SPAAC reactions between BCN-modified OTUB1 ligands and N3-FOXO-ODN to form FOXO-DUBTACs.
- the SPAAC reaction products were separated by 20% native PAGE.
- the arrow indicates the click reaction products, FOXO-DUBTACs, and the arrowhead indicates N3-FOXO-ODN.
- FIG.12G provices the chemical structures of BCN-linked OTUB1 binders, DUBL-X-BCN #5 and #6.
- FIG. 12H provides the chemical structure of of FOXO-DUBTAC #6.
- FIG.12I illustrates how FOXO- DUBTAC #6 stabilizes FOXO3A protein in HeLa cells.
- HeLa cells were treated with the indicated concentrations of FOXO-ODN, FOXO-DUBTAC #5, or #6 for 24 hours, followed by immunoblot analysis.
- FIGs.12J-K illustrate how FOXO-DUBTAC #6 stabilizes FOXO3A protein in HeLa cells. Repeat assay for FIG.12I.
- FIG.12L illustrates that the OTUB1 ligands, DUBL-X-BCN #5, #6, and #7 did not affect the basal expressions of FOXO3A or p53.
- FIG.12M shows that FOXO-DUBTAC #6 mediates the binding between OTUB1 and FOXO3A.
- Flag-OTUB1 protein was purified by Flag- bead immunoprecipitation and further used for pulldown of FOXO3A with or without adding FOXO3-DUBTAC #6.
- FIG.12N shows that FOXO-DUBTAC #6 treatment increases the expression of p27 and BIM. *, p ⁇ 0.05; **, p ⁇ 0.01.
- FIG.12O shows how FOXO-DUBTAC #6 treatment changes the proteome profile of HeLa cells.
- FIG.12P provides a diagram of the enrichment of MYC- related protein network in FOXO-DUBTAC #6-treated cells.
- FIG.12Q is a schematic diagram to showing that FOXO3A represses MYC expression and function, resulting in increase of NDRG1.
- FIG.12R illustrates that FOXO-DUBTAC #6 suppresses the tumorigenesis of HeLa cells. HeLa cells were treated with 1 ⁇ g/mL of FOXO-DUBTAC #6, followed by assessment in a colony formation assay. *, p ⁇ 0.05.
- FIG.12S shows that FOXO-DUBTAC #6 increases FOXO3A protein abundance in OTUB1 +/+ cells, but not in OTUB1 -/- cells.
- FIG. 12T shows that FOXO-DUBTAC #6 increases FOXO3A protein abundance in an OTUB1-dependent manner.
- FIG.12U illustrates FOXO-DUBTAC #6 treatment increases the expression of p27 and BIM in a OTUB1-dependent manner. *, p ⁇ 0.05; **, p ⁇ 0.01.
- FIG.12V illustrates how FOXO-DUBTAC #6 treatment represses colony formation in a OTUB1-dependent manner. ***, p ⁇ 0.001.
- FIG.13 (FIGs 13A-13L) illustrate how p53-DUBTACs such as p53-DUBTAC #6 and p53- DUBTAC #7 stabilize p53 in an OTUB1-dependent manner. These figures illustrate that in vitro SPAAC reactions between p53-ODN and DUBL-X-BCNs lead to the formation of p53-DUBTACs.
- FIG.13A is a schematic diagram for the p53 motif, p53-ODN, biotin-modified p53-ODN (biotin- p53-ODN) and azide-modified p53-ODN (N3-p53-ODN).
- FIG.13B shows that biotin-p53-ODN binds Flag-tagged p53.
- the cell lysates with p53 protein were extracted from HEK293T cells that expressed the Flag-p53 construct for 48 hours.
- FIG.13C provides the native PAGE analysis of the in vitro SPAAC reaction products between BCN-linked OTUB1 binders (DUBL-X-BCNs 1-9) and N3- p53-ODN.
- FIG.13D provides the chemical structure of p53-DUBTAC #6 (top), p53- DUBTAC #5 (middle) and p53-DUBTAC #7 (bottom).
- FIG.13E shows that p53-DUBTACs #5, #6 and #7 (1 ⁇ g/mL, 24 hours) increase the p53 protein level in HeLa cells.
- FIG.13F shows that p53- DUBTACs #5, #6 and #7 (0.1, 0.2 and 0.4 ⁇ g/mL, 24 hours) increase the p53 protein level in HeLa cells.
- FIG.13G provides the increase the p53 protein level in HeLa cells.
- FIG.13H provides that p53-DUBTACs #5, #6 and #7 increase the p53 protein level in HeLa cells.
- FIG 13I shows that p53-DUBTACs #6 and #7 mediate the binding between OTUB1 and p53.
- Flag-OTUB1 protein was purified by Flag-bead immunoprecipitation and further used for pulldown of p53 with or without adding of p53-DUBTACs.
- FIG.13J shows that p53-DUBTAC #6 treatment changes the proteome profile of HeLa cells.
- FIG.13K illustrates Enrichment of AURKA-related protein network in p53-DUBTAC #6-treated cells. Within the dotted box indicates increased proteins, and outside the box indicates decreased proteins.
- FIG.13L shows that p53-DUBTACs #6 and #7 (1 ⁇ g/mL, 24 hours) increase the p53 protein level in an OTUB1- dependent manner in HeLa cells. HeLa cells infected with either sgControl or sgOTUB1 virus and selected with puromycin, followed by treatment with indicated compound for 24 hours and immunoblotting analysis of p53 protein abundance.
- FIG.14 shows that IRF-DUBTACs, including IRF-DUBTAC #7, stabilizes IRF3 in vitro (e.g., in HeLa cells).
- FIG.14A is a schematic diagram for the motifs of IRF family members.
- FIG.14B is a schematic diagram for the IRF3 motif, IRF-ODN, biotin-modified IRF- ODN (biotin-IRF-ODN) and azide-modified IRF-ODN (N3-IRF-ODN).
- FIG.14C shows that Biotin-IRF-ODN binds all IRF family members, except IRF9. The cell lysates were extracted from HEK293T cells that overexpressed indicated Flag-IRF constructs.
- FIG.14D illustrates that Biotin- IRF-ODN binds IRF2 and IRF3 in a streptavidin-biotin pulldown assay.
- the cell lysates derived from HEK293T cells that expressed Flag-IRF2 or Flag-IRF3 were incubated with biotin or Biotin- IRF-ODN at 4 ⁇ C for 3 hours, followed by another 1-hour incubation with Streptavidin agarose beads and further analyzed with immunoblotting analysis.
- FIG.14E illustrates that DUBL-X-BCNs #5-7 are most effective in forming IRF-DUBTACs via in vitro SPAAC reactions with N3-IRF-ODN. The SPAAC reaction products were separated by 20% native PAGE.
- FIG.14F shows that IRF-DUBTAC #7 (2 ⁇ g/mL, 24 hours) is most effective in increasing the IRF3 protein level in HeLa cells. Vehicle stands for the treatment with transfection reagents.
- FIG.14G provides the chemical structure of IRF-DUBTAC #7.
- FIG.14H illustrates that IRF-DUBTAC #7 is most effective in increasing the IRF3 protein level in HeLa cells as compared to the other IRF-DUBTACs measured. Repeat assay for Figure 14F.
- the invention provides compositions and methods relating to Deubiquitinase-Targeting Chimera (DUBTACS) and their use for stabilizing proteins of interest.
- PROTACs typically adopts small molecule inhibitor (SMIs) as ligand for protein of interest (POI), but inhibitor cannot be used for DUBTAC, due to a high possibility of residual inhibitory effect on POI from these engineered DUBTACs.
- SMIs small molecule inhibitor
- POI protein of interest
- TF-DUBTAC degrade oncogenic transcription factors
- the present disclosure includes a generalizable platform termed TF-DUBTAC to selectively stabilize tumor suppressive transcription factors.
- a panel of clickable DUB ligands (referred to herein as DUBL1-10) were further conjugated onto the 5’-terminal of azide-modified DNA oligomers (N3-ODN) via a copper- free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, to generate the TF-DUBTACs for stabilizing tumor suppressor transcription factors in cells.
- TF-DUBTAC is composed of the OTUB1 ligand (e.g., EN523) and oligonucleotide corresponding to specific TF, linked by a series of linkers.
- DUBLs were chemically clicked on to the Azide-modifed oligonucleotide (N3-ODN) corresponding to the DNA binding motif of respective TF, in vitro using a copper free strain-promoted alkyne-azide cycloaddition (SPAAC) reaction.
- SPAAC copper free strain-promoted alkyne-azide cycloaddition
- Three series of TF-DUBTACs for FOXO3A, p53 and IRF3 were screened and validated, and identified agents such as compounds that efficiently stabilize FOXO3A, p53 and IRF3, respectively, in cells.
- This TF-DUBTAC is a generalizable platform for selectively stabilizing transcription factors with tumor suppressive function for the first time.
- the TF-DUBTAC may have an affinity for its Transcription Factor or binding motif thereof (K d ) of less than 1 mM (e.g. from 500 ⁇ M to 1 mM, etc.) or less than 500 ⁇ M (e.g., less than 450 ⁇ M, less than 400 ⁇ M, less than 350 ⁇ M, less than 300 ⁇ M, less than 250 ⁇ M, less than 200 ⁇ M, less than 150 ⁇ M, less than 100 ⁇ M, less than 100 ⁇ M, less than 50 ⁇ M, less than 10 ⁇ M, less than 1 ⁇ M, less than 500 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, etc.).
- K d Transcription Factor or binding motif thereof
- the TF-DUBTAC does not inhibit or has minimal inhibitory activity on the Transcpactor Factor of interest, such as, for example, having an IC50 of greater than (or up to 1M) 1 ⁇ M or greater than 10 ⁇ M or greater than 100 ⁇ M or greater than 1000 ⁇ M.
- the compounds, agents, methods and compositions described herein include the use of N- oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the disclosure, as well as metabolites and active metabolites of these compounds having the same type of activity.
- Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether, etc.) or alcohol (e.g., ethanol, etc.) solvates, acetates and the like.
- the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol.
- the compounds described herein exist in unsolvated form.
- the compounds and agents of the disclosure may exist as tautomers or mixtures of tautomers, racemates, mixtures of racemates, stereoisomers, mixtures of stereoisomers, or combinations thereof. All tautomers are included within the scope of the compounds presented herein.
- the DUBTACs of the present disclosure e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) typically include
- the target protein may be any class of protein, for example, any protein found in a cell (e.g., a mammalian cell, a plant cell, a fungal cell, an insect cell, a bacterial cell) or a viral particle.
- the protein is a soluble protein or a membrane protein.
- the protein is a soluble protein.
- the protein is a membrane protein.
- the target protein may comprise a post-translational modification, e.g., a sugar moiety, acyl moiety, lipid moiety.
- the target protein is glycosylated, e.g., at an asparagine, serine, threonine, tyrosine, or tryptophan residue.
- the target protein is modified with a ubiquitin or a ubiquitin-like protein (collectively referred to herein as “Ubls”).
- the Ubl is ubiquitin.
- the Ubl is SUMO, NEDD8, or Agp12.
- the target protein is monoubiquitinated or polyubiquitinated.
- the target protein may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Ubl chains, e.g., on a lysine amino acid residue.
- the target protein may comprise polyubiquitin chains linked in any manner, for example, K48-linked polyubiquitin chains, K63- linked polyubiquitin linked chains, K29-linked polyubiquitin chains, or K33-linked polyubiquitin chains.
- the target protein comprises a plurality of polyubiquitin chains.
- the target protein comprising a Ubl is capable of binding to a protein comprising a Ubl-binding domain (e.g., a ubiquitin binding domain).
- the target protein may comprise a feature that increases its instability or impairs its activity, e.g., relative to the wild-type target protein.
- the target protein may be mutated or misfolded.
- the target protein has a reduced capacity for binding to a binding partner, e.g., by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% relative to the wild type target protein.
- the target protein is less active than the wild type target protein, e.g., by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%.
- the target protein is more active than the wild type target protein, e.g., by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%.
- This ODN moietiy is conjugated, directly or indirectly to a moiety capable of binding to a deubiquitinase (DUB).
- DRB deubiquitinase
- Deubiquitinases comprise a large family of proteases responsible for hydrolyzing Ubl-Ubl bonds or Ubl-target protein bonds and play a role in numerous cellular processes.
- Deubiquitinases serve several functions, including generating free ubiquitin monomers from polyubiquitin chains, modulating the size of polyubiquitin chains, and reversing ubiquitin signaling by removal of a from a ubiquitinated target protein. Misregulation of deubiquitinase function is associated with many diseases, including cancer, metabolic diseases, genetic disorders, haploinsufficiency targets, and neurological diseases. Roughly 80 different functional deubiquitinases have been identified in human cells to date.
- the presently disclosed DUBTACs (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) comprise a
- the deubiquitinase may be any deubiquitinase, e.g., in a cell, including cysteine protease deubiquitinases and metalloprotease deubiquitinases.
- the deubiquitinase is a cysteine protease, e.g., comprising a catalytic site cysteine amino acid residue.
- the deubiquitinase may be a full-length protein or a fragment thereof.
- the deubiquitinase comprises a single active site.
- the deubiquitinase is one function of a multifunctional protein.
- Exemplary deubiquitinases include BAP1, CYLD, OTUB1, OTUB2, OTUD3, OTUD5, OTUD7A, OTUD7B, TNFAIP3, UCHL1, UCHL3, UCHL5, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP17L1, USP17L2, USP17L24, USP17L3, USP17L5, USP18, USP19, USP2, USP20, USP21, USP22, USP24, USP25, USP26, USP27X, USP28, USP3, USP30, USP31, USP33, USP34, USP35, USP36, USP37, USP38, USP4, USP40, USP41, USP42, USP43, USP44, USP45, USP46, USP47, USP48, USP49, USP5, USP50, USP51, USP54, USP
- the deubiquitinase is selected from the group consisting of WDR48, YOD1, OYUD3, OTUB1, USP8, USP5, USP16, UCHL3, UCHL1, and USP14, or a fragment thereof. In some embodiments, the deubiquitinase is selected from the group consisting of WDR48, YOD1, OYUD3, OTUB1, OTUD5, USP8, USP5, USP14, USP15, USP16, UCHL3, and UCHL1, or a fragment thereof. In some embodiments, the deubiquitinase comprises OTUB1 or a fragment or variant thereof. In some embodiments, the deubiquitinase comprises OTUD5 or a fragment or variant thereof.
- the deubiquitinase comprises USP15 or a fragment or variant thereof.
- the DUBTACs of the present disclosure e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (V), (
- the DUBTAC (e.g., via the DUB moiety) binds to a site other than a catalytic site within the deubiquitinase. In some embodiments, the DUBTAC (e.g., via the DUB moiety) binds to an allosteric site within the deubiquitinase.
- binding of the DUBTAC (e.g., DUB) to the deubiquitinase does not modulate the activity of the deubiquitinase more than 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%, relative to the activity of the deubiquitinase in the absence of the bifunctional compound.
- binding of the bifunctional compound (e.g., DUB) to the deubiquitinase does not modulate the activity of the deubiquitinase more than 0.1-50%, 1-50%, 1-25%, 1-10%, 0.1-10%, 1-5%, or 0.1-2%, relative to the activity of the deubiquitinase in the absence of the bifunctional compound.
- the binding of the bifunctional compound (e.g., DUB) to the deubiquitinase does not substantially modulate (e.g., inhibit) the activity (e.g., deubiquitinase activity) of the deubiquitinase.
- the DUBTACs of the present disclosure e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) may be
- the cysteine amino acid residue is an allosteric cysteine amino acid residue. In some embodiments, the cysteine amino acid residue is present on a surface of the deubiquitinase. In some embodiments, the cysteine amino acid residue is present on or in the interior of the deubiquitinase. In some embodiments, the cysteine amino acid residue is not a catalytic cysteine amino acid residue. In some embodiments, the bifunctional compound preferentially binds to an allosteric cysteine amino acid residue over a catalytic cysteine amino acid residue.
- the bifunctional compound does not substantially bind to a cysteine amino acid residue in the catalytic site of the deubiquitinase (e.g., a catalytic cysteine).
- the DUBTAC e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1),
- the DUB moiety comprises a heterocyclyl (e.g., a piperazinonyl). In some embodiments, DUB comprises an acrylamide moiety. In some embodiments, DUB comprises a heteroaryl (e.g., a furan moiety).
- compositions The disclosure includes a pharmaceutical composition comprising a DUBTAC described herein (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf),
- the composition is formulated for an administration route such as oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
- transdermal e.g., sublingual, lingual, (trans)buccal, (trans)urethral
- vaginal e.g., trans- and perivaginally
- intra)nasal and (trans)rectal intravesical, intrapulmonary, intraduodenal, intragastrical
- intrathecal subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial
- the pharmaceutical composition may comprise a compound disclosed herein (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) and
- the compound is formulated as a topical composition (e.g., ointment, gel, etc.).
- the composition comprises from 0.1%-90% (e.g., 0.1%-50%, 0.1%-20%, 0.1%- 10%, etc.) of the compound by weight of the composition.
- the pharmaceutical composition may be formulated to allow for or enhance transfection of the compounds of the present disclosure (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf
- Transfection may occur, for example, in vivo, ex vivo, or in vitro.
- the compounds of the present disclosure can be introduced by suitable transfection mechanisms such as electroporation, lipofection, particle gun acceleration.
- the method is a chemical method (e.g., calcium-phosphate transfection), physical method (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), or receptor-mediated endocytosis.
- a pharmaceutical composition may comprise one or more lipids with the compounds of the presente disclosure selected from cationic lipids, ionizable lipids, anionic lipids, sterols, pegylated lipids, and any combination of the foregoing.
- the pharmaceutical composition containing a translatable compound comprises a cationic lipid, a phospholipid, cholesterol, and a pegylated lipid.
- a pharmaceutical composition can be substantially free of liposomes.
- the pharmaceutical composition comprises liposomes.
- the pharmaceutical composition comprises lyophilized liposomes.
- a pharmaceutical composition can include nanoparticles. Lipid-based formulations have been increasingly recognized as one of the most promising delivery systems due to their biocompatibility with certain DNA and RNA and their ease of large-scale production. Cationic lipids, for example, have been widely studied as synthetic materials for compound.
- nucleic acids may be condensed by cationic lipids to form lipid/nucleic acid complexes known as lipoplexes. These lipid complexes may be able to protect compounds of the present disclosure from the action of nucleases and to deliver it into cells by interacting with the negatively charged cell membrane.
- Lipoplexes can be prepared by directly mixing positively charged lipids at physiological pH with negatively charged nucleic acids.
- Conventional liposomes typically consist of a lipid bilayer that can be composed of cationic, anionic, or neutral (phospho)lipids and cholesterol, which encloses an aqueous core. Both the lipid bilayer and the aqueous space can incorporate hydrophobic or hydrophilic compounds, respectively.
- Liposome characteristics and behaviour in vivo can be modified by addition of a hydrophilic polymer coating, e.g. polyethylene glycol (PEG), to the liposome surface to confer steric stabilization.
- a hydrophilic polymer coating e.g. polyethylene glycol (PEG)
- liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (Front Pharmacol.2015 Dec.1; 6:286, which is hereby incorporated by reference in its entirety).
- Pharmaceutical compostions of the present disclosure comprising liposomes may be colloidal lipid-based and surfactant-based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment.
- liposomes may be present as spherical vesicles and can range in size from 20 nm to a few microns.
- Cationic lipid-based liposomes are able to complex with negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required for in vivo clinical applications.
- Liposomes can fuse with the plasma membrane for uptake; once inside the cell, the liposomes are processed via the endocytic pathway and the genetic material is then released from the endosome/carrier into the cytoplasm.
- Cationic liposomes have been traditionally the most commonly used non-viral delivery systems for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA/small hairpin R A-shRNA).
- Cationic lipids such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids to form nanoparticles by electrostatic interaction, providing high transfection efficiency.
- neutral lipid-based nanoliposomes for compound delivery as e.g. neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomes may be well suited for delivery of the compounds of the present disclosure (Adv Drug Deliv Rev.2014 February; 66: 110-116, which is hereby incorporated by reference in its entirety).
- the compounds described herein are lipid formulated.
- the lipid formulation is preferably selected from, but not limited to, liposomes, lipoplexes, copolymers, such as PLGA, and lipid nanoparticles.
- Lipid nanoparticles may include a cationic lipid suitable for forming a lipid nanoparticle.
- the cationic lipid may be, for example, N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-Dioleyloxy-3- trimethylaminopropane chloride salt), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N
- cationic lipids include, but are not limited to, N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P- (N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(1-(2,3-dioleyloxy)propyl)-N- 2-(sperminecarboxamido)ethyl)-N,N-dimethyl- ammoniumtrifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(1,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), and 2,2-D
- cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and Lipofectamine (comprising DOSPA and DOPE, available from GIBCO/BRL).
- LIPOFECTIN including DOTMA and DOPE, available from GIBCO/BRL
- Lipofectamine comprising DOSPA and DOPE, available from GIBCO/BRL
- Other suitable cationic lipids are disclosed in International Publication Nos. WO 09/086558, WO 09/127060, WO 10/048536, WO 10/054406, WO 10/088537, WO 10/129709, and WO 2011/153493; U.S. Patent Publication Nos.2011/0256175, 2012/0128760, and 2012/0027803; U.S. Pat.
- Suitable amino lipids useful in the compositions of the present disclosure include those having alternative fatty acid groups and other dialkylamino groups, including those, in which the alkyl substituents are different (e.g., N-ethyl-N-methylamino-, and N-propyl-N-ethylamino-).
- amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below 0.3 microns, for purposes of filter sterilization.
- amino or cationic lipids used in lipid formulations of the present disclosure may have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH.
- physiological pH e.g., pH 7.4
- the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form.
- Lipids that have more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded from use in the invention.
- the protonatable lipids have a pKa of the protonatable group in the range of 4 to 11, e.g., a pKa of 5 to 7.
- the cationic lipid can comprise from 20 mol % to 70 or 75 mol % or from 45 to 65 mol % or 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, or 65-70 mol % of the total lipid present in the particle.
- the lipid nanoparticles include from 25% to 75% on a molar basis of cationic lipid, e.g., from 20 to 70%, from 35 to 65%, from 45 to 65%, on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle).
- the ratio of cationic lipid to nucleic acid is from 3 to 15, such as from 5 to 13 or from 7 to 11.
- the lipid-based formulations may als contain a non-cationic lipid.
- the non-cationic lipid can be a neutral lipid, an anionic lipid, or an amphipathic lipid.
- Neutral lipids when present, can be any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH.
- neutral lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.
- the selection of neutral lipids for use in the particles described herein is generally guided by consideration of, e.g., lipid particle size and stability of the lipid particle in the bloodstream.
- the neutral lipid is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).
- the neutral lipids contain saturated fatty acids with carbon chain lengths in the range of C10 to C20.
- neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of C10 to C20 are used.
- neutral lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
- Suitable neutral lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dimyristoyl phosphatidylcholine (D PC),
- Anionic lipids suitable for use in lipid particles of the invention include, but are not limited to, phosphatidyl lycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.
- the non-cationic lipid can be from 5 mol % to 90 mol %, 5 mol % to 10 mol %, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, or 85-90 mol % of the total lipid present in the particle.
- the lipid nanoparticles include from less than (or from 0.1% to) 15% or less than 45% on a molar basis of neutral lipid, e.g., from 3 to 12% or from 5 to 10% (based upon 100% total moles of lipid in the lipid nanoparticle).
- the lipid-based formulations may also contain a sterol such as cholesterol.
- the sterol can be from 10 mol % to 60 mol % or from 25 mol % to 40 mol % of the lipid particle. In one embodiment, the sterol is 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, or 55-60 mol % of the total lipid present in the lipid particle.
- the lipid nanoparticles include from 5% to 50% on a molar basis of the sterol (based upon 100% total moles of lipid in the lipid nanoparticle). When formulated as a nanoparticle, the lipid nanoparticles may have the structure of a liposome.
- a liposome is typically a structure having lipid-containing membranes enclosing an aqueous interior. Liposomes preferably have one or more lipid membranes. In certain embodiments, liposomes can be single-layered, referred to as unilamellar, or multi-layered, referred to as multilamellar. When complexed with nucleic acids (e.g., RNA, DNA, the ODN moiety of the compounds of the present disclosure), lipid particles may also be lipoplexes, which are preferably composed of cationic lipid bilayers sandwiched between nucleic acid layers.
- nucleic acids e.g., RNA, DNA, the ODN moiety of the compounds of the present disclosure
- Liposomes can further be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.
- liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
- Liposomes may contain a low (e.g. an acidic) or a high (e.g. a basic) pH in order to improve the delivery of the pharmaceutical formulations.
- liposomes such as synthetic membrane vesicles may be prepared by the methods, apparatus and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372, each of which are herein incorporated by reference in their entirety.
- ODN moiety or entire compound may be encapsulated by the liposome, and/or it may be contained in an aqueous core, which may then be encapsulated by the liposome as described in International Pub. Nos. WO2012031046, WO2012031043, WO2012030901 and WO2012006378 and US Patent Publication No. US20130189351, US20130195969 and US20130202684, each of which are herein incorporated by reference in their entirety).
- lung cancer including small cell lung cancer, non-small cell lung cancer, vulval cancer, thyroid cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric
- the cancer is at least one selected from the group consisting of ALL, T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma, B- cell ALL, Philadelphia chromosome positive ALL, Philadelphia chromosome positive CML, lymphoma, leukemia, multiple myeloma, myeloproliferative diseases, large B cell lymphoma, and B cell Lymphoma.
- the proliferative disease is melanoma, leukemia, lymphoma, or retinal blastoma.
- the methods of the disclosure may comprise administering to the subject a therapeutically effective amount of at least one compound of the disclosure, which is optionally formulated in a pharmaceutical composition.
- the method further comprises administering to the subject an additional therapeutic agent that treats or prevents cancer.
- the compound may be a compound for the treatment of a proliferative disease.
- the compound may be a compound for the manufacture of a medicament for the treatment of a proliferative disease.
- the method for the treatment of a proliferative disease may comprise the administration of a compound or pharmaceutical composition as disclosed herein.
- administering the compound of the disclosure to the subject allows for administering a lower dose of the additional therapeutic agent as compared to the dose of the additional therapeutic agent alone that is required to achieve similar results in treating or preventing a cancer in the subject.
- the compounds disclosed herein may enhance the anti-cancer activity of the additional therapeutic compound, thereby allowing for a lower dose of the additional therapeutic compound to provide the same effect.
- the compounds and the therapeutic agent are co-administered to the subject.
- the compound of the disclosure and the therapeutic agent are coformulated and co-administered to the subject.
- the subject is a mammal. In other embodiments, the mammal is a human.
- the therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of a cancer in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
- the therapeutically effective amount may be determined based on the amount of DUBTAC (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), a
- the therapeutically effective amount may be based on more than 20% or more than 30% or more than 40% or more than 50% or more than 60% or more than 70% or more than 80% or more than 90% or more than 95% or more than 95% or more than 99% or 100% being decaged following endocytosis f the compounds described herein by weight of the composition.
- a suitable dose of a compound of the present disclosure may be in the range of from 0.01 mg to 5,000 mg per day, such as from 0.1 mg to 1,000 mg, for example, from 1 mg to 500 mg, such as 5 mg to 250 mg per day.
- the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day.
- the amount of each dosage may be the same or different.
- a dose of 1 mg per day may be administered as two 0.5 mg doses, with, for example, a 12-hour interval between doses.
- the amount of compound dosed per day may be administered, in non- limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
- a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
- the administration of the inhibitor of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time.
- the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
- the dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
- a maintenance dose is administered if necessary.
- the dosage or the frequency of administration, or both is reduced to a level at which the improved disease is retained.
- patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
- the compounds for use in the method of the disclosure may be formulated in unit dosage form.
- unit dosage forms are physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
- the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
- Many applications of the compositions of the present disclosure may involve cell surface localization or targeting. However, the general methods of this invention may also be extended to include internalization of the compound in cells.
- constructs may incorporate an internalization or targeting moiety that causes the cell to internalize a DUBTAC (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd),
- Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD 50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
- the dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50.
- the data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity.
- the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
- TF-DUBTACs provide a generalizable technology to target any tumor suppressive TFs with known DNA binding motif.
- TF-DUBTACs include SMAD4, RFX7, REST, FOXP3, and FOXP1. This will create a TF-directed targeted chemotherapy platform to achieve better efficacy and target many previous untargetable tumor suppressive transcriptional factors for stabilization, which can present tremendous opportunity to expand our arsenal for anti-cancer therapies.
- TF-DUBTACs selectively stabilize tumor suppressor transcription factors.
- a panel of clickable DUB binders (DUBL-X-BCN 1-10) were synthesized for creation of the TF-DUBTACs.
- BCN-linked OTUB1 binders were conjugated onto the 5’-terminal of azide-modified DNA oligomers (N3-ODN) via a copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction (FIGs.1A and 1B), resulting in TF-DUBTACs for stabilizing tumor suppressor transcription factors in cells (FIG.1A).
- OTUB ligand EN523 Three tumor suppressor transcription factors, FOXO3A, p53 and IRF3, with well-defined DNA binding motifs were used to experimentally test the feasibility of the TF-DUBTAC approach.
- the OTUB ligand EN523 is described in the following reference, Henning et al., Nat. Chem. Biol.2022, 18:412-421, which is incorporated herein in its entirety particularly in relation to EN523, its use, and synthetic methods thereof. It will be understood that in the event of any inconsistency in chemical structure between the EN523 OTUB ligand in the present application and in Henning et al., Nat. Chem. Biol.2022, 18:412-421, both compounds will be considered embraced by the present disclosure.
- Example 1 DUBTAC Synthesis A general synthetic route for agents of the present disclosure is: ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (2-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)ethyl)carbamate (DUBL-X-BCN #1).
- Compound 1 was prepared as shown in Henning, N. J. et al.
- DUBL-X-BCN #2 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (3- aminopropyl)carbamate (3, 17.4 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #2 was obtained as white solid (19.6 mg, yield 37% for three steps).
- DUBL-X-BCN #3 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (4- aminobutyl)carbamate (4, 18.8 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #3 was obtained as white solid (7.9 mg, yield 15% for three steps).
- DUBL-X-BCN #4 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (5- aminopentyl)carbamate (5, 20.2 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #4 was obtained as white solid (16.3 mg, yield 30% for three steps).
- DUBL-X-BCN #5 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (6- aminohexyl)carbamate (6, 21.6 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #5 was obtained as white solid (15.5 mg, yield 27% for three steps).
- DUBL-X-BCN #6 ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (7-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)heptyl)carbamate.
- DUBL-X-BCN #6 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediates 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (7- aminoheptyl)carbamate (7, 23 mg, 0.01 mmol, 1.0 equiv).
- DUBL-X-BCN #7 ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (8-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)octyl)carbamate (DUBL-X-BCN #7).
- DUBL-X-BCN #7 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (8- aminooctyl)carbamate (8, 24.4 mg, 0.01 mmol, 1.0 equiv).
- DUBL-X-BCN #8 ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (9-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)nonyl)carbamate.
- DUBL-X-BCN #8 was synthesized following the standard procedure for preparing DUBL-X- BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (9- aminononyl)carbamate (9, 25.8 mg, 0.01 mmol, 1.0 equiv).
- DUBL-X-BCN #9 was synthesized following the standard procedure for preparing DUBL-X- BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (10- aminodecyl)carbamate (10, 27.2 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #9 was obtained as white solid (8.4 mg, yield 14% for three steps). 1 H NMR (600 MHz, CDCl 3 ) is shown in FIG.
- DUBL-X-BCN #10 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (11- aminoundecyl)carbamate (11, 28.6 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #10 was obtained as white solid (14.2 mg, yield 22% for three steps).
- FOXO3A is a member of FOXO family transcription factors, all of which have similar DNA binding motifs, also known as forkhead consensus binding sites (FIG.12A) and described in Brunet A. et al., Cell 96.6 (1999): 857-868, Tsai, K. et al. Nucleic Acids Res 35.20 (2007): 6984-6994, Pierrou S, et al.
- FOXO3A plays a tumor suppressive role.
- biotin-modified FOXO- ODN (Biotin-FOXO-ODN, FIG.12A) was also synthesized to test the binding and specificity of FOXO-ODN to FOXO family members.
- Biotin-FOXO-ODN bound both FOXO3A and FOXO1 to a similar extent, and this binding could be blocked by an excess amount of free FOXO- ODN (FIGs.12B-E).
- the azide-modified FOXO-ODN (N3-FOXO-ODN) was conjugated to a DUB ligand via a SPAAC reaction (FIG.1A and 12A).
- DUBL-X-BCNs #5-7 which contain 6-8 methylene groups as the linkers, are optimal for the SPAAC reaction to form FOXO-DUBTACs.
- the compounds with a longer linker such as DUBL-X-BCNs #9 and #10) displayed drastically reduced efficiency in the click reaction to generate FOXO-DUBTACs, while shorter linkers (in DUBL-X-BCNs #1-4) mildly disrupted the intra-molecular dimerization, potentially reducing the binding of these FOXO-DUBTACs to FOXO3A.
- FOXO-DUBTAC #5 and #6 were used for further experiments in cells (FIG.12F-G).
- FOXO-DUBTAC #6 (FIG.12H) was effective in stabilizing FOXO3A in a concentration- dependent manner in HeLa cells (0.2-1 ⁇ g/mL, FIGs.12I-K). Furthermore, the protein level of FOXO1, a close family member of FOXO3A, was not significantly elevated by the treatment with FOXO-DUBTAC #6 even at 1 ⁇ g/mL (FIG.12I), although FOXO-ODN displayed comparable binding to both FOXO3A and FOXO1 (FIGs.12B-E), indicating that FOXO-DUBTAC #6 preferentially stabilizes FOXO3A.
- This selectivity might be due to that: (1) the potential difference in FOXO3A and FOXO1 protein structures outside of the DNA binding pocket may impact the formation of OTUB1-DUBTAC-FOXO1 ternary complex, and/or (2) the distance between OTUB1 and polyubiquitin chain(s) on FOXO1 may not be optimal for its removal. In contrast, the OTUB1 ligands have no effect on the stability of FOXO3A (FIG.12L). In keeping with stabilization results, FOXO-DUBTAC #6 was capable of inducing the interaction between OTUB1 and FOXO3A (FIG. 12M).
- MYC is a well-defined FOXO3A downstream protein, and FOXO3A suppresses MYC in transcriptional, translational and post-translational level, and FOXO3A also antagonizes the function of MYC through directly binding to the promoters of MYC-target genes.
- WDR5 recruits MYC onto the chromatin and is essential for the oncogenic function of MYC.
- MYC forms a complex with TOP1 on chromatin to favor transcriptions.
- the reduction of WRD5 and TOP1 might be an indirect effect through MYC.
- MYC suppresses the transcription of a series of genes, including DNRG1, a cancer metastasis repressor, and FOXO-DUBTAC treatment led to an increase in NDRG1 level, which could also be due to the reduction of MYC.
- FOXO-DUBTAC stabilizes FOXO3A, which suppress the transcription and function of MYC, unleashing the repression of NDRG1 by MYC (FIGs.12P-Q).
- FOXO-DUBTAC #6 suppressed tumorigenesis of HeLa cells in vitro in a colony formation assay (FIG.12R).
- Endogenous OTUB1 was further depleted using CRISPR/Cas9 in HeLa cells to determine the dependence of DUBTAC on OTUB1.
- FOXO-DUBTAC #6 increased the protein level of FOXO3A in OTUB1 +/+ cells, but not in OTUB1 -/- cells (FIGs.12S-T).
- FOXO-DUBTAC #6 increased the transcription level of p27 and BIM only in OTUB1 +/+ cells but not in OTUB1 -/- cells (FIG.12U). Consistent with these findings, FOXO-DUBTAC #6 suppressed tumorigenesis of wild type HeLa cells but not OTUB1-KO cells (FIG.12V).
- Wild type p53 protein binds a DNA consensus sequence consisting of two tandem repeats of 10 base-pair motif 5'- PuPuPuC(A/T)(T/A)GPyPyPy-3' (Pu stands for purine and Py for pyrimidine) separated by 0-13 base-pairs as shown in Wang, Y. et al. Mol Cell Biol 15.4 (1995): 2157-2165 and El-Deiry, et al. Nat Genet 1.1 (1999): 45-49, which are hereby incorporated by reference in their entirety and particularly in relation to binding sites of p53.
- a double strand p53-ODN, with a sense strand of 5’- AGACATGCCTAGACATGCCT-3’ was synthesized.
- p53-DUBTAC #6 and #7 significantly increased the p53 protein level in HeLa cells (over 3-fold of increase, 1 ⁇ g/mL, 24 hours, FIGs.13E-H), while p53-DUBTAC #5 had a weaker effect.
- DUBL-X-BCNs #5-#7 or p53-ODN had minimal effects on the p53 protein level (FIGs.13E-F), thereby excluding the possibility of an indirect effect from ligands or oligomer.
- OTUB1 ligands have no effect on the stability of p53 (FIG.12L).
- p53-DUBTAC #6 and #7 were capable of guiding the interaction between OTUB1 and p53 (FIG.13I).
- 76 elevated and 33 decreased proteins were identified among the 6,333 quantified proteins in p53-DUBTAC-treated HeLa cell in comparison to control cells (FIG.13J). Similar to FOXO3A, p53 was also below the detection limit.
- Several ARUKA-related proteins were found among the proteins with reduced expression, while the level of several other p53 related proteins were increased, such as KAT5 (FIGs 13J-K).
- ARUKA has been determined as a p53 partner, and p53 suppresses AURKA function, by transcription regulation, direct inhibition through protein-protein interaction, as well as posttranslational regulation of ARUKA stability.
- KAT5 also known as Tip60, is another p53-binding protein that promotes p53 acetylation, and is essential for p53 signaling through direct association on the promoters of target genes.
- IRF-DUBTACs The generalizability of this TF-DUBTAC strategy was demonstrated by targeting IRF transcription factor family members, which have a conserved DNA binding motif (FIG.14). IRF transcription factor family members are responsible for the transcription of genes involving in inflammation, anti-bacteria and anti-virus protective pathways, such as interferons and interleukins.
- TF-DUBTACs could be a general platform for stabilizing TFs with known DNA binding motifs.
- proof-of-concept TF-DUBTACs that target and stabilize FOXO3A, p53 and IRF3 in cells were synthesized and characterized.
- FOXO-DUBTAC #6, p53-DUBTAC #6, #7 and IRF-DUBTAC #7 effectively stabilized FOXO3A, p53 and IRF3 tumor suppressor transcription factors, respectively, in an OTUB1-dependent manner.
- HPLC high-performance liquid chromatography
- the double stain p53-ODN was synthesized based on the well-defined p53-DNA binding consensus with two repeats of 10 base-pair motif.
- the sense stand of p53-ODN is 5’-AGACATGCCTAGACATGCCT-3’, and the antisense stand is 5’- AGGCATGTCTAGGCATGTCT-3’.
- the biotin modification of ODNs were synthesized by adding a biotin to the 5’ end of single strand ODN or the 5’ end of sense stand for double strand ODN using a spacer of NH 2 -(CH 2 ) 2 -O-(CH 2 ) 2 -OH.
- the azide modification of ODNs were synthesized by incorporating an azide group on the same site as biotin through the 5’ amino modifier C6. All oligomers were synthesized by Integrated DNA Technologies, Inc. The unmodified oligomers were purified by standard desalting, while both biotin- and azide-modified oligomers were purified by HPLC. The single FOXO-ODN and IRF-ODN were annealed by heating to 95 ⁇ C for 5 min, followed by cooled down to room temperature in 5 ⁇ C per min. Similarly, the sense and antisense p53 oligomers were mixed at 1:1 ratio and annealed before use.
- oligomers were separated by nageive PAGE at 100 V for 1 hour, followed by incubated in 0.2% EtBr solution in 1 x Tris-boric acid-EDTA (TBE, pH 8.3) buffer that consisted of 89 mM Tris, 89 mM boric acid, 2 mM EDTA.
- TBE Tris-boric acid-EDTA
- the native gels contain 20% acrylamide and 2.5% glycerol, 0.075 ammonium persulfate (APS), 0.05% tetramethylethylenediamine (TEMED) in 0.5 x TBE buffer.
- the gels were imaged with UV illumination with the ChemiDocTM Touch Imaging System (Bio-Rad).
- HEK293T Human embryonic kidney 293T
- DMEM Dulbecco's Modified Eagle's Medium
- FBS fetal bovine serum
- Flag- FOXO3A, HA-FOXO1, Flag-p53, Flag-IRF2, 3, 4, 5, 6, 8 and 9 were transfected into HEK293T cells, and harvested for lysis 48 hours after transfection.
- TF-DUBTACs treatment HeLa cells in 6-well plate were transfected with individual TF-DUBTAC for 24 hours, followed by harvest for western blot analysis.
- the sgRNA for OTUB1 (5’- TATCAACAGAAGATCAAGGT-3’) was synthesized and inserted into lenti-CRISPR-V2 construct as described in Sanjan, N. Nat Methods 11.8 (2014): 783-784, which is hereby incorporated by reference in its entirety.
- the sgOTUB1 lentivirus were generated in HEK293T cells as described in Liu, J. et al. Sci. Adv.6.8 (2020): eaay5154 and Liu, J. et al.
- HEK293T cells that ectopically expressed transcription factors were lysed by EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Pierce) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem).
- EBC buffer 50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40
- protease inhibitors Pieris
- phosphatase inhibitor cocktail set I and II, Calbiochem phosphatase inhibitor cocktail set I and II, Calbiochem
- the cell lysates (1 mg) were further incubated with biotin-ODN together with/without excess free ODN (3 or 10 folds) at 4 ⁇ C for 3 hours, followed by adding 10 ⁇ L of Streptavidin agarose beads (Thermo Fisher) for another 1 hour.
- the beads were washed 4 times with NETN buffer (100 mM NaCl, 20 mM Tris-Cl, pH 8.0, 0.5 mM EDTA, 0.5 % NP-40), boiled in SDS loading buffer and further separated by 10% SDS-PAGE and blotted with individual antibody.
- NETN buffer 100 mM NaCl, 20 mM Tris-Cl, pH 8.0, 0.5 mM EDTA, 0.5 % NP-40
- the lysates (60 ⁇ g protein) were then resolved by 10% SDS-PAGE at 130 V for 80 min and immunoblotted with indicated antibodies at 4°C overnight, washed 4 times with Tris-buffered saline with 0.1% Tween-20 (TBST), incubated with secondary antibody for 1 hour at room temperature, and then washed 4 times with TBST buffer.
- Anti-FOXO3A (#12829, 1:1,000), FOXO1 (#2880, 1:1000), OTUB1 (#3783, 1:1000) and IRF3(#11904, 1:1000) antibodies were purchased from Cell Signaling Technologies.
- Anti-HA antibody (clone 16B12, #901513, 1:1,000) was purchased from Biolegend.
- Anti-Flag F3165, 1:5,000
- anti-vinculin antibody V-4505, 1:50,000
- peroxidase-conjugated anti-mouse secondary antibody A-4416, 1:3,000
- peroxidase-conjugated anti-rabbit secondary antibody A-4914, 1:3,000
- Anti-p53 antibody clone DO-1, sc-126, 1:1000 was purchased from Santa Cruz. All primary antibodies were diluted in 1% bovine serum albumin (BSA) in TBST buffer, and secondary antibodies were diluted in 5% non-fat milk in TBST buffer. All western blot assays were repeated at least twice.
- BSA bovine serum albumin
- RNA quantification in immunoblot assay was performed with Quantity One software (Bio-Rad).
- RT-qPCR Total RNAs were extracted using Qiagen RNeasy mini kit (Qiagen. #74106) and reversed transcripted into cDNA using iScriptTM Reverse Transcription Supermix (Bio-Rad, # 1708841).
- RT- qPCR was performed with SYBR Select Master Mix (Thermo Fisher, #4472908) using primers as below: p27-forward: AACGTGCGAGTGTCTAACGG, p27-reverse: CCCTCTAGGGGTTTGTGATTCT, BIM-forward: TAAGTTCTGAGTGTGACCGAGA, BIM- reverse: GCTCTGTCTGTAGGGAGGTAGG, GAPDH-forward: TTGAGGTCAATGAAGGGGTC, GAPDH-reverse: GAAGGTGAAGGTCGGAGTCA.
- Proteomics sample preparation The cell pellets were resuspended in 8 M Urea, 50 mM Tris-HCl pH 8.0, reduced with dithiothreitol (5 mM final) for 30 min at room temperature, and alkylated with iodoacetamide (15 mM final) for 45 min in the dark at room temperature. Samples were diluted 4-fold with 25 mM Tris-HCl pH 8.0, 1 mM CaCl2 and digested with trypsin at 1:100 (w/w, trypsin:protein) ratio overnight at room temperature. There were two biological samples at each condition and total 8 samples.
- Peptides were cleaned by homemade C18 stage tips and the concentration was determined (Peptide assay, Thermo 23275).25 ⁇ g each was used for labeling with isobaric stable tandem mass tags (TMT10-126, 127C, 128N, 129N, 129C, 130N, 130C, 131, Thermo Fisher Scientific, San Jose, CA) following manufacture instruction.
- the mixture of labeled peptides was fractionated into 22 fractions on C18 stage tip with buffer 10 mM Trimethylammonium bicarbonate (TMAB), pH 8.5 containing 5 to 50% acetonitrile.
- TMAB Trimethylammonium bicarbonate
- Mass spectrometry analysis Dried peptides were dissolved in 0.1% formic acid, 2% acetonitrile.0.5 ⁇ g of peptides of each fraction was analyzed on a Q-Exactive HF-X coupled with an Easy nanoLC 1200 (Thermo Fisher Scientific, San Jose, CA). Peptides were loaded on to a nanoEase MZ HSS T3 Column (100 ⁇ , 1.8 ⁇ m, 75 ⁇ m ⁇ 250 mm, Waters). Analytical separation of all peptides was achieved with 110-min gradient.
- High energy collision-activated dissociation-MS/MS was used to dissociate peptides at a normalized collision energy of 32 eV in the presence of nitrogen bath gas atoms. Dynamic exclusion was 45 seconds.
- Raw proteomics data processing and analysis Peptide identification and quantification with TMT reporter ions were performed using the MaxQuant software version 1.6.10.43 (Max Planck Institute, Germany). Protein database searches were performed against the UniProt human protein sequence database (UP000005640). A false discovery rate (FDR) for both peptide-spectrum match (PSM) and protein assignment was set at 1%.
- FDR false discovery rate
- Search parameters included up to two missed cleavages at Lys/Arg on the sequence, oxidation of methionine, and protein N-terminal acetylation as a dynamic modification. Carbamidomethylation of cysteine residues was considered as a static modification. Peptide identifications are reported by filtering of reverse and contaminant entries and assigning to their leading razor protein. Data processing and statistical analysis were performed on Perseus (Version 1.6.10.50). Protein quantitation was performed on biological replicates and a two-sample t-test statistics was used with a p-value of 5% to report statistically significant protein abundance fold-changes.
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Abstract
The present disclosure features agents that bind and stabilize transcription factors of interest.
Description
AGENTS WITH TRANSCRIPTION FACTOR TARGETING MOIETIES STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant No. R35CA253027 awarded by the National Institutes of Health. The government has certain rights in this invention. CROSS REFERENCE TO RELATED APPLICATIONS The present application claims priority to and the benefit of U.S. App. No.63/359,710, filed July 8, 2022, which is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION Genetic regulation relies largely on the sequence-specific transcription factors (TFs) that recognize short DNA segments, also known as TF binding motifs, which typically locate in the enhancer and promotor regions of respective genes. These TFs promote or repress genetic transcription and play pivotal roles in the development of various human diseases, including cancer. The cancer dependency map project (DepMap) uncovered that TFs represent a major class of essential genes that maintain the proliferation and tumorigenesis of cancer cells, indicating a high potential of TFs as therapeutic targets. In the human genome, there are approximately 1600 putative TFs, which could be classified into tens of families largely based on their distinct DNA binding domains, such as C2H2, bZIP, bHLH and Homeodomain 21. Several low-throughput and high-throughput methods including protein binding microarray (PBM), SELEX, DAP-seq, HiTS-FLIP, EMSA and CHIP, have been used to define the specific DNA binding motif for each individual TF. To date, the DNA binding motif has been successfully defined for more than 600 putative human TFs by experimental approaches and more than 1300 by theoretical methods, which presents valueless treasury that may aid the development of TFs-targeting therapies. TFs typically lack active sites or allosteric regulatory pockets that normally exist in kinases or other types of enzymes, thereby making it difficult to design and screen for small molecule inhibitors (SMIs) of TFs. Given the key role of TFs in binding with specific DNA sequences to regulate genetic transcription, the DNA binding motif in theory may define the biological and potentially biochemical specificity of different TFs. Indeed, a large number of studies have theoretically and/or experimentally defined the unique DNA binding motif for most TFs. Nevertheless, identification of compounds capable of binding to TFs to modify their transcriptional activity has proved problematic such that TFs are often considered “undruggable.”
The Proteolysis-Targeting Chimeras (Pro-TAC) technology is an emerging novel class of bifunctional synthetic drug that engages the cell’s endogenous ubiquitin/proteasome system to target specific oncoprotein for proteasomal degradation in order to combat cancers. Compared to the conventional inhibitor-based drugs, PROTACs are more robust, high potency and durability, capable of degrading protein targets in a catalytic manner. Hence, proteins that are considered “undruggable” by traditional small molecule inhibitors will be targeted using the PROTAC technology, a process that is functioning like CRISPR-mediated knockout, but on the protein level. Thus, this new technology has drawn a lot of attention in the drug development field. Tumorigenesis is initiated by either induction/activation of oncoproteins or repression/inactivation of tumor suppressor proteins. Genetic alternations, including amplification or mutation of oncogenes, as well as deletion or mutation of tumor suppressor genes have been wildly detected and play a driver role in tumorigenesis and development. Small molecule degraders, such as molecule glues and the proteolysis targeting chimeras (PROTACs), have been developed for targeting these oncoproteins for proteasomal degradation. However, only a few agonists have been developed for tumor suppressors, including PPAR ^, AMPK (A769662 and Compound 991), and PP2A (iHAP). A recent preprint paper has identified a covalent ligand (EN523) for OTUB1, a OTU family member of deubiquitinase (DUB). EN523 binds the allosteric cysteine C23 rather than the catalytic C91 residue of OTUB1, thus does not interrupt its DUB enzymatic function. Among over 1500 transcription factors (TFs), the DNA binding motifs for 90% of which have been well defined by either experiments or theoretical methods. Although several targeted protein degradation strategies, such as molecule glue and PROTACs (PROteolysis TArgeting Chimeras), have been widely used for degrading oncogenic proteins, leveraging similar compounds including, TF-PROTACs, for stabilization of a protein of interest (POI), such as a tumor suppressor protein, have not been developed. SUMMARY Recently, a covalent ligand of OTUB1, an OTU family member of deubiquitinase (DUB), EN523, has been identified to develop a DUBTAC (Deubiquitinase-Targeting Chimera) platform for stabilization of CFTR (cystic fibrosis transmembrane conductance regulator), a protein responsible for cystic fibrosis (Henning et al., Nat. Chem. Biol.2022, 18:412-421 and WO 2022232634, which are incorporated herein by reference in its entirety). PROTACs typically adopt small molecule inhibitor (SMIs) as ligand for protein of interest (POI), but inhibitor moieties cannot be used for DUBTAC, due to a high possibility of residual inhibitory effect on protein of interest from these engineered DUBTACs. Moreover, it is still unknown whether the OTUB1 ligand could be used for stabilizing tumor suppressive proteins. For
example, transcription factor based TF-PROTAC platforms (Liu et al, JACS 143.23 (2021): 8902- 8910 and WO 2022251614, each of which are hereby incorporated by reference in their entirety and particularly in relation to transcription factor conjugation strategies), degrade oncogenic transcription factors (TFs), degrades oncogenic transcription factors (TFs). In the present disclosure, a nucleotide- based TF-DUBTAC platform for targeted stabilization of TFs with tumor suppressive function is provided. The TF-DUBTAC strategy avoids or minimizes the degradation of TFs to afford stabilization of certain POIs such as tumor suppressive transcription factors. By reducing this degradation and minimizing inhibition of the TF, the agents (e.g., DUBTACs) of the present disclosure may be able to stabilize the protein of interest such as tumor suppression propteins and result in therapeutic efficacy. The DUBTAC (or agent) may comprise an oligonucleotide that hybridizes to a transcription factor of interest (e.g., a tumor suppressor transcription factor), wherein the oligonucleotide is directly or indirectly bound to a ligand of a deubiquitinase. In some embodiments, the deubiquitinase is OTUB1. The oligonucleotide may be covalently bound to the ligand via a linker. In some embodiments, the linker comprises an azide/alkyne cycloaddition reaction product. For example, the linker may comprise a strained click chemistry reaction product. In some embodiments, the oligonucleotide has a double-strand hairpin structure formed via intra- dimerization. In various implementations, the oligonucleotide is a double-stranded DNA comprising a sense chain and an anti-sense chain. In some embodiments, the transcription factor is a tumor suppressor. For example, the oligonucleotide may be p53, FOXO3A, or IRF3. The DUBTACs (or agents) of the present disclosure may have the structure of formula (I): ODN–L1–DUB (I) wherein DUB is a deubiquitinase binding moiety; L1 is absent or a linker; ODN is a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing. Typically, conjugation occurs between the DNA oligomer protein and the DUB moiety via an azide/alkynyl cycloaddition reaction to form the functioning PROTAC. Accordingly, L1 often comprises an optionally substituted heteroarylene group (e.g., a five membered heteroarylene, a six membered heteroarylene, a divalent triazole, a divalent imidazole, a divalent pyrrole, a multicyclic (e.g., bicyclic, tricyclic) heteroarylene such as a five or six membered membered heteroaryl including triazolyl, imidazolyl, pyrrolyl which may be optionally fused to an mono or bicyclic cycloalkyl such as C3-C12 monocyclic cycloalkyl or C4-C12 bicyclic cycloalkyl including bicyclo[6.1.0] nonane). In certain aspects, L1 comprises a triazole
fused to a C6-C12 monocyclic or bicyclic cylcoalkyl (e.g., azide/alkynyl SPAAC reaction products). In some embodiments, L1 comprises an optionally substituted mono or multicyclic heteroarylene group or an optionally substituted mono or multicyclic heterocyclene group (e.g., a five membered heterocyclene, a six membered heterocycene, a mono or multicyclic heteroarylene (e.g., dihydroisoxazolyl which may be optionally fused to a mono or bicyclic cycloalkyl such as C3-C12 monocyclic cycloalkyl or C4-C12 bicyclic cycloalkyl including bicyclo[6.1.0] nonane). In some embodiments, the DUB binds to a deubiquitinase. The deubiquitinase may be any deubiquitinase, e.g., in a cell, including cysteine protease deubiquitinases and metalloprotease deubiquitinases. In some embodiments, the deubiquitinase is a cysteine protease, e.g., comprising a catalytic site cysteine amino acid residue. For example, the compound may have the structure of Formula (IIA), (IIB), (IIC), or (IID):
wherein Ring A is a cycloalkyl, heterocyclyl, aryl, or heteroaryl (and conjugation to L1 may occur through a substitution group) which may have 1-10 points of optional substitution which may be independently substituted with, for example, alkyl (e.g., C1-C6 alkyl), haloalkyl (e.g., C1-C6 haloalkyl), heteroalkyl (e.g., C1-C6 heteroalkyl), halo (e.g., F, Cl, Br);
R8 is hydrogen, alkyl (e.g., C1-C20 alkyl, C1-C6 alkyl, lower alkyl, C1-C4 alkyl), or an electrophilic moiety; n may be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; R9 is alkyl (e.g., C1-C6 alkyl), haloalkyl (e.g., C1-C6 haloalkyl), heteroalkyl (e.g., C1-C6 heteroalkyl, C1-C6 alkoxy C2-C6 alkenoxy, C2-C6 alkynoxy, C1-C6 haloalkoxy, cycloalkoxy, heterocyclyloalkoxy, aryloxy, heteroaryloxy), halo (e.g., F, Cl, Br). In some embodiments, Ring A is heteroaryl (e.g., monocyclic heteroaryl). In some embodiemnts, Ring A is a 5-membered heteroaryl (e.g., furanyl). In some embodiments, R8 is an electrophilic moiety which may be, for example, C2-C6 alkenyl, C2- C6 alkynyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, halo, cyano, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl which may be optionally substituted (e.g., have from 1-10 points of substitution which may be independently substituted with, for example, alkyl (e.g., C1-C6 alkyl), haloalkyl (e.g., C1-C6 haloalkyl), heteroalkyl (e.g., C1-C6 heteroalkyl), halo (e.g., F, Cl, Br). In various implementations, R8 is C2-C6 alkenyl (e.g., –CH=CH2). In various implementations the DUB may have the structure of:
wherein p is 0, 1, 2, or 3; and
indicates the point of attachment to L1. For example, DUB may have the structure of EN523 and the agent may have the structure of formula (III):
The ability to degrade the transcription factor may be strongly dependent on the linker between the chimera. In some embodiments, the agent has the structure of formula (IV): ODN–Y1–Y2–Y3–Y4–Y5–Y6–Y7–Y8–Y9–DUB (IV) wherein Y1-Y9 are independently selected from absent (i.e., it is a bond), an optionally substituted heteroarylene group or optionally substituted heterocycloalkylene (e.g., a five membered heteroarylene, a six membered heteroarylene, a triazolylene, a imidazole, a divalent pyrrole) optionally fused to an mono or bicyclic cycloalkyl (e.g., C3-C10 monocyclic cycloalkyl, C4-C10 bicyclic cycloalkyl such as bicyclo[6.1.0] nonane) –C(O)–, –O–, –OC(O)–, –NRa–, –N(Ra)C(O)–, –(C(Ra)(Ra))1-12, –(C(Ra)(Ra)C(Ra)(Ra)O)1-12–, –S–S–; and Ra is independently selected at each occurrence from hydrogen and optionally substitued alkyl (e.g., C1-C4 alkyl). In some embodiments, at least one of Y1-Y9 (e.g., Y2, Y3) comprises a linker moiety that is a click chemistry reaction product including click chemistry reaction products synthesized via another reaction mechanism. For example, at least one of Y1-Y9 may include a moiety that could be formed from Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction product (e.g., a divalent triazole), a strain-promoted azide-alkyne cycloaddition (SPAAC) product (e.g., a divalent fused triazole), a strain-promoted alkyne-nitrone cycloaddition (SPANC) product (e.g., a divalent fused dihydroisoxazole), or another product of strained alkene reactions such as alkene-azide cycloaddition. Click-chemistry compatible reactions (and products thereof for use as linker moieties in one of Y1-Y9) may also be considered to include alkene-tetrazine inverse-demand Diers-Alder reactions, alkene-tetrazole photoclick reactions, Michael additions of thiols, nucleophilic substitution of thiols with amines, and certain Diels-Alder reactions, such as those disclosed by Becer, et al. Angew. Chem. Int. Ed.48 (2009): 4900-4908, which is hereby incorporated by reference in its entirety. In some embodiments, one of Y1-Y9 includes a triazole product (e.g., similar to those produced from a Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, or a Strain- promoted azide-alkyne cycloaddition (SPAAC)). In some embodiments, one of Y1-Y9 includes an isoxazole product such as fused dihydroisoxazole (e.g., similar to those produced from a Strain-
promoted alkyne-nitrone cycloaddition (SPANC) reaction). In some embodiments, the compound has the structure of formula (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), or (IVt): ODN–Y1–Y2–Y3–NH–(CH2)1-12–NH–C(O)–DUB (IVa) ODN–Y1–Y2–Y3–(CH2)1-12–NH–C(O)–(CH2)1-12–DUB (IVb) ODN–Y1–Y2–Y3–(CH2)1-12–NH–(CH2)1-12–DUB (IVc) ODN–Y1–Y2–Y3–NH–(CH2)1-12–NH–C(O)–DUB (IVd) ODN–Y1–Y2–Y3–C(O)–(CH2)1-12–DUB (IVf) ODN–Y1–Y2–Y3–NH–(CH2)1-12–DUB (IVg) ODN–Y1–Y2–Y3–NH–(CH2CH2O)1-12–NH–C(O)–DUB (IVh) ODN–Y1–Y2–Y3–(CH2CH2O)1-12–NH–C(O)–(CH2CH2O)1-12–DUB (IVi) ODN–Y1–Y2–Y3–(CH2CH2O)1-12–NH–(CH2CH2O)1-12–DUB (IVj) ODN–Y1–Y2–Y3–NH–(CH2CH2O)1-12–NH–C(O)–DUB (IVk) ODN–Y1–Y2–Y3–C(O)–(CH2CH2O)1-12–DUB (IVl) ODN–Y1–Y2–Y3–NH–(CH2CH2O)1-12–DUB (IVm) ODN–Y1–Y2–Y3–(CH2)1-12–C(O)–NH–(CH2)1-12–DUB (IVn) ODN–Y1–Y2–Y3–(CH2)1-12–OC(O)–NH–(CH2)1-12–DUB (IVo) ODN–Y1–Y2–Y3–(CH2)1-12–OC(O)–NH–(CH2CH2O)1-12–DUB (IVp) ODN–Y1–Y2–Y3–(CH2)1-12–OC(O)–NH–(CH2)1-12–(CH2CH2O)1-12–DUB (IVq) ODN–Y1–Y2–Y3–(CH2)1-12–OC(O)–NH–(CH2CH2O)1-12–(CH2)1-12–DUB (IVr) ODN–Y1–Y2–Y3–(CH2)1-12–OC(O)–NH–(CH2)1-12–NHC(O)–(CH2)1-12–DUB (IVs) ODN–Y1–Y2–Y3–(CH2)1-12–OC(O)–NH–(CH2CH2O)1-12–NHC(O)–(CH2)1-12–DUB (IVt) wherein Y1-Y8 are independently selected from absent, an optionally substituted heterocycloalkylene or heteroarylene group (e.g., a five membered heteroarylene, a six membered heteroarylene, a divalent triazole, a divalent imidazole, a divalent pyrrole) optionally fused to an mono or bicyclic cycloalkyl (e.g., C3-C10 monocyclic cycloalkyl, C4-C10 bicyclic cycloalkyl such as bicyclo[6.1.0] nonane), –C(O)–, –O–, –OC(O)–, –NRa–, –N(Ra)C(O)–, –(C(Ra)(Ra))1-12, –(C(Ra)(Ra)C(Ra)(Ra)O)1-12–, and –S–S–; and Ra is independently selected at each occurrence from hydrogen and optionally substitued alkyl (e.g., C1-C4 alkyl). In various implementations, the compound has the structure of formula, (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), or (IVt1): ODN–NHCO–(CH2)1-12–Y3–NH–(CH2)1-12–NH–C(O)–DUB (IVa1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–NH–C(O)–(CH2)1-12–DUB (IVb1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–NH–(CH2)1-12–DUB (IVc1)
ODN–NH–(CH2)1-12–Y3–NH–(CH2)1-12–NH–C(O)–DUB (IVd1) ODN–NH–(CH2)1-12–Y3–C(O)–(CH2)1-12–DUB (IVf1) ODN–NH–(CH2)1-12–Y3–NH–(CH2)1-12–DUB (IVg1) ODN–NHCO–(CH2)1-12–Y3–NH–(CH2CH2O)1-12–NH–C(O)–DUB (IVh1) ODN–NHCO–(CH2)1-12–Y3–(CH2CH2O)1-12–NH–C(O)–(CH2CH2O)1-12–DUB (IVi1) ODN–NHCO–(CH2)1-12–Y3–(CH2CH2O)1-12–NH–(CH2CH2O)1-12–DUB (IVj1) ODN–NHCO–(CH2)1-12–Y3–NH–(CH2CH2O)1-12–NH–C(O)–DUB (IVk1) ODN–NHCO–(CH2)1-12–Y3–C(O)–(CH2CH2O)1-12–DUB (IVl1) ODN–NHCO–(CH2)1-12–Y3–NH–(CH2CH2O)1-12–DUB (IVm1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–C(O)–NH–(CH2)1-12–DUB (IVn1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–OC(O)–NH–(CH2)1-12–DUB (IVo1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–OC(O)–NH–(CH2CH2O)1-12–DUB (IVp1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–OC(O)–NH–(CH2)1-12–(CH2CH2O)1-12–DUB (IVq1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–OC(O)–NH–(CH2CH2O)1-12–(CH2)1-12–DUB (IVr1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–OC(O)–NH–(CH2)1-12–(CH2CH2O)1-12–DUB (IVq1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–OC(O)–NH–(CH2CH2O)1-12–(CH2)1-12–DUB (IVr1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–OC(O)–NH–(CH2)1-12–NHC(O)–(CH2)1-12–DUB (IVs1) ODN–NHCO–(CH2)1-12–Y3–(CH2)1-12–OC(O)–NH–(CH2CH2O)1-12–NHC(O)–(CH2)1-12–DUB (IVt1) Particularly due to synthetic routes involving azide/alkynyl cycloaddition, in certain embodiments, Y2 or Y3 may be optionally substituted heterocycloalkylene or heteroarylene group (e.g., a five membered heteroarylene, a six membered heteroarylene, a divalent triazole, a divalent imidazole, a divalent pyrrole) optionally fused to an mono or multicyclic cycloalkyl (e.g., C3-C10 monocyclic cycloalkyl, C4-C10 bicyclic cycloalkyl such as bicyclo[6.1.0] nonane). For example, at least one of Y1-Y7 (e.g., Y2, Y3) may be a linker thaty may be formed from a strained click chemistry reaction (also referred to herein as –LSCC–) such as those having the structure:
wherein each indicates the point of attachment to the neighboring linker group (e.g., Y1, Y2, Y3
, Y4); m is an integer from 0-12 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12); p is an integer from 0-10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); Z1 is –N– or –CR3–; Z2 is O, C(O), or C(R3)2; and R3 is independently selected at each occurrence from hydrogen, –N(Ra)(Ra), alkyl (e.g., C1-C7 alkyl, C1-C3 alkyl, etc.), or alkoxy (e.g., C1-C7 alkoxy, C1-C3 alkoxy, etc.) and wherein any two vicinal R3 groups of the C8 ring may together form a five or six membered optionally substituted optionally
aromatic ring (e.g., aryl including phenyl) fused to the C8 ring. In some embodiments, at least one of Y1-Y7 (e.g., Y2, Y3) comprises the structure:
In particular embodiments, the compound has the structure of formula (V): ODN–Y1–Y2–LSCC–Y4–Y5–Y6–Y7–Y8– Y9–DUB. (V) For example, the compound of the present disclosure may have the structure: ODN–NHCO–(CH2)1-12–LSCC–NH–(CH2)1-12–NH–C(O)–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2)1-12–NH–C(O)–(CH2)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2)1-12–NH–(CH2)1-12–DUB ODN–NH–(CH2)1-12– LSCC –NH–(CH2)1-12–NH–C(O)–DUB ODN–NH–(CH2)1-12– LSCC –C(O)–(CH2)1-12–DUB ODN–NH–(CH2)1-12– LSCC –NH–(CH2)1-12–DUB
ODN–NHCO–(CH2)1-12– LSCC –NH–(CH2CH2O)1-12–NH–C(O)–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2CH2O)1-12–NH–C(O)–(CH2CH2O)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2CH2O)1-12–NH–(CH2CH2O)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –NH–(CH2CH2O)1-12–NH–C(O)–DUB ODN–NHCO–(CH2)1-12– LSCC –C(O)–(CH2CH2O)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –NH–(CH2CH2O)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2)1-12–C(O)–NH–(CH2)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2)1-12–OC(O)–NH–(CH2)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2)1-12–OC(O)–NH–(CH2CH2O)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2)1-12–OC(O)–NH–(CH2)1-12–(CH2CH2O)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2)1-12–OC(O)–NH–(CH2CH2O)1-12–(CH2)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2)1-12–OC(O)–NH–(CH2)1-12–NHCO–(CH2)1-12–DUB ODN–NHCO–(CH2)1-12– LSCC –(CH2)1-12–OC(O)–NH–(CH2CH2O)1-12–NHCO–(CH2)1-12–DUB The compounds of the present disclosure may have a linker length and be characterized with a click efficiency (e.g., as measured by reaction between a synthetic intermediate of the present disclosure with 10 fold excess of an azide linked ODN group) may be above 10%, above 20% or above 30% or above 40% or above 60% or above 70% or above 80% or above 90% (e.g., from 10%-99%, from 20%-99%, from 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, from 10%-99%, from 20%- 99%, from 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, from 20%-90%, from 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%, from 20%-90%, from 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%) efficient. In some embodiments, the compound may have the structure of formula (Va):
The linker constituents and ultimate length alter the activity of the DUBTAC. For example, in some embodiments, the compound has the structure of formula (Vb), (Vc), (Vd), or (Ve):
wherein Y7, Y8, and Y9 are independently selected from absent (i.e., it is a bond), –(C(Ra)(Ra))1-12, – N(Ra)C(O)– and –(C(Ra)(Ra)C(Ra)(Ra)O)1-12–; and Ra is independently selected at each occurrence from hydrogen and optionally substitued alkyl (e.g., C1-C4 alkyl). In some embodiments, Y8 is absent and Y7 is –(CH2)1-12–. In various implementations, Y7 is –(CH2CH2O)1-12– and Y8 is –(CH2)1-12–. In some embodiments, Y7 is –(CH2)1-12–, Y8 is – NH(Ra)–, and Y9 is –(CH2)1-12–. In certain embodiments, –Y7–Y8–Y9– (particulalry in compounds having the structure of formula (Va), (Vb), (Vc), (Vd), or (Ve)) may be part of a linker as shown in Table 1. Table 1
In particular embodiments, the compound comprises linkers 19, 20, 21, 22, 23, 24, 25, 26, or 27 from Table 1. In some embodiments any one of Y1-Y9 (e.g., Y7) may be a linear or branched linker, wherein the linking portion between adjacent linking moieties has from 1-12 atoms. For example, the compound may have the structure of formula (Vf):
wherein r and s are independently 1-12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) and the wavy bond illustrates conjugation to ODN (e.g., via (e.g., directly to) the 5’ end of the oligonucleotide). In particular embodiments, s is 5. The selection of the ODN targetting factor is typically guided by those DNA oligomer protein sequences (e.g., double stranded DNA oligomers) which bind to a transcription factor binding motif (e.g., tumor suppressor transcription factors). In some embodiments, ODN may bind to SMAD4, RFX7, REST, FOXP3, FOXP1, p53, FOXO3A, or IRF3. For example, the ODN may bind to p53, FOXO3A, or IRF3. In various aspects, ODN has a double-band hairpin structure optionally formed via intra-dimerization. In some embodiments, ODN is a double-stranded DNA comprising a sense chain and an anti-sense chain. In some embodiments, the transcription factor has one or more forkhead consensus binding sites. The DNA oligomer protein of ODN may have from 1-30 bases (e.g., 10-20 bases, 1-10 bases, 20-30 bases) and comprises transcription factor binding motif. In some embodiments, the transcription factor binding motif is G/TAAAC/TA. Suitable binding motifs (particularly for FOXO3A) are described in Brunet, A. Cell 96.6 (1999): 857-868 and Tsai, K. L., et al, Nucleic Acids Res 35.20 (2007): 6984-6994, which are hereby incorporated by reference in their entirety and particularly in relation to suitable binding motifs. In some embodiments, ODN comprises 5'- PuPuPuC(A/T)(T/A)GPyPyPy-3' (where Pu stands for purine and Py for pyrimidine) separated by 0-
13 base-pairs. In some embodiments, ODN comprises two tandem repeates of 10 base-pair motif 5'- PuPuPuC(A/T)(T/A)GPyPyPy-3' (where Pu stands for purine and Py for pyrimidine) separated by 0- 13 base-pairs separated by 0-13 base pairs. Suitable binding motifs (particularly for p53) are described in Wang, Y., et al Mol Cell Biol 15.4 (1995): 2157-2165 and El-Deiry, eta l. Nat Genet 1.1 (1992): 45-49, which are hereby incorporated by reference in their entirety and particularly in relation to suitable binding motifs. In some embodiments, ODN comprises AANNGAAA (N stands for any nucleotide). Suitable binding motifs (particularly for IRF3) are described in Panne D., et al, Cell 129.9 (2007): 1111-1123 and Panne, D., et al. EMBO J 23.22 (2004): 4384-4393, which are hereby incorporated by reference in their entirety and particularly in relation to suitable binding motifs. wherein R is purine, Y is pyrimidine, and N is any base. ODN may comprise, be substantially identical to, or is the sequence CTATGTAAACAACTTTGTTGTTTACATAG, AGACATGCCTAGACATGCCT, TCCGTACAGATCCGTACAGA, GAAACTGAAACTTTTAGTTTCAGTTTC, 5’-CTATGTAAACAACTTTGTTGTTTACATAG-3’, 5’-AGACATGCCTAGACATGCCT-3’, 5’-TCCGTACAGATCCGTACAGA-3’, or 5’-GAAACTGAAACTTTTAGTTTCAGTTTC-3’; each of which may form a double stranded DNA hairpin structure via interdimerization. In some embodiments, ODN is a double stranded DNA having a sense chain and an anti-sense chain, wherein the sense chain. For example, the sense chain may be 5’-AGACATGCCTAGACATGCCT- 3’; and the anti-sense chain may be 5’-TCCGTACAGATCCGTACAGA-3’. The ODN oligonucleotide may be conjugated to L1 through the 5’ end of the oligonucleotide (e.g., via the amino modifier C6). The compound may be, for example, any compound in Table 2 and formula (Vg) (where the ODN is conjugated through the 5’ end of the oligonucleotide).
. In some embodiments Y7 may be a linear or branched linker, wherein the linking portion between adjacent linking moieties has from 1-12 atoms. In some embodiments Y7 is –(CH2)2-11– and ODN is 5’-CTATGTAAACAACTTTGTTGTTTACATAG-3’, 5’-GAAACTGAAACTTTTAGTTTCAGTTTC-3’, or 5’-AGACATGCCTAGACATGCCT-3’ (bound to 5’-TCCGTACAGATCCGTACAGA-3’) Table 2
Pharmaceutical compositions comprising the DUBTACs (or agents) are also provided. These compositions may comprise one or more pharmaceutically acceptable salts, carriers, or diluents, and a DUBTAC. In some embodiments, the composition comprises an agent comprising an oligonucleotide that hybridizes to a transcription factor of interest (e.g., a tumor suppressor transcription factor), wherein the oligonucleotide is directly or indirectly bound to a ligand of a deubiquitinase. For example, the composition may comprise an agent having the structure of formula (I): ODN–L1–DUB (I) wherein DUB is a deubiquitinase binding moiety; L1 is absent or a linker; ODN is a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing. Methods for reducing the proliferation or survival of a neoplastic cell comprising a transcription factor of interest (e.g., a tumor suppressor) are also provided comprising contacting the cell with a DUBTAC. Contact may stabilize the transcription factor in the cell. In some embodiments, the comprises comprises contacting the cell with an agent comprising an oligonucleotide that hybridizes to the transcription factor of interest (e.g., a tumor suppressor transcription factor), wherein the oligonucleotide is directly or indirectly bound to a ligand of a deubiquitinase. For example, the composition may comprise an agent having the structure of formula (I): ODN–L1–DUB (I) wherein DUB is a deubiquitinase binding moiety; L1 is absent or a linker;
ODN is a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing. Methods for the treatment or prophylaxis of a disease (e.g., a proliferative disease) in a subject in need thereof are also provided. These methods may comprise administering a DUBTAC of the present disclosure (or a pharmaceutical composition comprising a DUBTAC of the present disclosure) to the subject in need thereof. In some embodiments, the method comprises administering an agent comprising an oligonucleotide that hybridizes to a transcription factor of interest (e.g., a tumor suppressor transcription factor), wherein the oligonucleotide is directly or indirectly bound to a ligand of a deubiquitinase. For example, the composition may comprise an agent having the structure of formula (I): ODN–L1–DUB (I) wherein DUB is a deubiquitinase binding moiety; L1 is absent or a linker; ODN is a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof (e.g., a tumor suppressor transcription factor); or pharmaceutically acceptable salts of the foregoing. Compounds for synthesis of the DUBTACs as described herein are also provided. These synthetic intermediates may be a compound having the structure of formula (VI): Y2–Y3–Y4–Y5–Y6–Y7–Y8–Y9–DUB (VI) wherein DUB is a deubiquitinase binding moiety; and wherein Y2 is optionally substituted alkynyl (e.g., C1-C12 alkynyl), optionally substituted monocyclic or bicyclic cycloalkynyl (e.g., C3-C12 monocyclic or multicyclic cycloalkynyl, C3-C12 bicyclic cycloalkynyl), optionally substituted heteroalkynyl (e.g., C1-C12 heteroalkynyl), or optionally substituted monocyclic or bicyclic heterocycloalkynyl (e.g., C3-C12 monocyclic heterocycloalkynyl, C3-C12 bicyclic heterocycloalkynyl); Y3-Y9 are independently selected from absent, an optionally substituted heteroarylene group (e.g., a five membered heteroarylene, a six membered heteroarylene, a divalent triazole, a divalent imidazole, a divalent pyrrole) optionally fused to an mono or bicyclic cycloalkyl (e.g., C3-C10 monocyclic cycloalkyl, C4-C10 bicyclic cycloalkyl such as bicyclo[6.1.0] nonane), –C(O)–, –O–, –OC(O)–, –NRa–, –N(Ra)C(O)–, –(C(Ra)(Ra))1-12, –(C(Ra)(Ra)C(Ra)(Ra)O)1-12–, and –S–S–; and Ra is independently selected at each occurrence from hydrogen and optionally substitued alkyl (e.g., C1-C4 alkyl). For example Y2 may be:
wherein indicates the point of attachment to the neighboring linker group (Y3); m is an integer from 0-12 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12); p is an integer from 0-10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); Z1 is –N– or –CR3–; Z2 is O, C(O), or C(R3)2; and
R3 is independently selected at each occurrence from hydrogen, –N(Ra)(Ra), alkyl (e.g., C1-C7 alkyl, C1-C3 alkyl, etc.), or alkoxy (e.g., C1-C7 alkoxy, C1-C3 alkoxy, etc.) and wherein any two vicinal R3 groups of the C8 ring may together form a five or six membered optionally aromatic ring (e.g., aryl such as phenyl) fused to the C8 ring. In some embodiments, the compound may have the structure of formula (VIa):
The compound may, for example, have the structure of formula (VIb):
. wherein Y7 and Y9 are independently selected from absent (i.e., it is a bond), –(C(Ra)(Ra))1-12, and –(C(Ra)(Ra)C(Ra)(Ra)O)1-12–; and Ra is independently selected at each occurrence from hydrogen and optionally substitued alkyl (e.g., C1-C4 alkyl). In some embodiments, Y9 is –(CH2)2–. In particular embodiments, the synthetic intermediate is a compound having the structure of formula (VIb) with the Y7, Y8, and Y9 linkers in Table 1. The synthetic intermediates may have a linker length and be characterized with a click efficiency (e.g., as measured by reaction between a synthetic intermediate of the present disclosure with 10 fold excess of an azide linked ODN group) may be above 10%, above 20% or above 30% or above 40% or above 60% or above 70% or above 80% or above 90% (e.g., from 10%-99%, from 20%-99%, from 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, from 10%-99%, from 20%- 99%, from 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, from 20%-90%, from 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%, from 20%-90%, from 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%) efficient. Methods for forming the DUBTACs of the present disclosure a compound ((e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) are provided as well. Typically, these methods comprise reacting (e.g. via an azide-alkyne
cycloaddition reaction) a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof; where said DNA oligomer protein is conjugated to an azide with a compound having the structure of formula (VI): Y2–Y3–Y4–Y5–Y6–Y7–Y8–Y9–DUB (VI) wherein DUB is a deubiquitinase binding moiety; and wherein Y2 is optionally substituted alkynyl (e.g., C1-C12 alkynyl), optionally substituted monocyclic or bicyclic cycloalkynyl (e.g., C3-C12 monocyclic or multicyclic cycloalkynyl, C3-C12 bicyclic cycloalkynyl), optionally substituted heteroalkynyl (e.g., C1-C12 heteroalkynyl), or optionally substituted monocyclic or bicyclic heterocycloalkynyl (e.g., C3-C12 monocyclic heterocycloalkynyl, C3-C12 bicyclic heterocycloalkynyl); Y3-Y8 are independently selected from absent, an optionally substituted heteroarylene group (e.g., a five membered heteroarylene, a six membered heteroarylene, a divalent triazole, a divalent imidazole, a divalent pyrrole) optionally fused to an mono or bicyclic cycloalkyl (e.g., C3-C10 monocyclic cycloalkyl, C4-C10 bicyclic cycloalkyl such as bicyclo[6.1.0] nonane), –C(O)–, –O–, –OC(O)–, –NRa–, –N(Ra)C(O)–, –(C(Ra)(Ra))1-12, –(C(Ra)(Ra)C(Ra)(Ra)O)1-12–, and –S–S–; and Ra is independently selected at each occurrence from hydrogen and optionally substitued alkyl (e.g., C1-C4 alkyl). The method may further include the step of conjugating an azide to the DNA oligomer protein. For example, the azide can be introduced to an oligonucleotide by attaching an azide-NHS ester functional group to 5', 3' or through internal amino modified base or amino linkers such as amino C6 or amino C7 for the 3' end of an oligo. Synthetic steps may occur in physiological conditions such as in phosphate buffered saline (PBS) at from 30°C-40°C. These synthetic methods be characterized with a click efficiency (e.g., as measured by reaction between a synthetic intermediate of the present disclosure with 10 fold excess of an azide linked ODN group) may be above 10%, above 20% or above 30% or above 40% or above 60% or above 70% or above 80% or above 90% (e.g., from 10%-99%, from 20%-99%, from 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%-99%, from 10%-99%, from 20%-99%, from 30%-99%, 40%-99%, 50%-99%, 60%-99%, 70%- 99%, from 20%-90%, from 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%, from 20%-90%, from 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%) efficient. Definitions Detailed embodiments of the present disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative and may be embodied in various
forms. In addition, each of the examples given in connection with the various embodiments of the disclosure is intended to be illustrative, and not restrictive. All terms used herein are intended to have their ordinary meaning in the art unless otherwise provided. All concentrations are in terms of percentage by weight of the specified component relative to the entire weight of the topical composition, unless otherwise defined. As used herein, “a” or “an” shall mean one or more. As used herein when used in conjunction with the word “comprising,” the words “a” or “an” mean one or more than one. As used herein “another” means at least a second or more. As used herein, all ranges of numeric values include the endpoints and all possible values disclosed between the disclosed values. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. For example, the exact values of all half-integral numeric values are also contemplated as specifically disclosed and as limits for all subsets of the disclosed range. For example, a range of from 0.1% to 3% specifically discloses a percentage of 0.1%, 1%, 1.5%, 2.0%, 2.5%, and 3%. Additionally, a range of 0.1 to 3% includes subsets of the original range including from 0.5% to 2.5%, from 1% to 3%, from 0.1% to 2.5%, etc. It will be understood that sum of all weight percentages does not exceed 100% unless otherwise specified. By “OTUB1 polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_060140.2 or a fragment thereof having deubiquitinase activity. An exemplary OTUB1 amino acid sequence follows: 1 maaeepqqqk qeplgsdseg vnclaydeai maqqdriqqe iavqnplvse rlelsvlyke 61 yaeddniyqq kikdlhkkys yirktrpdgn cfyrafgfsh leallddske lqrfkavsak 121 skedlvsqgf teftiedfhn tfmdlieqve kqtsvadlla sfndqstsdy lvvylrllts 181 gylqreskff ehfieggrtv kefcqqevep mckesdhihi ialaqalsvs iqveymdrge 241 ggttnphifp egsepkvyll yrpghydily k By “OTUB1 polynucleotide” is meant a polynucleotide encoding an OTUB1 polypeptide. An exemplary OTUB1 polynucleotide sequence is provided at NCBI Accession No. NM_017670, which is reproduced below: 1 agtgcggcgc tgtttaaaga tggcggcgga ggaacctcag cagcagaagc aggagccgct 61 gggcagcgac tccgaaggtg ttaactgtct ggcctatgat gaagccatca tggctcagca 121 ggaccgaatt cagcaagaga ttgctgtgca gaaccctctg gtgtcagagc ggctggagct 181 ctcggtccta tacaaggagt atgctgaaga tgacaacatc tatcaacaga agatcaagga 241 cctccacaaa aagtactcgt acatccgcaa gaccaggcct gacggcaact gtttctatcg 301 ggctttcgga ttctcccact tggaggcact gctggatgac agcaaggagt tgcagcggtt 361 caaggctgtg tctgccaaga gcaaggaaga cctggtgtcc cagggcttca ctgaattcac 421 aattgaggat ttccacaaca cgttcatgga cctgattgag caggtggaga agcagacctc 481 tgtcgccgac ctgctggcct ccttcaatga ccagagcacc tccgactacc ttgtggtcta 541 cctgcggctg ctcacctcgg gctacctgca gcgcgagagc aagttcttcg agcacttcat 601 cgagggtgga cggactgtca aggagttctg ccagcaggag gtggagccca tgtgcaagga 661 gagcgaccac atccacatca ttgcgctggc ccaggccctc agcgtgtcca tccaggtgga 721 gtacatggac cgcggcgagg gcggcaccac caatccgcac atcttccctg agggctccga 781 gcccaaggtc taccttctct accggcctgg acactacgat atcctctaca aatagggctg
841 gctccagccc gctgctgccc tgctgccccc ctctgccagg cgctagacat gtacagaggt 901 ttttctgtgg ttgtaaatgg tcctatttca cccccttctt cctgtcacat gacccccccc 961 catgttttat taaagggggt gctggtggtg agccgtgtgt gcgtgtccct gctctgctgc 1021 ccgcctggct gctctgtctg ctgccccctc cccccaggtg ggtccccctg cttttcacct 1081 atctactcct gagcttcccc aacaggagca ggtttgaggg gccaggcctc ttggaggccc 1141 ctcctgcttc gttgggttct gcttccttcc cttcttagct ggctcagggg cttctatggg 1201 atcctggaag ttccttaggg acttgcccag ggtcccaggg ccacccacac ttcatctgct 1261 ccctcatagg ccccacctcc acgtcccggc tgggccccag accccagctt cctgccctcc 1321 accgggagtc tgcatggttg ggagtcctgg gtggaggggc ctttgtgagg ctggacccgg 1381 ctcagggcag gtggaggagc tgggcctccc acagggtgcc cgggcagtgc catcctggtg 1441 ggggagggca gccttcaaac gtgtggggtc tacagtcctc aggtctaggc agggctgccg 1501 gttctccacc tccccatccg ccccaggccc cctgcctgtg cctgccttgc accccctctg 1561 cttgggccac ggtgtctctg cattgcctgc ctttttgcct tcacctcttt tcttccccgc 1621 cccctgcaca ttcggggtct cagcccccag gctgtgagct ccttgggggc aggccctcaa 1681 taaatgtgaa ctgctgctgc c By “p53 polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NG_017013.2 or a fragment thereof having DNA binding activity. An exemplary p53 amino acid sequence follows: 1 meepqsdpsv epplsqetfs dlwkllpenn vlsplpsqam ddlmlspddi eqwftedpgp 61 deaprmpeaa prvapapaap tpaapapaps wplsssvpsq ktyqgsygfr lgflhsgtak 121 svtctyspal nkmfcqlakt cpvqlwvdst pppgtrvram aiykqsqhmt evvrrcphhe 181 rcsdsdglap pqhlirvegn lrveylddrn tfrhsvvvpy eppevgsdct tihynymcns 241 scmggmnrrp iltiitleds sgnllgrnsf evhvcacpgr drrteeenlr kkgephhelp 301 pgstkralsn ntssspqpkk kpldgeyftl qirgrerfem frelnealel kdaqagkepg 361 gsrahsshlk skkgqstsrh kklmfktegp dsd By “p53 polynucleotide” is meant a polynucleotide encoding a p53 polypeptide. An exemplary p53 polynucleotide sequence is provided at NCBI Accession No. NM_001276760.3, which is reproduced below: 1 ctcaaaagtc tagagccacc gtccagggag caggtagctg ctgggctccg gggacacttt 61 gcgttcgggc tgggagcgtg ctttccacga cggtgacacg cttccctgga ttggcagcca 121 gactgccttc cgggtcactg ccatggagga gccgcagtca gatcctagcg tcgagccccc 181 tctgagtcag gaaacatttt cagacctatg gaaactactt cctgaaaaca acgttctgtc 241 ccccttgccg tcccaagcaa tggatgattt gatgctgtcc ccggacgata ttgaacaatg 301 gttcactgaa gacccaggtc cagatgaagc tcccagaatg ccagaggctg ctccccccgt 361 ggcccctgca ccagcagctc ctacaccggc ggcccctgca ccagccccct cctggcccct 421 gtcatcttct gtcccttccc agaaaaccta ccagggcagc tacggtttcc gtctgggctt 481 cttgcattct gggacagcca agtctgtgac ttgcacgtac tcccctgccc tcaacaagat 541 gttttgccaa ctggccaaga cctgccctgt gcagctgtgg gttgattcca cacccccgcc 601 cggcacccgc gtccgcgcca tggccatcta caagcagtca cagcacatga cggaggttgt 661 gaggcgctgc ccccaccatg agcgctgctc agatagcgat ggtctggccc ctcctcagca 721 tcttatccga gtggaaggaa atttgcgtgt ggagtatttg gatgacagaa acacttttcg 781 acatagtgtg gtggtgccct atgagccgcc tgaggttggc tctgactgta ccaccatcca 841 ctacaactac atgtgtaaca gttcctgcat gggcggcatg aaccggaggc ccatcctcac 901 catcatcaca ctggaagact ccagtggtaa tctactggga cggaacagct ttgaggtgcg 961 tgtttgtgcc tgtcctggga gagaccggcg cacagaggaa gagaatctcc gcaagaaagg 1021 ggagcctcac cacgagctgc ccccagggag cactaagcga gcactgccca acaacaccag 1081 ctcctctccc cagccaaaga agaaaccact ggatggagaa tatttcaccc ttcagatccg 1141 tgggcgtgag cgcttcgaga tgttccgaga gctgaatgag gccttggaac tcaaggatgc 1201 ccaggctggg aaggagccag gggggagcag ggctcactcc agccacctga agtccaaaaa 1261 gggtcagtct acctcccgcc ataaaaaact catgttcaag acagaagggc ctgactcaga 1321 ctgacattct ccacttcttg ttccccactg acagcctccc acccccatct ctccctcccc 1381 tgccattttg ggttttgggt ctttgaaccc ttgcttgcaa taggtgtgcg tcagaagcac 1441 ccaggacttc catttgcttt gtcccggggc tccactgaac aagttggcct gcactggtgt 1501 tttgttgtgg ggaggaggat ggggagtagg acataccagc ttagatttta aggtttttac 1561 tgtgagggat gtttgggaga tgtaagaaat gttcttgcag ttaagggtta gtttacaatc 1621 agccacattc taggtagggg cccacttcac cgtactaacc agggaagctg tccctcactg 1681 ttgaattttc tctaacttca aggcccatat ctgtgaaatg ctggcatttg cacctacctc 1741 acagagtgca ttgtgagggt taatgaaata atgtacatct ggccttgaaa ccacctttta
1801 ttacatgggg tctagaactt gacccccttg agggtgcttg ttccctctcc ctgttggtcg 1861 gtgggttggt agtttctaca gttgggcagc tggttaggta gagggagttg tcaagtctct 1921 gctggcccag ccaaaccctg tctgacaacc tcttggtgaa ccttagtacc taaaaggaaa 1981 tctcacccca tcccacaccc tggaggattt catctcttgt atatgatgat ctggatccac 2041 caagacttgt tttatgctca gggtcaattt cttttttctt tttttttttt ttttttcttt 2101 ttctttgaga ctgggtctcg ctttgttgcc caggctggag tggagtggcg tgatcttggc 2161 ttactgcagc ctttgcctcc ccggctcgag cagtcctgcc tcagcctccg gagtagctgg 2221 gaccacaggt tcatgccacc atggccagcc aacttttgca tgttttgtag agatggggtc 2281 tcacagtgtt gcccaggctg gtctcaaact cctgggctca ggcgatccac ctgtctcagc 2341 ctcccagagt gctgggatta caattgtgag ccaccacgtc cagctggaag ggtcaacatc 2401 ttttacattc tgcaagcaca tctgcatttt caccccaccc ttcccctcct tctccctttt 2461 tatatcccat ttttatatcg atctcttatt ttacaataaa actttgctgc ca By “IRF3 polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NG_031810.1 or a fragment thereof having DNA binding activity. An exemplary IRF3 amino acid sequence follows: 1 mgtpkprilp wlvsqldlgq legvawvnks rtrfripwkh glrqdaqqed fgifqawaea 61 tgayvpgrdk pdlptwkrnf rsalnrkegl rlaedrskdp hdphkiyefv nsgvgdfsqp 121 dtspdtnggg stsdtqedil dellgnmvla plpdpgppsl avapepcpqp lrspsldnpt 181 pfpnlgpsen plkrllvpge ewefevtafy rgrqvfqqti scpeglrlvg sevgdrtlpg 241 wpvtlpdpgm sltdrgvmsy vrhvlsclgg glalwragqw lwaqrlghch tywavseell 301 pnsghgpdge vpkdkeggvf dlgpfivgsw aprsdylhgr krtlttlcpl vlcggvmapg 361 pavdqeardg qgcahvpqgl grngpgrgcl lpgeycgpah fqqpptlphl rpvqglpagl 421 ggghgfpgpw gelsprsswc asnppvphhl nq By “IRF3 polynucleotide” is meant a polynucleotide encoding a IRF3 polypeptide. An exemplary IRF3 polynucleotide sequence is provided at NCBI Accession No. NG_
31810.1, which is reproduced below: 1 tcaagaagtc gatcaaaaag aaagccccag cgctctagag ctcagctgac gggaaagggg 61 gtgcgcagcc tcgagtttga gagctacccg gagctccaag acaggggtgg gttccagctg 121 cccgcacgcc ccgaccttcc atcgtaggcc ggaccatggg aaccccaaag ccacggatcc 181 tgccctggct ggtgtcgcag ctggacctgg ggcaactgga gggcgtggcc tgggtgaaca 241 agagccgcac gcgcttccgc atcccttgga agcacggcct acggcaggat gcacagcagg 301 aggatttcgg aatcttccag gcctgggccg aggccactgg tgcatatgtt cccgggaggg 361 ataagccaga cctgccaacc tggaagagga atttccgctc tgccctcaac cgcaaagaag 421 ggttgcgttt agcagaggac cggagcaagg accctcacga cccacataaa atctacgagt 481 ttgtgaactc aggagttggg gacttttccc agccagacac ctctccggac accaatggtg 541 gaggcagtac ttctgatacc caggaagaca ttctggatga gttactgggt aacatggtgt 601 tggccccact cccagatccg ggacccccaa gcctggctgt agcccctgag ccctgccctc 661 agcccctgcg gagccccagc ttggacaatc ccactccctt cccaaacctg gggccctctg 721 agaacccact gaagcggctg ttggtgccgg gggaagagtg ggagttcgag gtgacagcct 781 tctaccgggg ccgccaagtc ttccagcaga ccatctcctg cccggagggc ctgcggctgg 841 tggggtccga agtgggagac aggacgctgc ctggatggcc agtcacactg ccagaccctg 901 gcatgtccct gacagacagg ggagtgatga gctacgtgag gcatgtgctg agctgcctgg 961 gtgggggact ggctctctgg cgggccgggc agtggctctg ggcccagcgg ctggggcact 1021 gccacacata ctgggcagtg agcgaggagc tgctccccaa cagcgggcat gggcctgatg 1081 gcgaggtccc caaggacaag gaaggaggcg tgtttgacct ggggcccttc attgtaggct 1141 cctgggcccc cagatctgat taccttcacg gaaggaagcg gacgctcacc acgctatgcc 1201 ctctggttct gtgtggggga gtcatggccc caggaccagc cgtggaccaa gaggctcgtg 1261 atggtcaagg ttgtgcccac gtgcctcagg gccttggtag aaatggcccg ggtagggggt 1321 gcctcctccc tggagaatac tgtggacctg cacatttcca acagccaccc actctccctc 1381 acctccgacc agtacaaggc ctacctgcag gacttggtgg agggcatgga tttccagggc 1441 cctggggaga gctgagccct cgctcctcat ggtgtgcctc caacccccct gttccccacc 1501 acctcaacca ataaactggt tcctgctatg aaaaaaaaaa aaaaaaaaa By “FOXO3a polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_963853.1 or a fragment thereof having DNA binding activity. An exemplary FOXO3a amino acid sequence follows:
1 maeapaspap lspleveldp efepqsrprs ctwplqrpel qaspakpsge taadsmipee 61 eddeddedgg gragsamaig ggggsgtlgs gllledsarv lapggqdpgs gpataaggls 121 ggtqallqpq qplpppqpga aggsgqprkc ssrrnawgnl syadlitrai esspdkrltl 181 sqiyewmvrc vpyfkdkgds nssagwknsi rhnlslhsrf mrvqnegtgk sswwiinpdg 241 gksgkaprrr avsmdnsnky tksrgraakk kaalqtapes addspsqlsk wpgsptsrss 301 deldawtdfr srtnsnastv sgrlspimas teldevqddd aplspmlyss saslspsvsk 361 pctvelprlt dmagtmnlnd gltenlmddl ldnitlppsq psptgglmqr sssfpyttkg 421 sglgsptssf nstvfgpssl nslrqspmqt iqenkpatfs smshygnqtl qdlltsdsls 481 hsdvmmtqsd plmsqastav saqnsrrnvm lrndpmmsfa aqpnqgslvn qnllhhqhqt 541 qgalggsral snsvsnmgls essslgsakh qqqspvsqsm qtlsdslsgs slystsanlp 601 vmghekfpsd ldldmfngsl ecdmesiirs elmdadgldf nfdslistqn vvglnvgnft 661 gakqassqsw vpg By “FOXO3a polynucleotide” is meant a polynucleotide encoding a FOXO3a polypeptide. An exemplary FOXO3a polynucleotide sequence is provided at NCBI Accession No. NM_001455.4, which is reproduced below: 1 ggtgtctgct gcgccaggtt cgctggccgc acgtcttcag gtcctcctgt tcctgggagg 61 cgggcgcggc aggactggga ggtggcggca gcgggcgagg actcgccgag gacggggctc 121 cggcccggga taaccaactc tccttctctc ttctttggtg cttccccagg cggcggcggc 181 ggcgcccggg agccggagcc ttcgcggcgt ccacgtccct cccccgctgc accccgcccc 241 ggcgcgagag gagagcgcga gagccccagc cgcgggcggg cgggcggcga agatggcaga 301 ggcaccggct tccccggccc cgctctctcc gctcgaagtg gagctggacc cggagttcga 361 gccccagagc cgtccgcgat cctgtacgtg gcccctgcaa aggccggagc tccaagcgag 421 ccctgccaag ccctcggggg agacggccgc cgactccatg atccccgagg aggaggacga 481 tgaagacgac gaggacggcg ggggacgggc cggctcggcc atggcgatcg gcggcggcgg 541 cgggagcggc acgctgggct ccgggctgct ccttgaggac tcggcccggg tgctggcacc 601 cggagggcaa gaccccgggt ctgggccagc caccgcggcg ggcgggctga gcgggggtac 661 acaggcgctg ctgcagcctc agcaaccgct gccaccgccg cagccggggg cggctggggg 721 ctccgggcag ccgaggaaat gttcgtcgcg gcggaacgcc tggggaaacc tgtcctacgc 781 ggacctgatc acccgcgcca tcgagagctc cccggacaaa cggctcactc tgtcccagat 841 ctacgagtgg atggtgcgtt gcgtgcccta cttcaaggat aagggcgaca gcaacagctc 901 tgccggctgg aagaactcca tccggcacaa cctgtcactg catagtcgat tcatgcgggt 961 ccagaatgag ggaactggca agagctcttg gtggatcatc aaccctgatg gggggaagag 1021 cggaaaagcc ccccggcggc gggctgtctc catggacaat agcaacaagt ataccaagag 1081 ccgtggccgc gcagccaaga agaaggcagc cctgcagaca gcccccgaat cagctgacga 1141 cagtccctcc cagctctcca agtggcctgg cagccccacg tcacgcagca gtgatgagct 1201 ggatgcgtgg acggacttcc gttcacgcac caattctaac gccagcacag tcagtggccg 1261 cctgtcgccc atcatggcaa gcacagagtt ggatgaagtc caggacgatg atgcgcctct 1321 ctcgcccatg ctctacagca gctcagccag cctgtcacct tcagtaagca agccgtgcac 1381 ggtggaactg ccacggctga ctgatatggc aggcaccatg aatctgaatg atgggctgac 1441 tgaaaacctc atggacgacc tgctggataa catcacgctc ccgccatccc agccatcgcc 1501 cactggggga ctcatgcagc ggagctctag cttcccgtat accaccaagg gctcgggcct 1561 gggctcccca accagctcct ttaacagcac ggtgttcgga ccttcatctc tgaactccct 1621 acgccagtct cccatgcaga ccatccaaga gaacaagcca gctaccttct cttccatgtc 1681 acactatggt aaccagacac tccaggacct gctcacttcg gactcactta gccacagcga 1741 tgtcatgatg acacagtcgg accccttgat gtctcaggcc agcaccgctg tgtctgccca 1801 gaattcccgc cggaacgtga tgcttcgcaa tgatccgatg atgtcctttg ctgcccagcc 1861 taaccaggga agtttggtca atcagaactt gctccaccac cagcaccaaa cccagggcgc 1921 tcttggtggc agccgtgcct tgtcgaattc tgtcagcaac atgggcttga gtgagtccag 1981 cagccttggg tcagccaaac accagcagca gtctcctgtc agccagtcta tgcaaaccct 2041 ctcggactct ctctcaggct cctccttgta ctcaactagt gcaaacctgc ccgtcatggg 2101 ccatgagaag ttccccagcg acttggacct ggacatgttc aatgggagct tggaatgtga 2161 catggagtcc attatccgta gtgaactcat ggatgctgat gggttggatt ttaactttga 2221 ttccctcatc tccacacaga atgttgttgg tttgaacgtg gggaacttca ctggtgctaa 2281 gcaggcctca tctcagagct gggtgccagg ctgaaggatc actgaggaag gggaagtggg 2341 caaagcagac cctcaaactg acacaagacc tacagagaaa accctttgcc aaatctgctc 2401 tcagcaagtg gacagtgata ccgtttacag cttaacacct ttgtgaatcc cacgccattt 2461 tcctaaccca gcagagactg ttaatggccc cttaccctgg gtgaagcact tacccttgga 2521 acagaactct aaaaagtatg caaaatcttc cttgtacagg gtggtgagcc gcctgccagt 2581 ggaggacagc acccctcagc accacccacc ctcattcaga gcacaccgtg agcccccgtc 2641 ggccattctg tggtgtttta atattgcgat ggtttatggg acgttttaag tgttgttctt 2701 gtgtttgttt tcctttgact ttctgagttt ttcacatgca ttaacttgcg gtatttttct
2761 gttaaaatgt taaccgtcct tcccctagca aatttaaaaa cagaaagaaa atgttgtacc 2821 agttaccatt ccgggttcga gcatcacaag cttttgagcg catggaactc cataaactaa 2881 caaattacat aaactaaagg gggattttct ttcttctttt gtttggtaga aaattatcct 2941 tttctaaaaa ctgaacaatg gcacaattgt ttgctatgtg cacccgtcca ggacagaacc 3001 gtgcataggc aaaaggagtg gagcacagcg tccggcccag tgtgtttccg gttctgagtc 3061 agggtgatct gtggacggga ccccagcacc aagtctacgg gtgccagatc agtagggcct 3121 gtgatttcct gtcagtgtcc tcagctaatg tgaacagtgt tggtctgctg gttagaaact 3181 agaatattga tattttcagg aaagaaatca gctcagctct ccactcattg ccaaatgtca 3241 ctaaagggtt tagttttaag gagaaagaaa aggaaaaaaa aaaaaaacaa aaaagtcctg 3301 ttttgctttg cagaacaaat gaacttacag gtgagcatta agcttgcagt gagaaatgtg 3361 cgaagagtaa aaacccaagt caatgctgag gcagttctaa cttcactgtt ttcctaaata 3421 cacatccttg attattttca gccttgctat ataatctgat ctgctagaag tgtatgagtg 3481 agaggcaata gcatacaaac tgatttttta aatataagct taggttgtaa ttgtacaagt 3541 gactcaatgg aagtacaaaa tagggcagtt ttaacttttt tttctgcttc tatggatttc 3601 attttgttgt gttttcaaaa agttatggtg ctgtataggt gctttctgtt taacctggaa 3661 agtgtgatta tattcgttac cttctttggt agacggaata gttgggacca cctttggtac 3721 ataagaaatt ggtataacga tgctctgatt agcacagtat atgcatactt ctccaaagtg 3781 atatatgaag actcttttct ttgcataaaa agcattaggc atataaatgt ataaatatat 3841 tttatcatgt acagtacaaa aatggaacct tatgcatggg ccttaggaat acaggctagt 3901 atttcagcac agacttccct gcttgagttc ttgctgatgc ttgcaccgtg acagtgggca 3961 ccaacacaga cgtgccaccc aaccccctgc acacaccacc ggccaccagg ggcccccttg 4021 tgcgccttgg ctttataact cctctggggg tgatattggt ggtgatcaca gctcctagca 4081 taatgagagt tccatttggt attgtcacac gtctcctgcc tcgcttgggt tgccatgttt 4141 gagcgatggc cctgttgatt tcaccctgcc ttttactgaa tctgtaaatt gttgtgcaat 4201 tgtggttata gtagactgta gcacattgcc ttttctaaac tgctacatgt ttataatctt 4261 catttttaaa gtatgtgtaa tttttttaag tatgtattct attcatatgg tctgcttgtc 4321 agtgagccag acttgcttac tatattcctt tataataatg ctagccactt cctggattct 4381 ttagtaatgt gctgtatgca agaactttcc agtagcagtg aaggagggtt gcctctccaa 4441 gcttcctaag ggatgctgcc ctgtgtgggg atgcattgca gaggcactag tagcatgggg 4501 gctagagtgg ggagcgagat gtaaaagggt ggggggatag gagaattcca gagtgcttcc 4561 agcattaggg tcctgagaac ttctgagttc agagaaacat gcaaagtgac taacaaaata 4621 gctacttacc tttgcagttt tacagaccct gggagctgct ttgggagtga gaaaggcaac 4681 cctccaatgt gtttcaactt taaaatgttg aattcttttc agacatggta tctcatttat 4741 tctccttttc tagcgtttgt tgaatttcag gcagaatgtc ttacagaatg tcctagaacc 4801 agattatcat ttaatctgaa acagctgagg aagggacaga gaaggtacaa gggcaaggca 4861 gcacaaaaca gatcaggaga atgaagaggg aatgctttgg ttttttgttt tgttttgttt 4921 tttctttttc aagtaactaa aacagcatct acatgtagag tgttgtggag agctgagacc 4981 agggtaaagt caagtgcagc atcagtactg cgagacccac cagcccctgg agagggtcag 5041 ccgagaatct ggtagtgaag cctgtctagg gtcccggcac cctcaccctc agccacctgc 5101 agagaggcca gggccccaga gactagcctg gttctgaagt gggcaggggt gctgccagag 5161 ccctctgccc cttatgttga gaccctgctt tcaggacagg ccagccgttg gccaccatgt 5221 cacattctga gtgagtgtca caggtcccta acaataattt tctgatctgg agcatatcag 5281 cagaatgctt agcctcaagg ggcctggcag ctgtaatgtt tgatttatga tgagaactat 5341 ccgaggccac ccttggcctc taaataagct gctctaggga gccgcctact ttttgatgag 5401 aaattagaag agtacctaat gttgaaaaca tgacatgcgc tcttgggatc tgctgttctc 5461 tccagggctc cagaacctga tacctgttac caaagctagg aaagagcttt atcacaagcc 5521 ttcactgtcc tggcatgaga actggctgcc aggctcagtg taccccatta actgtgaatg 5581 aatctgagct tggtttcctt tattgcttcc tctgcaatat gattgctgaa acacatttta 5641 aaaattcaga agcttgtcac tcctgttaat gggaggatca gtcacacatg tgtagtacaa 5701 ggcggacttt gtgtttgttt ttggtgttaa tttttagcat tgtgtgtgtt gcttccccac 5761 cctgaggaga ggacaccatg gcttactact caggacaagt atgccccgct cagggtgtga 5821 tttcaggtgg cttccaaact tgtacgcagt ttaaagatgg tggggacaga ctttgcctct 5881 acctagtgaa ccccacttaa agaataagga gcatttgaat ctcttggaaa aggccatgaa 5941 gaataaagca gtcaaaaaga agtcctccat gttggtgcca aggacttgcg aggggaaata 6001 aaaatgttat ccagcctgac caacatggag aaaccccgtc tccattaaaa atacaaaatt 6061 agcctggcat ggtggcgcat gcctgtaatc ccagctactc tggaggctga ggcaggagaa 6121 tcgcttgaac ccaggaggcg gaggtcgcag tgagccgaga tcatgccagt gcactccagc 6181 ctgggtaaca agagtgaaac tccgtgtcaa aaaaaaaaaa aaaatgttac tcatcctctc 6241 tgaaagcaaa aaggaaaccc taacagctct gaactctggt tttatttttc ttgctgtatt 6301 tgggtgaaca ttgtatgatt aggcataatg ttaaaaaaaa aaattttttt ttggtagaaa 6361 tgcaatcacc agtaaagagg tacgaaaaag ctagcctctc tcagagaccg gggaggcaga 6421 gtactactag aggaagtgaa gttctgatgg aatcatgcct gtcaaatgag gtcttgaagc 6481 ggatgcccaa ataaaagagt atattttatc taaatcttaa gtgggtaaca ttttatgcag 6541 tttaaatgaa tggaatattt tcctcttgtt tagttgtatc tgtttgtatt tttctttgat
6601 gaatgattgg tcatgaggcc tcttgccaca ctccagaaat acgtgtgcgg ctgcttttaa 6661 gaactatgtg tctggtcact tatttctcta aaattatctc attgcctggc aatcagtctt 6721 ctcttgtata cttgtcctag cacattatgt acatgggaaa tgtaaacaaa tgtgaaggag 6781 gaccagaaaa attagttaat atttaaaaaa atgtattgtg cattttggct tcacatgttt 6841 aacttttttt aagaaaaaag ttgcatgaat ggaaaaaaaa atctgtatac agtatctgta 6901 aaaactatct tatctgtttc aattccttgc tcatatccca tataatctag aactaaatat 6961 ggtgtgtggc catatttaaa cacctgagag tcaagcagtt gagactttga tttgaagcac 7021 ctcatccttc tttcaatgcg aacactatca tatggcattc ttactgagga ttttgtctaa 7081 ccatatgttg ccatgaatta actctgccgc ctttcttaag gatcaaaacc agtttgattt 7141 gggaatcttc ccctttccaa atgaaataga gatgcagtac ttaactttcc ttggtgtttg 7201 tagatattgc cttgtgtatt ccacttaaaa ccgtaatcta gtttgtaaaa gagatggtga 7261 cgcatgtaaa taaagcatca gtgacactct atctta By “SMAD4 polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_001393970.1 or a fragment thereof having DNA binding activity. An exemplary SMAD4 amino acid sequence follows: 1 mdnmsitntp tsndaclsiv hslmchrqgg esetfakrai eslvkklkek kdeldslita 61 ittngahpsk cvtiqrtldg rlqvagrkgf phviyarlwr wpdlhknelk hvkycqyafd 121 lkcdsvcvnp yhyervvspg idlsgltlqs napssmmvkd eyvhdfegqp slsteghsiq 181 tiqhppsnra stetystpal lapsesnats tanfpnipva stsqpasilg gshsegllqi 241 asgpqpgqqq ngftgqpaty hhnstttwtg srtapytpnl phhqnghlqh hppmpphpgh 301 ywpvhnelaf qppisnhpap eywcsiayfe mdvqvgetfk vpsscpivtv dgyvdpsggd 361 rfclgqlsnv hrteaierar lhigkgvqle ckgegdvwvr clsdhavfvq syyldreagr 421 apgdavhkiy psayikvfdl rqchrqmqqq aataqaaaaa qaaavagnip gpgsvggiap 481 aislsaaagi gvddlrrlci lrmsfvkgwg pdyprqsike tpcwieihlh ralqlldevl 541 htmpiadpqp ld By “SMAD4 polynucleotide” is meant a polynucleotide encoding a SMAD4 polypeptide. An exemplary SMAD4 polynucleotide sequence is provided at NCBI Accession No. NM_001407041.1, which is reproduced below: 1 atgctcagtg gcttctcgac aagttggcag caacaacacg gccctggtcg tcgtcgccgc 61 tgcggatcaa aattgcttca gaaattggag acatatttga tttaaaagga aaaacttgaa 121 caaatggaca atatgtctat tacgaataca ccaacaagta atgatgcctg tctgagcatt 181 gtgcatagtt tgatgtgcca tagacaaggt ggagagagtg aaacatttgc aaaaagagca 241 attgaaagtt tggtaaagaa gctgaaggag aaaaaagatg aattggattc tttaataaca 301 gctataacta caaatggagc tcatcctagt aaatgtgtta ccatacagag aacattggat 361 gggaggcttc aggtggctgg tcggaaagga tttcctcatg tgatctatgc ccgtctctgg 421 aggtggcctg atcttcacaa aaatgaacta aaacatgtta aatattgtca gtatgcgttt 481 gacttaaaat gtgatagtgt ctgtgtgaat ccatatcact acgaacgagt tgtatcacct 541 ggaattgatc tctcaggatt aacactgcag agtaatgctc catcaagtat gatggtgaag 601 gatgaatatg tgcatgactt tgagggacag ccatcgttgt ccactgaagg acattcaatt 661 caaaccatcc agcatccacc aagtaatcgt gcatcgacag agacatacag caccccagct 721 ctgttagccc catctgagtc taatgctacc agcactgcca actttcccaa cattcctgtg 781 gcttccacaa gtcagcctgc cagtatactg gggggcagcc atagtgaagg actgttgcag 841 atagcatcag ggcctcagcc aggacagcag cagaatggat ttactggtca gccagctact 901 taccatcata acagcactac cacctggact ggaagtagga ctgcaccata cacacctaat 961 ttgcctcacc accaaaacgg ccatcttcag caccacccgc ctatgccgcc ccatcccgga 1021 cattactggc ctgttcacaa tgagcttgca ttccagcctc ccatttccaa tcatcctgct 1081 cctgagtatt ggtgttccat tgcttacttt gaaatggatg ttcaggtagg agagacattt 1141 aaggttcctt caagctgccc tattgttact gttgatggat acgtggaccc ttctggagga 1201 gatcgctttt gtttgggtca actctccaat gtccacagga cagaagccat tgagagagca 1261 aggttgcaca taggcaaagg tgtgcagttg gaatgtaaag gtgaaggtga tgtttgggtc 1321 aggtgcctta gtgaccacgc ggtctttgta cagagttact acttagacag agaagctggg 1381 cgtgcacctg gagatgctgt tcataagatc tacccaagtg catatataaa ggtctttgat 1441 ttgcgtcagt gtcatcgaca gatgcagcag caggcggcta ctgcacaagc tgcagcagct 1501 gcccaggcag cagccgtggc aggaaacatc cctggcccag gatcagtagg tggaatagct 1561 ccagctatca gtctgtcagc tgctgctgga attggtgttg atgaccttcg tcgcttatgc 1621 atactcagga tgagttttgt gaaaggctgg ggaccggatt acccaagaca gagcatcaaa 1681 gaaacacctt gctggattga aattcactta caccgggccc tccagctcct agacgaagta 1741 cttcatacca tgccgattgc agacccacaa cctttagact gaggtctttt accgttgggg
1801 cccttaacct tatcaggatg gtggactaca aaatacaatc ctgtttataa tctgaagata 1861 tatttcactt ttgttctgct ttatcttttc ataaagggtt gaaaatgtgt ttgctgcctt 1921 gctcctagca gacagaaact ggattaaaac aatttttttt ttcctcttca gaacttgtca 1981 ggcatggctc agagcttgaa gattaggaga aacacattct tattaattct tcacctgtta 2041 tgtatgaagg aatcattcca gtgctagaaa atttagccct ttaaaacgtc ttagagcctt 2101 ttatctgcag aacatcgata tgtatatcat tctacagaat aatccagtat tgctgatttt 2161 aaaggcagag aagttctcaa agttaattca cctatgttat tttgtgtaca agttgttatt 2221 gttgaacata cttcaaaaat aatgtgccat gtgggtgagt taattttacc aagagtaact 2281 ttactctgtg tttaaaaagt aagttaataa tgtattgtaa tctttcatcc aaaatatttt 2341 ttgcaagtta tattagtgaa gatggtttca attcagattg tcttgcaact tcagttttat 2401 ttttgccaag gcaaaaaact cttaatctgt gtgtatattg agaatccctt aaaattacca 2461 gacaaaaaaa tttaaaatta cgtttgttat tcctagtgga tgactgttga tgaagtatac 2521 ttttcccctg ttaaacagta gttgtattct tctgtatttc taggcacaag gttggttgct 2581 aagaagccta taagaggaat ttcttttcct tcattcatag ggaaaggttt tgtatttttt 2641 aaaacactaa aagcagcgtc actctaccta atgtctcact gttctgcaaa ggtggcaatg 2701 cttaaactaa ataatgaata aactgaatat tttggaaact gctaaattct atgttaaata 2761 ctgtgcagaa taatggaaac attacagttc ataataggta gtttggatat ttttgtactt 2821 gatttgatgt gacttttttt ggtataatgt ttaaatcatg tatgttatga tattgtttaa 2881 aattcagttt ttgtatcttg gggcaagact gcaaactttt ttatatcttt tggttattct 2941 aagccctttg ccatcaatga tcatatcaat tggcagtgac tttgtataga gaatttaagt 3001 agaaaagttg cagatgtatt gactgtacca cagacacaat atgtatgctt tttacctagc 3061 tggtagcata aataaaactg aatctcaaca tacaaagttg aattctaggt ttgattttta 3121 agattttttt tttcttttgc acttttgagt ccaatctcag tgatgaggta ccttctacta 3181 aatgacaggc aacagccagt tctattgggc agctttgttt ttttccctca cactctaccg 3241 ggacttcccc atggacattg tgtatcatgt gtagagttgg tttttttttt ttttaatttt 3301 tattttacta tagcagaaat agacctgatt atctacaaga tgataaatag attgtctaca 3361 ggataaatag tatgaaataa aatcaaggat tatctttcag atgtgtttac ttttgcctgg 3421 agaactttta gctatagaaa cacttgtgtg atgatagtcc tccttatatc acctggaatg 3481 aacacagctt ctactgcctt gctcagaagg tcttttaaat agaccatcct agaaaccact 3541 gagtttgctt atttctgtga tttaaacata gatcttgatc caagctacat gacttttgtc 3601 tttaaataac ttatctacca cctcatttgt actcttgatt acttacaaat tctttcagta 3661 aacacctaat tttcttctgt aaaagtttgg tgatttaagt tttattggca gttttataaa 3721 aagacatctt ctctagaaat tgctaacttt aggtccattt tactgtgaat gaggaatagg 3781 agtgagtttt agaataacag atttttaaaa atccagatga tttgattaaa accttaatca 3841 tacattgaca taattcattg cttctttttt ttgagatatg gagtcttgct gtgttgccca 3901 ggcaggagtg cagtggtatg atctcagctc actgcaacct ctgcctcccg ggttcaactg 3961 attctcctgc ctcagcctcc ctggtagcta ggattacagg tgcccgccac catgcctggc 4021 taacttttgt agttttagta gagacggggt tttgcctgtt ggccaggctg gtcttgaact 4081 cctgacctca agtgatccat ccaccttggc ctcccaaagt gctgggatta cgggcgtgag 4141 ccactgtccc tggcctcatt gttccctttt ctactttaag gaaagttttc atgtttaatc 4201 atctggggaa agtatgtgaa aaatatttgt taagaagtat ctctttggag ccaagccacc 4261 tgtcttggtt tctttctact aagagccata aagtatagaa atacttctag ttgttaagtg 4321 cttatatttg tacctagatt tagtcacacg cttttgagaa aacatctagt atgttatgat 4381 cagctattcc tgagagcttg gttgttaatc tatatttcta tttcttagtg gtagtcatct 4441 ttgatgaata agactaaaga ttctcacagg tttaaaattt tatgtctact ttaagggtaa 4501 aattatgagg ttatggttct gggtgggttt tctctagcta attcatatct caaagagtct 4561 caaaatgttg aatttcagtg caagctgaat gagagatgag ccatgtacac ccaccgtaag 4621 acctcattcc atgtttgtcc agtgcctttc agtgcattat caaagggaat ccttcatggt 4681 gttgccttta ttttccgggg agtagatcgt gggatatagt ctatctcatt tttaatagtt 4741 taccgcccct ggtatacaaa gataatgaca ataaatcact gccatataac cttgcttttt 4801 ccagaaacat ggctgttttg tattgctgta accactaaat aggttgccta taccattcct 4861 cctgtgaaca gtgcagattt acaggttgca tggtctggct taaggagagc catacttgag 4921 acatgtgagt aaactgaact catattagct gtgctgcatt tcagacttaa aatccatttt 4981 tgtggggcag ggtgtggtgt gtaaaggggg gtgtttgtaa tacaagttga aggcaaaata 5041 aaatgtcctg tctcccagat gatatacatc ttattatttt taaagtttat tgctaattgt 5101 aggaaggtga gttgcaggta tctttgacta tggtcatctg gggaaggaaa attttacatt 5161 ttactattaa tgctccttaa gtgtctatgg aggttaaaga ataaaatggt aaatgtttct 5221 gtgcctggtt tgatggtaac tggttaatag ttactcacca ttttatgcag agtcacatta 5281 gttcacaccc tttctgagag ccttttggga gaagcagttt tattctctga gtggaacaga 5341 gttctttttg ttgataattt ctagtttgct cccttcgtta ttgccaactt tactggcatt 5401 ttatttaatg atagcagatt gggaaaatgg caaatttagg ttacggaggt aaatgagtat 5461 atgaaagcaa ttacctctaa agccagttaa caattatttt gtaggtgggg tacactcagc 5521 ttaaagtaat gcattttttt ttcccgtaaa ggcagaatcc atcttgttgc agatagctat 5581 ctaaataatc tcatatcctc ttttgcaaag actacagaga ataggctatg acaatcttgt
5641 tcaagccttt ccattttttt ccctgataac taagtaattt ctttgaacat accaagaagt 5701 atgtaaaaag tccatggcct tattcatcca caaagtggca tcctaggccc agccttatcc 5761 ctagcagttg tcccagtgct gctaggttgc ttatcttgtt tatctggaat cactgtggag 5821 tgaaattttc cacatcatcc agaattgcct tatttaagaa gtaaaacgtt ttaattttta 5881 gccttttttt ggtggagtta tttaatatgt atatcagagg atatactaga tggtaacatt 5941 tctttctgtg cttggctatc tttgtggact tcaggggctt ctaaaacaga caggactgtg 6001 ttgcctttac taaatggtct gagacagcta tggttttgaa tttttagttt ttttttttta 6061 acccacttcc cctcctggtc tcttccctct ctgataatta ccattcatat gtgagtgtta 6121 gtgtgcctcc ttttagcatt ttcttcttct ctttctgatt cttcatttct gactgcctag 6181 gcaaggaaac cagataacca aacttactag aacgttcttt aaaacacaag tacaaactct 6241 gggacaggac ccaagacact ttcctgtgaa gtgctgaaaa agacctcatt gtattggcat 6301 ttgatatcag tttgatgtag cttagagtgc ttcctgattc ttgctgagtt tcaggtagtt 6361 gagatagaga gaagtgagtc atattcatat tttccccctt agaataatat tttgaaaggt 6421 ttcattgctt ccacttgaat gctgctctta caaaaactgg ggttacaagg gttactaaat 6481 tagcatcagt agccagaggc aataccgttg tctggaggac accagcaaac aacacacaac 6541 aaagcaaaac aaaccttggg aaactaaggc catttgtttt gttttggtgt cccctttgaa 6601 gccctgcctt ctggccttac tcctgtacag atatttttga cctataggtg cctttatgag 6661 aattgagggt ctgacatcct gccccaagga gtagctaaag taattgctag tgttttcagg 6721 gattttaaca tcagactgga atgaatgaat gaaacttttt gtcctttttt tttctgtttt 6781 tttttttcta atgtagtaag gactaaggaa aacctttggt gaagacaatc atttctctct 6841 gttgatgtgg atacttttca caccgtttat ttaaatgctt tctcaatagg tccagagcca 6901 gtgttcttgt tcaacctgaa agtaatggct ctgggttggg ccagacagtt gcactctcta 6961 gtttgccctc tgccacaaat ttgatgtgtg acctttgggc aagtcattta tcttctctgg 7021 gccttagttg cctcatctgt aaaatgaggg agttggagta gattaattat tccagctctg 7081 aaattctaag tgaccttggc taccttgcag cagttttgga tttcttcctt atctttgttc 7141 tgctgtttga gggggctttt tacttatttc catgttattc aaaggagact aggcttgata 7201 ttttattact gttcttttat ggacaaaagg ttacatagta tgcccttaag acttaatttt 7261 aaccaaaggc ctagcaccac cttaggggct gcaataaaca cttaacgcgc gtgcgcacgc 7321 gcgcgcgcac acacacacac acacacacac acacacacag gtcagagttt aaggctttcg 7381 agtcatgaca ttctagcttt tgaattgcgt gcacacacac acgcacgcac acactctggt 7441 cagagtttat taaggctttc gagtcatgac attatagctt ttgagttggt gtgtgtgaca 7501 ccaccctcct aagtggtgtg tgcttgtaat tttttttttc agtgaaaatg gattgaaaac 7561 ctgttgttaa tgcttagtga tattatgctc aaaacaagga aattcccttg aaccgtgtca 7621 attaaactgg tttatatgac tcaagaaaac aataccagta gatgattatt aactttattc 7681 ttggctcttt ttaggtccat tttgattaag tgacttttgg ctggatcatt cagagctctc 7741 ttctagccta cccttggatg agtacaatta atgaaattca tattttcaag gacctgggag 7801 ccttccttgg ggctgggttg agggtggggg gttggggagt cctggtagag gccagctttg 7861 tggtagctgg agaggaaggg atgaaaccag ctgctgttgc aaaggctgct tgtcattgat 7921 agaaggactc acgggcttgg attgattaag actaaacatg gagttggcaa actttcttca 7981 agtattgagt tctgttcaat gcattggaca tgtgatttaa gggaaaagtg tgaatgctta 8041 tagatgatga aaacctggtg ggctgcagag cccagtttag aagaagtgag ttgggggttg 8101 gggacagatt tggtggtggt atttcccaac tgtttcctcc cctaaattca gaggaatgca 8161 gctatgccag aagccagaga agagccactc gtagcttctg ctttggggac aactggtcag 8221 ttgaaagtcc caggagttcc tttgtggctt tctgtatact tttgcctggt taaagtctgt 8281 ggctaaaaaa tagtcgaacc tttcttgaga actctgtaac aaagtatgtt tttgattaaa 8341 agagaaagcc aactaaa By “RFX7 polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_001355002.1 or a fragment thereof having DNA binding activity. An exemplary RFX7 amino acid sequence follows: 1 msssraqqmh afswirntle ehpetslpkq evydeyksyc dnlgyhplsa adfgkimknv 61 fpnmkarrlg trgkskycys glrkkafvhm ptlpnldfhk tgdglegaep sgqlqnidee 121 vissacrlvc ewaqkvlsqp fdtvlelarf lvkshyigtk smaaltvmaa apagmkgitq 181 psafiptaes nsfqpqvktl pspidakqql qrkiqkkqqe qklqsplpge saakksesat 241 sngvtnlpng npsilspqpi givvaavpsp ipvqrtrqlv tspspmsssd gkvlplnvqv 301 vtqhmqsvkq apktpqnvpa spggdrsarh rypqilpkpa ntsaltirsp ttvlftsspi 361 ktavvpashm sslnvvkmtt isltpsnsnt plkhsasvss atgtteesrs vpqikngsvv 421 slqspgsrss saggtsavev kvepetssde hpvqcqensd eakapqtpsa llgqksntdg 481 alqkpsnegv ieikatkvcd qrtkcksrcn emlpgtstgn nqstitlsva sqnltftsss 541 sppngdsink dpklctkspr krlsstlqet qvppvkkpiv eqlsaatieg qkqgsvkkdq 601 kvphsgkteg stagaqipsk vsvnvsshig anqplnssal visdsaleqq ttpssspdik 661 vklegsvfll dsdsksvgsf npngwqqitk dsefisasce qqqdisvmti pehsdindle
721 ksvwelegmp qdtysqqlhs qiqesslnqi qahssdqlpl qselkefeps vsqtnesyfp 781 fddeltqdsi veelvlmeqq msmnnshsyg nclgmtlqsq svtpgapmss htssthfyhp 841 ihsngtpiht ptptptptpt ptptptptse miagsqslsr espcsrlaqt tpvdsalgss 901 rhtpigtphs ncsssvppsp vecrnpfaft pisssmayhd asivssspvk pmqrpmathp 961 dktklewmnn gysgvgnssv sghgilpsyq elvedrfrkp hafavpgqsy qsqsrhhdth 1021 fgrltpvspv qhqgatvnnt nkqegfavpa pldnkgtnss assnfrcrsv spavhrqrnl 1081 sgstlypvsn iprsnvtpfg spvtpevhvf tnvhtdacan niaqrsqsvp ltvmmqtafp 1141 nalqkqansk kitnvllskl dsdnddavrg lgmnnlpsny tarmnltqil epstvfpsan 1201 pqnmidssts vyefqtpsyl tksnstgqin fspgdnqaqs eigeqqldfn stvkdllsgd 1261 slqtnqqlvg qgasdltnta sdfssdirls selsgsindl ntldpnllfd pgrqqgqdde 1321 atleelkndp lfqqicsesm nsmtssgfew ieskdhptve mlg By “RFX7 polynucleotide” is meant a polynucleotide encoding a RFX7 polypeptide. An exemplary RFX7 polynucleotide sequence is provided at NCBI Accession No. NM_001368073.2, which is reproduced below: 1 ggctgcgcgc actgggctgc ctccggggct ggcagggcag cggcggccgc ggcggcggcg 61 gggccgggag cgagcggcgg cggcggcggc ggcgtgggga gtggcgtgga gcggtgcctg 121 gggctgcagc agcacgggga cccggcagta gaaagccccg gggacaagga aggaaggctt 181 ggccgccaaa tccctgagca accgttcttc ttggattttg gggggagcct gcccccccca 241 cttcgtcttc aaacgccccc atctcacccc cccacagccc cggccgctga cgggaggagg 301 cggcggcgcc agcggggggc tgaggggcgc cgccatgcct ctcccgcggt gaagcgcccc 361 ggccgtgagg agccgctggt ctccccggtg atgttcccca ggcggcaggc gaaagcgact 421 cactcgagcc ctgggcgatg gcagaggaac aacaacagcc gccaccacag cagcctgatg 481 cccatcagca gcttcccccc agcgccccca actcgggggt ggccctgcca gcccttgtgc 541 ccgggctgcc agggacagag gccagcgcgc tgcaacacaa gatcaagaac tccatctgca 601 agaagttgag aagtttacag acctagagaa actctacctc taccttcagc tgccttctgg 661 tctcagcaat ggagagaaaa gtgatcagaa tgccatgtca tctagtcggg cacaacaaat 721 gcatgccttt tcctggattc ggaataccct agaggaacat ccggagactt cactgcccaa 781 acaggaagtc tatgatgagt acaagagcta ttgtgacaat cttggttacc atccattaag 841 tgctgctgat tttggaaaga tcatgaaaaa cgtctttcca aacatgaagg cacgtcgttt 901 gggcacaaga ggcaaatcta aatattgcta cagtggacta agaaaaaaag cttttgttca 961 tatgccaaca ctgcccaacc ttgactttca caaaactgga gatgggttgg aaggagctga 1021 accttctggg cagcttcaaa atattgatga agaagttatc tcttctgctt gccgtcttgt 1081 gtgtgagtgg gcccagaaag tgttaagcca accatttgac accgtcttgg aattagcccg 1141 cttccttgta aaaagtcact atataggcac caagtcaatg gcagctctaa ctgtaatggc 1201 agcagcacca gcaggaatga aaggaattac ccagccttct gcttttatac ctacagctga 1261 aagtaattcc tttcagcctc aggtgaagac tttgccatct ccaattgatg ctaaacagca 1321 gttgcaacgg aaaatccaga agaagcagca agaacagaaa ctacaatccc ctttgccagg 1381 agaatctgca gcaaaaaagt cagaaagtgc tacaagcaat ggagtgacta atcttcctaa 1441 tggaaatcct tcaatccttt ctcctcaacc tattggtatc gttgtggcag ctgtccctag 1501 tcccattccg gtccagcgga ctaggcaatt ggtaacttca ccgagtccaa tgagttcttc 1561 tgacggcaaa gttcttcccc tcaatgtaca ggtggtcact cagcacatgc agtctgtgaa 1621 acaggcacca aagactcccc agaacgttcc agccagtcct ggtggggatc gttctgcccg 1681 gcaccgttac cctcagatct tacccaaacc agcgaacacc agtgcactca ccattcgctc 1741 tccaactact gtcctcttta ctagtagtcc catcaaaact gctgttgtac ccgcttcaca 1801 catgagttct ctaaatgtgg tgaaaatgac aacaatatcc ctcacaccca gcaacagtaa 1861 cacccctctt aaacattctg cctcagtcag cagtgctaca ggaacaacag aagaatcaag 1921 gagtgttcca cagatcaaga atggttctgt cgtgtcgctt cagtctcctg ggtccaggag 1981 cagcagtgcg gggggaacat ctgctgtgga agtcaaagtg gaacccgaaa catcatcaga 2041 tgagcatcct gtacagtgcc aagagaactc tgatgaggct aaagctcccc agacacctag 2101 tgcccttttg gggcagaaaa gtaatacaga cggagcactg cagaaacctt caaatgaagg 2161 tgtcattgaa ataaaagcaa ctaaggtctg tgaccagagg accaaatgta aaagtcgctg 2221 taatgaaatg ctgccaggca cgtcaacagg caataatcaa agcactatca ctctatcagt 2281 tgcttctcag aacttaactt tcaccagcag cagctcacca cctaatggtg actcaatcaa 2341 taaagaccct aaattatgca ctaaaagccc aagaaaacga ctgtcttcta cattgcagga 2401 gacccaggtg cctcctgtaa agaaaccaat tgtggaacag ctttcagcag ctaccataga 2461 agggcagaaa caaggcagtg ttaagaagga ccaaaaggtt ccacattcag ggaaaacaga 2521 aggttcaaca gcaggtgctc agattcctag caaggtatca gtaaatgtca gttcacacat 2581 aggagcaaat caacccttga attcctctgc ccttgttatc agtgattcag ctttggaaca 2641 gcaaacaacc ccatcatcat ctccagatat aaaagtaaaa cttgaaggaa gtgtctttct 2701 cttggacagt gattcaaagt cagttggcag ctttaatcca aatggatggc aacaaatcac 2761 taaagattct gagtttatat ctgccagttg tgaacaacag caagatatca gtgttatgac
2821 aattcctgag cactctgata tcaatgactt agagaaatct gtttgggaat tagaaggaat 2881 gccacaggac acatatagcc agcagctaca tagccagata caggaatctt ctttaaatca 2941 aatacaagca cattcttcag atcagttacc tctgcaatct gaactgaagg agtttgagcc 3001 ttctgtttcc cagacaaatg aaagctactt tccttttgat gatgaactta cacaagatag 3061 tattgtggaa gagctggtgc ttatggagca gcaaatgtca atgaacaatt ctcattctta 3121 cggcaactgt ttgggaatga cccttcagag tcagtcagta actccaggag ctccaatgtc 3181 atctcacact tccagcaccc acttctatca tccaatccac agcaatggca ctccaatcca 3241 cacacccaca cccacaccca cacccactcc tactccaacc ccaaccccaa ccccgacatc 3301 tgaaatgatt gctggatctc agagtctgtc acgggagagc ccttgctcca ggctagccca 3361 gactacacct gtggatagtg ctttaggaag tagccgacat acacccattg gtactccaca 3421 ttctaactgc agcagtagtg tcccccccag ccctgttgaa tgcaggaatc cgtttgcatt 3481 cactccaata agctccagta tggcatatca tgacgccagc attgtctcaa gtagtcctgt 3541 gaaaccgatg caaagaccca tggccacaca ccctgacaaa accaagcttg aatggatgaa 3601 taatgggtat agtggggttg gtaattcatc agtttctggc catggtattc tcccaagcta 3661 tcaggaacta gtggaagacc gtttcaggaa acctcatgct tttgctgtgc ctggacagtc 3721 ttatcagtct caatccagac atcatgacac tcattttggt cgtttgactc ctgtctctcc 3781 tgtgcagcat caaggtgcca ctgtaaataa caccaacaaa caggagggtt ttgcagtccc 3841 tgcccctctt gataataaag gaactaattc atctgccagc agcaacttca gatgccggag 3901 tgtgagccct gctgttcatc gccaacgtaa tcttagtgga agcaccctct atccagtatc 3961 taatatccca cgatctaatg tgaccccctt tggaagtcca gttaccccag aagttcatgt 4021 tttcacaaat gttcacacag acgcatgtgc caacaacata gctcaaagaa gccaatcagt 4081 tccattgaca gtcatgatgc agacagcctt cccaaacgct cttcagaagc aagcaaacag 4141 taaaaaaata accaatgttt tgttgagtaa acttgattcc gacaatgatg atgcagtgag 4201 aggtttggga atgaacaacc tgccctctaa ttatacagcc cggatgaatc tcactcagat 4261 tttggaacct tccactgttt ttcctagtgc caacccacaa aatatgatcg attccagcac 4321 ttctgtttat gagttccaaa caccatctta cctcaccaaa agtaatagca ccggtcagat 4381 caatttttct cctggagata atcaagcaca atcagaaatt ggagagcaac aattagattt 4441 caatagcact gttaaagacc tgttgagtgg agacagcttg caaaccaacc agcagctggt 4501 aggtcaggga gcatctgatc tcactaatac tgcatctgat ttctctagcg atatcaggtt 4561 gtcttctgag ctctcaggca gcatcaatga tttgaacact ttagacccaa atctactgtt 4621 tgatccaggt cgtcagcagg gacaagatga tgaagctaca ctggaagaat taaagaatga 4681 cccattattt caacaaattt gcagtgaatc catgaattct atgacttcat caggttttga 4741 atggatagaa agcaaggacc atcctactgt tgaaatgttg ggttaaattg tgttttataa 4801 catgtagcac actgtatcta aagacatatg tattgtattt gtcttaatgg aagtgcctcc 4861 cgcagcagaa atactattaa ttgtgacatt ttaaaagcgt ttgctgcaga agtggtttct 4921 tcagtaggaa atggttagac ttgcctcagt tcttaacaag tgtctaaaga cagtaggctg 4981 tagaagaaca agtattaggt tttaaatttt ataatgtgcc ccatttattc acattgattg 5041 actatccagc tgttacaaga gcagtcgatc tttattttgt gtaagttagt ttcatttcat 5101 cagatgatct gcacagaact cagaataaac catcaatttt aggtataggc tgttttgttg 5161 ttggcattat cttttagata taaaatcata gctagggtga actgtagaca agaaagtctt 5221 ccttgaaaca gggataatat tcaggttatt ataaatattt aggcttgaag catagagcta 5281 ctgtagggtg aaaaatctct gtaggacttt ctggggcttc agtaccattt acacccacga 5341 tgaagggctt aaatagaaaa taatggaaaa ttaaatatta ctttaaggaa agcaaagctg 5401 cactaaaatt tttaagggaa aaaaaacaag tagccattta tatttgtgac cttgcattaa 5461 tatttaaatg ttgattttaa tatggagcta gcacacatag atgcgcaggc acgcacacac 5521 aaaacaagca aatgtaactc caggcatttg taatttaata taataaatac aagcatgtta 5581 gaaaaatgct ttatgtctta ttatttgtta agttagggga atgagaagga aaacaaaagc 5641 attctcttag tttcatcaca atggctgcat gtacatattg agattctaac tgagatttac 5701 tttgaaattt ttcattaaaa tattccacaa taatcagttt ttttcctctt caattgagtg 5761 actgactccc tcactcctcc tgaatatagt aatgtagttt gatcatagga tccctttgaa 5821 aaacaggaag gaggataaca taaaaaacaa caaaaaacat ttcaccacct aaggtctgaa 5881 gccacaaatc tttatatctc tgatgaaaca aaggggatat caatagtact tgaagaagaa 5941 gagcatttta ttacttgctt aatttattta ttgtgataac cacaattgca aaagaagaac 6001 tcaggatttg ttttaaaatg tttctctcta tggctcccat tttgttttgt gaataattat 6061 ttatgagttg catgttgaca caaacctact cactggttga aagctccatt taagtgagtt 6121 tcaagatcac tttcaaagag atttttaaat tgccattgtt tttaaagcta acataaaacc 6181 atttgctatg ctaccttgaa cctagctaag ctttccttta ccttagtaag ccaatcagaa 6241 gactacaatc aatgaatatt tagcaaatgt gctgaagggt ttaataaggt tttgttgttc 6301 aataaatttg caaggtaggg tattaacata ttttgataaa taaatggtgc tagagttggc 6361 gcagaggttt atatattgat atgtcttagt tattggcatt tgtgtgtgtt tgctgttatc 6421 ttgatttcat gatttgttag gtattttttt cttgatgcta gttatttctt cactgtacat 6481 tgagacacag cactactgca caccaaagta tgcaataccc aaaggaaact gtgtgatttc 6541 tcctgacaaa tgatgggagc ctttctttat gagatactct gaaaaggaga ttttgaggcc 6601 attctaatcc cttgtttaca ttattagctt cctactaata taaaaataat ggaaatcttt
6661 tttgataaaa atattaagac tggaggattt ttaacccctt gtacagtatt gcagcaaact 6721 ctttaaattt ggttgaattc gtccagcaaa ctttctgggg ttcatatatt gcactaaatt 6781 tgttgaacct gtggtaggca catctcttag gataaatgca accaatacag tccacagatt 6841 aaacttttta aatgtgtttc attatgaaaa gacaaaatgt catcaaagtg acagataaaa 6901 ttgtcttatg tagcaatgtg cattgatctc aatgagatat ttctattatt ctcttacatg 6961 agaatattac agcaggaaga attaagttta gtttgcttca ccatgttaat acatgatcac 7021 aaaatgtgca tgagtgttat tgacttatct tgtacagtac taggattcct gtaaccactc 7081 ttttttttct tcgctgtatt gaaactggtt cagtgttaac caggcaagca tagttattca 7141 caggttattt actttatgtt tactaattct gcttagttat tgttgaatat gttctgttcc 7201 agattatttt tattgttttg atgtttaaaa cattttgcat actgtttatc ataaaacttc 7261 atgaagtaaa taattttgca tataattgca ataagcactg acttttgaac tgaaaagaag 7321 gaaagtgttt ttttgtgcag tgcttctgag gttcatataa tagtcttgta ctataatgtg 7381 ctttactaca cgataaatga caaaaacaag ccctgtttca tgtttcattt agtgcaaaaa 7441 gtcagatttc cccagtgttt tgttgtctgt tgtacctgct gaaactacag tagttgaata 7501 ttgcggtagc atttcagttc tcttttttta ttaaagtgct caaaaattgt tttttaaatg 7561 ttttcaaaaa caattaaaaa cattttatat taaactgact ggatctgaag gtgttttatg 7621 ggacatattt tatataggca acacattttg taaacagacc caatgttatt cttcctttga 7681 tgtcattaac tttagggagt gaataaaaat gagcagaagg cagaacagtg ctgctgatct 7741 gctgtgtgct attaatccag aagaatacac agatcttagt tcattcatca aaactctgtt 7801 gttcacttaa gattattacc aatgaatggt gcatggcata gaactttatg ttgtccaaaa 7861 agccatcttt ttctctttca catagtgttt aaatgttagc ctatggagta cctaggtaag 7921 tatatttctg gagctctgaa tagaaaatga gaaaatttta agcatagctg tgttcaaaag 7981 cagtatagac atttccaggt accagaaata acaaactgat aagtgtaatg aatttgtaag 8041 ccagaagctg ggctacaggt ctggaagcag gccgaagtag ccagaaatgt cagctcctgc 8101 aaagtaatga aaccagggtc ttgaatggag tttcctcact cttgaaactg agaagcaaca 8161 acaaccagtg ccagggacta tatattgtag acagatcttc tcaaacagcc tactcctctg 8221 tttacataga tctttgtaga tctgtgatag ttccactatc accatagatc tggagccctc 8281 agtttgaaca ggtggtagaa gcaattacaa aaggaaagga gtggagatta ggaagggaga 8341 ggtaaagagc aggaaggctc ccatgaaagc tgagcctgca gatcaaaatt caaaagtact 8401 tgaagaaaaa ctaatcccaa gaaaggcaat cagtcactca agagaaaatg aaagtaatga 8461 actacctaga taatactata aaattagtat gttaggatcc tcagtgctca atgacatgga 8521 agggtaaaat gtcagagcag gcaaacataa gaaccagtgc atagagtcaa ccagtaagaa 8581 attctggggg tgaaaatact gtgtaacacc tgaggtgatt ttggtttcaa gtaattgaaa 8641 acccaacact ggcttaaacc ataaggacac ttacttttag cttaataata gtggagatag 8701 gaggttcaag ggttcattca atggttcatt gtttacaggc accagatcct gtttgtgttt 8761 ctgttccatc atcttcagct ttgctttgct gtgtctatta cctcatagtc acaagatggc 8821 tgccacagct cagatgttat accctcataa catcccaagc aggatagtaa gagcagttct 8881 tagcctttta tcagagagaa aaaaaaatct cagaatcttc cattaagcac tccatacaca 8941 cacactgtta gtctcattgt attaaacaaa ggctcatcct aaggccaatt acttagctta 9001 gatccttaat taatgcttta taggtaggag ggggggccca tttcctggag catgtggtca 9061 ttcatcttaa atgagttcca tcacctaggg aagatattag ggacttgctc tttgggaggc 9121 aaccagtagt ggtcactgca aaaaatagct aggtaagatg agtaagatct tgtacccacc 9181 tttgaggggc ttacaatcat aaagagttaa gtacatgttc tttgttaagt gccaacagta 9241 tgtatactac actatgtaga agaaaaaata agaatttgaa atctgccgaa ctaagtttac 9301 tggtgctaac tgttaactgg tatcttgcct tccccctatg agctgaaaaa tcaggtatta 9361 ttgagtatca caaatgcaag ttgcctcagc tcctacagca taagaaaaga ccaaactttt 9421 tattttgtta aatctgaagt acagatttat tttgacatgc attgttataa atgaattttt 9481 aaataattaa agttataaat ttatggatgg gatcacttat taatattgca tcatctgaaa 9541 ttttaaaaat atgtatgcca gtcttgttgc tataatctgc ttagaaaaaa gaacaaaaca 9601 aaaagactac atttttcaag atttgatttt taagaagtac ttaattcttg gagaggagaa 9661 gagagtcata taaataaaat atatcatgag agtgatatta ctgtggtata tagagtattt 9721 ctttgcatta gacccagtgt tagtttgcat atattctcat gtcctcattg taacaatttt 9781 tagcgagtat tatttttcag gtgagaaaat tttacttcag agattaactg gtggaggtaa 9841 cataataaca ggaagagcca ggattatctt aatttcaaat cccacttcat accattatgc 9901 tatacttaag atatgtgttc acagtgtacc tactgcagtt gtatgatctg gttgcttctt 9961 tgaaattttc attctggttt taaatttaga attgagtttg atttttgtgg agttctttat 10021 ttaacctccc atccttatat gtcaacaatt ctttttcatt tttattgctt ttagatttac 10081 ctgtagtttg taattttcct ccaaattact tgaatccttt tccctccctt ctctccatag 10141 aaataccctg atgtattagg agaatgaaga tttaaaaaca aaattttgtt ttccacacag 10201 cttttcaagc atatttcata caggtatact ctagtcatat ttatgtttaa atcacttaaa 10261 atgtcatcaa aataaaaatg ggactttttt tttaacagat gataaagtgc ctaaaatatg 10321 tcagtctctg tacaaggtgc tagagaagac acctgccatt ttgaaatttt gcaatataat 10381 gaaaaagaca cagtgaatgt ctttgcagga tgcaatgagg tcactatagc agaggcggat 10441 acttaccagt agtatgcatt ctccccttct tctttactgt aaaagtcctt gatttttagc
10501 tgagcacaca gccatctgac caaaaggctg catctgccaa cttcccttgc agttaagtgg 10561 agacacttac caagtactca aagagatgta accaaaaggt ttgggtgcta cttctttgca 10621 gtgttcttgt ccttcctctc tgctggctgg aatgcaaatg tgatggctag aactcaagca 10681 gccctcctgg acttcatgtt aaagacacat gctgaggatg attgagcaac aagatacaag 10741 gagcccaggg catgatgact ttggaaggga agatgcccaa ctagtcctga ctgcttacct 10801 ctggatgtgt tttacatgag agaaataaat gtctttctgg tttaactcac a By “REST polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_001180437.1 or a fragment thereof having DNA binding activity. An exemplary REST amino acid sequence follows: 1 matqvmgqss gggglftssg nigmalpndm ydlhdlskae laapqlimla nvaltgevng 61 sccdylvgee rqmaelmpvg dnnfsdseeg egleesadik gephglenme lrslelsvve 121 pqpvfeasga pdiyssnkdl ppetpgaedk gkssktkpfr ckpcqyeaes eeqfvhhirv 181 hsakkffvee saekqakare sgsstaeegd fskgpircdr cgyntnrydh ytahlkhhtr 241 agdnervykc iictyttvse yhwrkhlrnh fprkvytcgk cnyfsdrknn yvqhvrthtg 301 erpykcelcp ysssqkthlt rhmrthsgek pfkcdqcsyv asnqhevtrh arqvhngpkp 361 lncphcdykt adrsnfkkhv elhvnprqfn cpvcdyaask kcnlqyhfks khptcpnktm 421 dvskvklkkt kkreadlpdn itnekteieq tkikgdvagk kneksvkaek rdvskekkps 481 nnvsviqvtt rtrksvtevk emdvhtgsns ekfsktkksk rklevdshsl hgpvndeess 541 tkkkkkvesk sknnsqevpk gdskveenkk qntcmkkstk kktlknkssk ksskppqkep 601 vekgsaqmdp pqmgpaptea vqkgpvqvep pppmehaqme gaqirpapde pvqmevvqeg 661 paqkellppv epaqmvgaqi vlahmelppp metaqtevaq mgpapmepaq mevaqvesap 721 mqvvqkepvq melsppmevv qkepvqiels ppmevvqkep vkielsppie vvqkepvqme 781 lsppmgvvqk epaqrepppp repplhmepi skkpplrkdk keksnmqser arkeqvliev 841 glvpvkdswl lkesvstedl sppspplpke nlreeasgdq kllntgegnk eaplqkvgae 901 eadeslpgla aninesthis ssgqnlntpe getlngkhqt dsivcemkmd tdqntrenlt 961 ginstveepv spmlppsave ereavsktal asppatmaan esqeidedeg ihshegsdls 1021 dnmsegsdds glhgarpvpq essrknakea lavkaakgdf vcifcdrsfr kgkdyskhln 1081 rhlvnvyyle eaaqgqe By “REST polynucleotide” is meant a polynucleotide encoding a REST polypeptide. An exemplary REST polynucleotide sequence is provided at NCBI Accession No. NM_001193508.1, which is reproduced below: 1 cggcgctcgg agcccgacgc ctcgcgaggg cgcccgcgga gcctccccgg ccctgggcgt 61 tgggcgagcc ccggggcggt cggaggggcc cgggggcggt cggagaagcc cggacgccgg 121 ctgcgcgaat acagttatgg ccacccaggt aatggggcag tcttctggag gaggagggct 181 gtttaccagc agtggcaaca ttggaatggc cctgcctaac gacatgtatg acttgcatga 241 cctttccaaa gctgaactgg ccgcacctca gcttattatg ctggcaaatg tggccttaac 301 tggggaagta aatggcagct gctgtgatta cctggtcggt gaagaaagac agatggcaga 361 actgatgccg gttggggata acaacttttc agatagtgaa gaaggagaag gacttgaaga 421 gtctgctgat ataaaaggtg aacctcatgg actggaaaac atggaactga gaagtttgga 481 actcagcgtc gtagaacctc agcctgtatt tgaggcatca ggtgctccag atatttacag 541 ttcaaataaa gatcttcccc ctgaaacacc tggagcggag gacaaaggca agagctcgaa 601 gaccaaaccc tttcgctgta agccatgcca atatgaagca gaatctgaag aacagtttgt 661 gcatcacatc agagttcaca gtgctaagaa attttttgtg gaagagagtg cagagaagca 721 ggcaaaagcc agggaatctg gctcttccac tgcagaagag ggagatttct ccaagggccc 781 cattcgctgt gaccgctgcg gctacaatac taatcgatat gatcactata cagcacacct 841 gaaacaccac accagagctg gggataatga gcgagtctac aagtgtatca tttgcacata 901 cacaacagtg agcgagtatc actggaggaa acatttaaga aaccattttc caaggaaagt 961 atacacatgt ggaaaatgca actatttttc agacagaaaa aacaattatg ttcagcatgt 1021 tagaactcat acaggagaac gcccatataa atgtgaactt tgtccttact caagttctca 1081 gaagactcat ctaactagac atatgcgtac tcattcaggt gagaagccat ttaaatgtga 1141 tcagtgcagt tatgtggcct ctaatcaaca tgaagtaacc cgccatgcaa gacaggttca 1201 caatgggcct aaacctctta attgcccaca ctgtgattac aaaacagcag atagaagcaa 1261 cttcaaaaaa catgtagagc tacatgtgaa cccacggcag ttcaattgcc ctgtatgtga 1321 ctatgcagct tccaagaagt gtaatctaca gtatcacttc aaatctaagc atcctacttg 1381 tcctaataaa acaatggatg tctcaaaagt gaaactaaag aaaaccaaaa aacgagaggc 1441 tgacttgcct gataatatta ccaatgaaaa aacagaaata gaacaaacaa aaataaaagg 1501 ggatgtggct ggaaagaaaa atgaaaagtc cgtcaaagca gagaaaagag atgtctcaaa 1561 agagaaaaag ccttctaata atgtgtcagt gatccaggtg actaccagaa ctcgaaaatc
1621 agtaacagag gtgaaagaga tggatgtgca tacaggaagc aattcagaaa aattcagtaa 1681 aactaagaaa agcaaaagga agctggaagt tgacagccat tctttacatg gtcctgtgaa 1741 tgatgaggaa tcttcaacaa aaaagaaaaa gaaggtagaa agcaaatcca aaaataatag 1801 tcaggaagtg ccaaagggtg acagcaaagt ggaggagaat aaaaagcaaa atacttgcat 1861 gaaaaaaagt acaaagaaga aaactctgaa aaataaatca agtaagaaaa gcagtaagcc 1921 tcctcagaag gaacctgttg agaagggatc tgctcagatg gaccctcctc agatggggcc 1981 tgctcccaca gaggcggttc agaaggggcc cgttcaggtg gagccgccac ctcccatgga 2041 gcatgctcag atggagggtg cccagatacg gcctgctcct gacgagcctg ttcagatgga 2101 ggtggttcag gaggggcctg ctcagaagga gctgctgcct cccgtggagc ctgctcagat 2161 ggtgggtgcc caaattgtac ttgctcacat ggagctgcct cctcccatgg agactgctca 2221 gacggaggtt gcccaaatgg ggcctgctcc catggaacct gctcagatgg aggttgccca 2281 ggtagaatct gctcccatgc aggtggtcca gaaggagcct gttcagatgg agctgtctcc 2341 tcccatggag gtggtccaga aggagcctgt tcagatagag ctgtctcctc ccatggaggt 2401 ggtccagaag gaacctgtta agatagagct gtctcctccc atagaggtgg tccagaagga 2461 gcctgttcag atggagttgt ctcctcccat gggggtggtt cagaaggagc ctgctcagag 2521 ggagccacct cctcccagag agcctcccct tcacatggag ccaatttcca aaaagcctcc 2581 tctccgaaaa gataaaaagg aaaagtctaa catgcagagt gaaagggcac ggaaggagca 2641 agtccttatt gaagttggct tagtgcctgt taaagatagc tggcttctaa aggaaagtgt 2701 aagcacagag gatctctcac caccatcacc accactgcca aaggaaaatt taagagaaga 2761 ggcatcagga gaccaaaaat tactcaacac aggtgaagga aataaagaag cccctcttca 2821 gaaagtagga gcagaagagg cagatgagag cctacctggt cttgctgcta atatcaacga 2881 atctacccat atttcatcct ctggacaaaa cttgaatacg ccagagggtg aaactttaaa 2941 tggtaaacat cagactgaca gtatagtttg tgaaatgaaa atggacactg atcagaacac 3001 aagagagaat ctcactggta taaattcaac agttgaagaa ccagtttcac caatgcttcc 3061 cccttcagca gtagaagaac gtgaagcagt gtccaaaact gcactggcat cacctcctgc 3121 tacaatggca gcaaatgagt ctcaggaaat tgatgaagat gaaggcatcc acagccatga 3181 aggaagtgac ctaagtgaca acatgtcaga gggtagtgat gattctggat tgcatggggc 3241 tcggccagtt ccacaagaat ctagcagaaa aaatgcaaag gaagccttgg cagtcaaagc 3301 ggctaaggga gattttgttt gtatcttctg tgatcgttct ttcagaaagg gaaaagatta 3361 cagcaaacac ctcaatcgcc atttggttaa tgtgtactat cttgaagaag cagctcaagg 3421 gcaggagtaa tgaaactttg aacaaggttt cagttcttag tttgtaaggt atattacatt 3481 ttatattcat ttatgatagc agacaacctt ttaagattgc tttaattagt atctgatgtt 3541 gatttttaag tggcattctt ttccttagga ctttttatgt atacctgttg attgttgtgt 3601 aaattttagt aaatctaaga gagtgtacta aaccagcagg tatctgttag cttatgtgtt 3661 taattgaaat tagaaggcta agatggtata acagcatttt attgctttgt ccagctacaa 3721 cttgtcattt ttttctccat gtcttatctt cctgtttcac tttagtttat tcttcgtttt 3781 ttattgagat ctataaaaaa ttggcttact taatagcaaa ttacttgaag aatttgcctg 3841 ctttatataa agttagcact ttaagatttt tttttttaga gatgagaaga catttaaatt 3901 gaagaaaaat tcccccagca atagacagtc tatcagtcca agtatttact tcctgagttt 3961 tgatcaatat tttttatttg tgtatgttaa tcgtcataaa aacagtgatt ttggtgtgtt 4021 ttttattttg gtgctttaat ggcttaagat gttgcacatt ttttttttct tttggtttct 4081 gtttatgttt ttttgcctat gcagttaaat ttttcctaga aatagcattt gtgttgaaca 4141 gtaacacttt atacatatat atatgcatgt ttattttgtt tggcgtcttt ggagggatgc 4201 ttttagactt gtttgcaaaa gggcagtttt ctttttcttt gctgcagttg tctattttgc 4261 agaataatag tgtgtgcaag tttgtgagca aatgaaatat gcaggttcaa tctattgatt 4321 ttgattttta catcttatat ctatgccaga atctgtattt catataactt atttatttcg 4381 aatggatgta gtaaattcac agctatcagt tttgattttg caataaataa accactaggt 4441 tgcatgtcga acaaattttt atctcaaata ccaaccatca gttttttttt tcatgtgttt 4501 tggtacagct aattcctaat tgtagagtgt taaatgtttg aggagaacct tttctcatag 4561 atggttggtg ttcatatggc tactttacaa taaagagaac tgtaagtgat atttggaaac 4621 tacaaacctg gaattaggag atataattat tccttcaagt tttatagaat atcacttggg 4681 agattccaaa gccatagcta ttacgcggca aacctaggat aagaaaggta gtatgagtgc 4741 tggtagacca gctgcaactt tcctatacag tgaaaaaggc tggtgaaaca agtacagtcc 4801 agatttttta aaatcatact ttctcaggga tctccacaaa ctggtgggtg tcctggctgt 4861 ctgtgtgata gcctctttct ataggtgagg cctcaaatga attgcagcta tcctggtgtt 4921 cctatgaggg cactttgtat gaaaaagggc atgtactcca aaacattttt gtaggttctt 4981 tggccagttg ccaaagagtg tgaaagaatc caatagagga tttttcttac tgatagcagt 5041 cattcattgc agtaaaataa aatatgatcc cattagggaa tcttgaattc tgacctccca 5101 tactccgttt tgaaataacc actttatatt tcatttttta aaaatctgat gatctctttg 5161 aggcaggttt cagatttggc agtacaacat gaaagattag gaaaagcatt aataacgtgt 5221 gggtggaaag cttgttaaaa atctgagagt gaagtttgag ttaaaagttg tttgaacatg 5281 gcattgactg ggaggccaaa gatttaaaga agcggaagat tcttctctta agacatgagg 5341 agtaagttgt gtgataatgg tatgtgtttt gtgtgcatga atggacattg taaatgttga 5401 attctaggct ccgacaatca ttgtcaacag aagatcaagc tgcaaatatt tatgttttaa
5461 aacttaaatt ataaagctag ttaagtcttt ctaatgacta gttttaatgt tcatgggtac 5521 attttaccta agttaccgtt tacattgtat agaaaaagat acatcttaag cacagattgg 5581 ttattaggaa ttagtttggg gaagaggttt ttttgtggat tctttcatac tgcaaagaaa 5641 aaccatttgc cttttgggga attgagctaa cttctaatct agtcttaaga ctagaatgct 5701 aaaaacaaaa acatgaagga aattaaaacc ccttattatt aaattgattt gtaaaaacat 5761 tgttactgga aatttattgg acttgaggcc ttcctccaga aaataaggac ttgattgtca 5821 ggcctatatt aggttctgaa ccttaatgcc atgtatttgt acttactaaa aattgtttca 5881 atgaaaagta cattagcagt atgaacttct ggtccagttg gaagtttttc catttgaaaa 5941 atgtgatgtt tgcatggaac tgtttgaaac ttttttattt tctagtcccc ctcccccaca 6001 ctggatagaa tttagcctag aattttccct ttggataaaa gaacaaaaat tgaacatgtt 6061 atttgtaaat tgatgtttag taattagtga taaacttgaa atactagcat atattataag 6121 ccttaatctt aggtagtctt atgaaaatga atctcttaac tatcttttga acctgtattc 6181 acattggttt tcaagatatt ttaagttata ttttttcctc ttttcagagc tgcttcttat 6241 tctggggcta cttttttttt tagttgtgta attcacaaag ggctgcattt tttttttttt 6301 ttaataaggc ttataactat ggctggatct tttgctctag tcttctaaga agggccattt 6361 tattttttag agtcacttct aaagtcatgt ggtaattaac tttggagact gttttgcgta 6421 tgagtgctga tacaaattaa aacccaagta gacctcattg catgtcaccc tatgaatgtt 6481 gacaatggaa ggaatacctt gcctgtagta tactgtcact tctggattga taagctgagg 6541 aagaaagtta agtttctttt ttacataagt cagaaaaact tacagctggt gttcctagtt 6601 tcctggttga cctcagcaga tgaagtgaac agatagtgtt aattcagatt gaagaaatta 6661 tctgaatctt ggtttgtgta gatttacaat ctacatgcaa tattaactaa atcagatagc 6721 ttttacagtt tcacatgtgt acataggttc cctcccggtc ccttccatat ccattagtta 6781 ttgaactttc taaactggca ttgaaacatt acaacaatgt tttgttgcac caattttata 6841 aacttaagca gtgcaatacg tgttactttt ctgaggcaaa ccaaaggtaa atttctcaag 6901 gttcttgctg ccttctttag cagcatttga tggaagatct tttatacatt tgtaatagat 6961 aaaaataaac cagattgcaa atcctttttt aaaatcctaa accatgtacc aagtttttgg 7021 tccaaattat gtaggataag ttaaacttaa attgcattct attaaccaat atgagtgtat 7081 ttctgtaagc atagttatgt tgaaataaag ttttaaaaac ca By “FOXP3 polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_001393970.1 or a fragment thereof having DNA binding activity. An exemplary FOXP3 amino acid sequence follows: 1 mpnprpgkps apslalgpsp gaspswraap kasdllgarg pggtfqgrdl rggahassss 61 lnpmppsqlq lstvdahart pvlqvhples pamisltppt tatgvfslka rpglppginv 121 aslewvsrep allctfpnps aprkdstlsa vpqssyplla ngvckwpgce kvfeepedfl 181 khcqadhlld ekgraqcllq remvqsleqq lvlekeklsa mqahlagkma ltkassvass 241 dkgsccivaa gsqgpvvpaw sgpreapdsl favrrhlwgs hgnstfpefl hnmdyfkfhn 301 mrppftyatl irwaileape kqrtlneiyh wftrmfaffr nhpatwknai rhnlslhkcf 361 vrvesekgav wtvdelefrk krsqrpsrcs nptpgp By “FOXP3 polynucleotide” is meant a polynucleotide encoding a FOXP3 polypeptide. An exemplary FOXP3 polynucleotide sequence is provided at NCBI Accession No. NM_001114377.2, which is reproduced below: 1 agtttcccac aagccaggct gatccttttc tgtcagtcca cttcaccaag cctgcccttg 61 gacaaggacc cgatgcccaa ccccaggcct ggcaagccct cggccccttc cttggccctt 121 ggcccatccc caggagcctc gcccagctgg agggctgcac ccaaagcctc agacctgctg 181 ggggcccggg gcccaggggg aaccttccag ggccgagatc ttcgaggcgg ggcccatgcc 241 tcctcttctt ccttgaaccc catgccacca tcgcagctgc agctctcaac ggtggatgcc 301 cacgcccgga cccctgtgct gcaggtgcac cccctggaga gcccagccat gatcagcctc 361 acaccaccca ccaccgccac tggggtcttc tccctcaagg cccggcctgg cctcccacct 421 gggatcaacg tggccagcct ggaatgggtg tccagggagc cggcactgct ctgcaccttc 481 ccaaatccca gtgcacccag gaaggacagc accctttcgg ctgtgcccca gagctcctac 541 ccactgctgg caaatggtgt ctgcaagtgg cccggatgtg agaaggtctt cgaagagcca 601 gaggacttcc tcaagcactg ccaggcggac catcttctgg atgagaaggg cagggcacaa 661 tgtctcctcc agagagagat ggtacagtct ctggagcagc agctggtgct ggagaaggag 721 aagctgagtg ccatgcaggc ccacctggct gggaaaatgg cactgaccaa ggcttcatct 781 gtggcatcat ccgacaaggg ctcctgctgc atcgtagctg ctggcagcca aggccctgtc 841 gtcccagcct ggtctggccc ccgggaggcc cctgacagcc tgtttgctgt ccggaggcac 901 ctgtggggta gccatggaaa cagcacattc ccagagttcc tccacaacat ggactacttc 961 aagttccaca acatgcgacc ccctttcacc tacgccacgc tcatccgctg ggccatcctg
1021 gaggctccag agaagcagcg gacactcaat gagatctacc actggttcac acgcatgttt 1081 gccttcttca gaaaccatcc tgccacctgg aagaacgcca tccgccacaa cctgagtctg 1141 cacaagtgct ttgtgcgggt ggagagcgag aagggggctg tgtggaccgt ggatgagctg 1201 gagttccgca agaaacggag ccagaggccc agcaggtgtt ccaaccctac acctggcccc 1261 tgacctcaag atcaaggaaa ggaggatgga cgaacagggg ccaaactggt gggaggcaga 1321 ggtggtgggg gcagggatga taggccctgg atgtgcccac agggaccaag aagtgaggtt 1381 tccactgtct tgcctgccag ggcccctgtt cccccgctgg cagccacccc ctcccccatc 1441 atatcctttg ccccaaggct gctcagaggg gccccggtcc tggccccagc ccccacctcc 1501 gccccagaca caccccccag tcgagccctg cagccaaaca gagccttcac aaccagccac 1561 acagagcctg cctcagctgc tcgcacagat tacttcaggg ctggaaaagt cacacagaca 1621 cacaaaatgt cacaatcctg tccctcactc aacacaaacc ccaaaacaca gagagcctgc 1681 ctcagtacac tcaaacaacc tcaaagctgc atcatcacac aatcacacac aagcacagcc 1741 ctgacaaccc acacacccca aggcacgcac ccacagccag cctcagggcc cacaggggca 1801 ctgtcaacac aggggtgtgc ccagaggcct acacagaagc agcgtcagta ccctcaggat 1861 ctgaggtccc aacacgtgct cgctcacaca cacggcctgt tagaattcac ctgtgtatct 1921 cacgcatatg cacacgcaca gccccccagt gggtctcttg agtcccgtgc agacacacac 1981 agccacacac actgccttgc caaaaatacc ccgtgtctcc cctgccactc acctcactcc 2041 cattccctga gccctgatcc atgcctcagc ttagactgca gaggaactac tcatttattt 2101 gggatccaag gcccccaacc cacagtaccg tccccaataa actgcagccg agctcccca By “FOXP1 polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence NP_001012523.1 or a fragment thereof having DNA binding activity. An exemplary FOXP1 amino acid sequence follows: 1 mmqesgtetk sngsaiqngs ggsnhllecg glregrsnge tpavdigaad lahaqqqqqq 61 whlinhqpsr spsswlkrli sspwelevlq vplwgavaet kmsgpvcqpn pspf By “FOXP1 polynucleotide” is meant a polynucleotide encoding a FOXP1 polypeptide. An exemplary FOXP1 polynucleotide sequence is provided at NCBI Accession No. NM_001012505.2, which is reproduced below: 1 gccagcgccc cggcgaacgg caaagaggga gccgctcccg ctcggggggc cgctggagtg 61 cccagcggga acccgaaagt ttgtaagagg aagagagcgc gcggcgagcg agcgagcggg 121 ccgggggcag cggcagcggc gccggggacc atggtgctgc cggcgcctcc tccgcgggcg 181 tgaaggcggc gctcctactc cctccccgga ctccgcggtg tcccagaagc ttttgttgac 241 aattccagtt tccgaacaaa acatttcggc aatggtgagg gcttcgatcc cttctctgat 301 ttgctgtcag ccatgaacgg atggatgtga tgcctgctag ccaaaaggct tccctctgtg 361 tgttgcagtc ctgtggcatt atgcatgccc cctcccagtg accccaggct ttttatggct 421 gtgagacacg ttaaaatttc aggggtaaga cgtgaccttt tgaggtgact ataactgaag 481 attgctttac agaagccaaa aaaggttttt gagtcatgat gcaagaatct gggactgaga 541 caaaaagtaa cggttcagcc atccagaatg ggtcgggcgg cagcaaccac ttactagagt 601 gcggcggtct tcgggagggg cggtccaacg gagagacgcc ggccgtggac atcggggcag 661 ctgacctcgc ccacgcccag cagcagcagc aacagtggca tctcataaac catcagccct 721 ctaggagtcc cagcagttgg cttaagagac taatttcaag cccttgggag ttggaagtcc 781 tgcaggtccc cttgtgggga gcagttgctg agacgaagat gagtggacct gtgtgtcagc 841 ctaacccttc cccattttga ataaaattat tctttggaga aa By “agent” is meant a peptide, nucleic acid molecule, or small compound. By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. By “alteration” is meant a change (increase or decrease) in the expression levels, structure, or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels. “
By “analog” is meant a molecule that is not identical but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid. As used herein, the term “antisense strand” refers to a polynucleotide that is substantially or 100% complementary to a target nucleic acid of interest. For example, an antisense strand may be complementary, in whole or in part, to a molecule of mRNA (messenger RNA), an RNA sequence that is not mRNA (e.g., microRNA, piwiRNA, tRNA, rRNA and hnRNA) or a sequence of DNA that is either coding or non-coding. The terms “antisense strand” and “guide strand” are used interchangeably herein. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. By “complementary” is meant capable of pairing to form a double-stranded nucleic acid molecule or portion thereof. In one embodiment, an antisense molecule is in large part complementary to a target sequence. The complementarity need not be perfect, but may include mismatches at 1, 2, 3, or more nucleotides. By “corresponds” is meant comprising at least a fragment of a double-stranded gene, such that a strand of the double-stranded inhibitory nucleic acid molecule is capable of binding to a complementary strand of the gene. By “decreases” is meant a reduction by at least about 5% relative to a reference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more and any intervening percentages “Detect” refers to identifying the presence, absence or amount of the analyte to be detected. By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. The term “expression” or “expressed” as used herein in reference to a gene means the transcriptional and/or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88). Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell. By “effective amount” is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. The term “effective amount” or “therapeutically effective amount” of an agent, as used herein, may be that amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. In one embodiment, an effective amount is the amount of an irradiated compound described herein sufficient to affect the degradation of a protein of interest. In another embodiment, in the context of administering an agent that is an anticancer agent, an effective amount of an agent is, for example, an amount sufficient to achieve alleviation or amelioration or prevention or prophylaxis of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition (e.g., cancer, etc.); and remission (whether partial or total), whether detectable or undetectable, as compared to the response obtained without administration of the agent. In some embodiments, the effective amount assumes that more than 50% of the compounds administered release the photolabile group under irradiation conditions (e.g., more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%, 100%, etc.) to achieve the active compound capable of degrading a protein of interest. \
The invention provides a number of targets that are useful for the development of highly specific drugs to treat, or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity. By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. A “host cell” or “cell” is any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell. The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography (HPLC). The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant
DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%. By “reference” is meant a standard or control condition. A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween. By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid
molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol.152:507). For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will
occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence. By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or
symptoms associated therewith be completely eliminated. The present disclosure features a method of treating or preventing a disease, disorder or condition that can be suppressed by a transcription factor, e.g., a tumor suppressing transcription factor and, unstable tumor suppressing transcription factors, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the disease, disorder, or condition is selected from the group consisting of a respiratory disorder, a proliferative disorder, an autoimmune disorder, an autoinflammatory disorder, an inflammatory disorder, a metabolic disorder, a neurological disorder, and an infectious disease. In some embodiments, the disease, disorder, or condition is selected from the group consisting of a respiratory disorder, a proliferative disorder, an autoimmune disorder, an autoinflammatory disorder, an inflammatory disorder, a neurological disorder, and an infectious disease. In some embodiments, the disease, disorder, or condition comprises a respiratory disorder. In some embodiments, the disease, disorder, or condition comprises a proliferative disorder. In some embodiments, the disease, disorder, or condition comprises an autoinflammatory disorder. In some embodiments, the disease, disorder, or condition comprises an inflammatory disorder. In some embodiments, the disease, disorder, or condition comprises a metabolic disorder. In some embodiments, the disease, disorder, or condition comprises a neurological disorder. In some embodiments, the disease, disorder, or condition comprises an infectious disease. In some embodiments, the disease, disorder, or condition is cancer. In some embodiments, the disease, disorder, or condition is cystic fibrosis. In some embodiments, the disease, disorder, or condition is diabetes. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural. By “consist essentially” it is meant that the ingredients include only the listed components along with the normal impurities present in commercial materials and with any other additives present at levels which do not affect operation, for instance at levels less than 5% by weight or less than 1% or even 0.5% by weight. Typically, alkyl groups described herein refer to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of 1-30 carbon atoms (e.g., 1-16 carbon atoms, 6-20 carbon atoms, 8-16 carbon atoms, or 4-18 carbon atoms, 4-12 carbon atoms, 1-4 carbon atoms, 1-8 carbon atoms) which are optionally substituted. In some embodiments, the alkyl group may be substituted with 1, 2, 3, or 4 substituent groups as defined herein. Alkyl groups may have from 1-26 carbon atoms. In other embodiments, alkyl groups will have from 6-18 or from 1-8 or from 1-6 or from 1-4 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five,
six, seven, eight, nine, or ten carbon atoms. Any alkyl group may be substituted or unsubstituted. Examples of alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl groups. Heteroalkyl groups may refer to branched or straight-chain monovalent optionally saturated aliphatic hydrocarbon radicals with one or more heteroatoms (e.g., N, O, S, etc.) in the carbon chain. Heteroalkyl groups may have 1-30 carbon atoms (e.g., 1-16 carbon atoms, 6-20 carbon atoms, 8-16 carbon atoms, or 4-18 carbon atoms, 4-12 carbon atoms, etc.) with one or more heteroatoms (e.g., N, O, S, etc.) replacing a carbon in the chain. In some embodiments, the heteroalkyl group may be substituted with 1, 2, 3, or 4 substituent groups as defined herein. Heteroalkyl groups may have from 1-26 carbon atoms. In other embodiments, heteroalkyl groups will have from 6-18 or from 1-8 or from 1-6 or from 1-4 or from 1-3 carbon atoms, including for example, embodiments having one, two, three, four, five, six, seven, eight, nine, or ten carbon atoms. In some embodiments, the heteroalkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups. Examples of heteroalkyl groups are an “alkoxy” which, as used herein, refers alkyl-O-; and “alkoyl” which, as used herein, refers to alkyl-CO-. Alkoxy substituent groups or alkoxy-containing substituent groups may be substituted by, for example, one or more alkyl groups. Alkenyl groups include alkyl groups having a double bond between any two carbon atoms. Alkynyl groups include alkyl groups having a triple bond between carbon atoms. Aryl groups may be aromatic mono- or polycyclic radicals of 6 to 12 carbon atoms having at least one aromatic ring. Aryl groups may be optionally substituted. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalyl, 1,2- dihydronaphthalyl, indanyl, and 1H-indenyl. Aryl groups as used herein may be substituted or unsubstitued. Typically, heteroaryls include mono- or polycyclic radical of 5 to 12 atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, and S, with the remaining ring atoms being C. One or more ring carbon atoms of the heteroaryl group may be replaced with a carbonyl group. Examples of heteroaryl groups are pyridyl, triazolyl, benzooxazolyl, benzoimidazolyl, and benzothiazolyl. Linking groups, when present, may be referred to as divalent hydrocarbon groups (e.g., alkylene, heteroalkylene, alkynylene, alkeneylene, heteroalkynylene, heteroalkeneylene, arylene, heteroarylene), where each radical position is the point of attachment to the remaining portions of the compound. Alkylene groups may refer to a straight or branched chain divalent hydrocarbon radical having from one to ten carbon atoms, optionally substituted with substituents, for example, selected from the group which includes lower alkyl (e.g., C1-C4, etc.), lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by
alkyl, nitro, cyano, halogen and lower perfluoroalkyl, multiple degrees of substitution being allowed. Examples of alkylene as used herein include, but are not limited to, methylene, ethylene, n- propylene, n-butylene, and the like. Alkylene groups may be saturated or unsaturated. Heteroalkylene groups may be alkylene groups comprising one or more heteroatoms (e.g., N, S, O) in the carbon chain. Cycloalkylene groups may be divalent hydrocarbons comprising one or more saturated or unsaturated cycloalkyl groups. The two points of attachment on cycloalkylene groups may be at two points in the ring, for example, at vicinal positions or at geminal positions. Cycloalkylene groups may be divalent saturated mono- or multicyclic ring system, in certain embodiments of 3 to 10 carbon atoms, in other embodiments 3 to 6 carbon atoms; cycloalkenylene and cycloalkynylene refer to divalent mono- or multicyclic unsaturated ring systems that respectively include at least one double bond and at least one triple bond. Cycloalkylene, Cycloalkenylene and cycloalkynylene groups may, in certain embodiments, contain 3 to 10 carbon atoms, with cycloalkenylene groups in certain embodiments containing 4 to 7 carbon atoms and cycloalkynylene groups in certain embodiments containing 8 to 10 carbon atoms. It will be understood that any divalent linking moiety with multiple points of attachment (each typically indicated with “
) may be attached to the specified moieties in either direction to the extent permitted by valency, unless otherwise indicated (e.g., by indicating the attachments on each side). For example, a linking moiety having the structure –Y1–Y2– may be used to link two portions of a compound in the –Y1–Y2– orientation or in the –Y2–Y1– orientation. The ring systems of the saturated, partially saturated, or unsaturated cycloalkyl, aryl, heterocylcoalkyl, heteroaryl, cycloalkylene, cycloalkenylene cycloalkynylene, heterocycloalkylene, heterocycloalkenylene and heterocycloalkynylene groups may be composed of one ring, two rings, or three or more rings (e.g., from 1 to 5 rings) which may be joined together in a fused, bridged or spiro-connected fashion, each of which may be optionally substituted. Heteocyclo and Heterocyclene groups may be monocyclic or multicyclic non-aromatic ring system, in certain embodiments of 3 to 10 members, in one embodiment 4 to 7 members, in another embodiment 5 to 6 members, where one or more, including 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur. Aryl and Arylene groups may be monocyclic or polycyclic, in certain embodiments monocyclic, aromatic group, in some embodiments having from 5 to 20 carbon atoms and at least one aromatic ring, in another embodiment 5 to 12 carbons. Arylene groups include, but are not limited to, 1,2-, 1,3- and 1,4-phenylene. Heteroarylene groups are typically divalent monocyclic or multicyclic aromatic ring systems, in one embodiment of 5 to 15 atoms in the ring(s), where one or more, in certain embodiments 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur. In some embodiments,
heteroarylene or heterocyclene groups (particularly in linking moieties) are divalent optionally substituted multicyclic heteroaryl or heterocyclyl fused with a monocyclic or multicyclic (e.g., bicyclic, tricyclic) optionally saturated optionally substituted cycloalkyl, wherein one point of attachment to the rest of the compound is on the heteroaryl moiety and the other point of attachment to the rest of the compound is on the cycloalkyl moiety. For example, the multicyclic heteroarylene or heterocyclene groups (particularly as a linking moiety such as Y2 or Y3 as a a strained click chemistry reaction product –LSCC–) may have have the structure:
wherein wherein each
indicates a point of attachment to the compound; ring “A” is optionally aromatic five or six membered ring; r and s are independently 0, 1, 2, 3, 4, or 5; t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; X1-X4 are independently selected at each occurrence from absent, CR, C(R)2, N, NR, O, S or SR; one of X1-X4 comprises a
(e.g., in place of an R group); and at least one of X1-X4 is not CR or C(R)2; X5 is CR or N; X6 is absent, C(R)2, NR, or O; R is independently selected at each occurrence from hydrogen, halogen, –ORa, –N(Ra)(Ra), alkyl (e.g., C1-C7 alkyl, C1-C3 alkyl, etc.), or alkoxy (e.g., C1-C7 alkoxy, C1-C3 alkoxy, etc.) wherein any two vicinal R groups may together form a fused ring (e.g., five membered fused ring, six membered fused ring, five membered fused aromatic ring, six membered fused aromatic ring); and Ra is independently selected at each occurrence from hydrogen, or alkyl (e.g., C1-C7 alkyl, C1-C4 alkyl, etc.). The term “substituent” refers to a group “substituted” on, e.g., an alkyl, at any atom of that group, replacing one or more hydrogen atoms therein. In some aspects, the substituent(s) on a group are independently any one single, or any combination of two or more of the permissible atoms or groups of atoms delineated for that substituent. In another aspect, a substituent may itself be substituted with any one of the substituents described herein.
A substituted hydrocarbon group may have as a substituent one or more hydrocarbon radicals, substituted hydrocarbon radicals, or may comprise one or more heteroatoms. Examples of substituted hydrocarbon radicals include, without limitation, heterocycles, such as heteroaryls. Unless otherwise specified, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-20 heteroatoms. In other embodiments, a hydrocarbon substituted with one or more heteroatoms will comprise from 1-12 or from 1-8 or from 1-6 or from 1-4 or from 1-3 or from 1-2 heteroatoms. Examples of heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorous, halogen (e.g., F, Cl, Br, I, etc.), boron, silicon, etc. In some embodiments, heteroatoms will be selected from the group consisting of oxygen, nitrogen, sulfur, phosphorous, and halogen (e.g., F, Cl, Br, I, etc.). In some embodiments, a heteroatom or group may substitute a carbon. In some embodiments, a heteroatom or group may substitute a hydrogen. In some embodiments, a substituted hydrocarbon may comprise one or more heteroatoms in the backbone or chain of the molecule (e.g., interposed between two carbon atoms, as in “oxa”). In some embodiments, a substituted hydrocarbon may comprise one or more heteroatoms pendant from the backbone or chain of the molecule (e.g., covalently bound to a carbon atom in the chain or backbone, as in “oxo”). In addition, the phrase “substituted with a[n],” as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is “substituted with an unsubstituted C1-C20 alkyl, or unsubstituted 2 to 20 membered heteroalkyl,” the group may contain one or more unsubstituted C1- C20 alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls. Moreover, where a moiety is substituted with an R substituent (e.g., a hydrocarbon), the group may be referred to as “R- substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. Unless otherwise noted, all groups described herein (e.g., alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, alkylene, heteroalkylene, cylcoalkylene, arylene, heteroaryelene, heterocycloalkylene) may optionally contain one or more common substituents, to the extent permitted by valency. Common substituents include halogen (e.g., F, Cl, etc.), C1-12 straight chain or branched chain alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C6-12 aryl, C3-12 heteroaryl, C3-12 heterocyclyl, C1-12 alkylsulfonyl, nitro, cyano, –COOR, –C(O)NRR’, –OR, –SR, –NRR’, and oxo, such as mono- or di- or tri-substitutions with moieties such as halogen, fluoroalkyl, perfluoroalkyl, perfluroalkoxy, trifluoromethoxy, chlorine, bromine, fluorine, methyl, methoxy, pyridyl, furyl, triazyl, piperazinyl, pyrazoyl, imidazoyl, and the like, each optionally containing one or more heteroatoms such as halo, N, O, S, and P. R and R’ are independently hydrogen, C1-12 alkyl, C1-12 haloalkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C4-24 cycloalkylalkyl, C6-12 aryl, C7-24 aralkyl, C3-12 heterocyclyl, C3-24 heterocyclylalkyl, C3-12 heteroaryl, or C4-24 heteroarylalkyl. Further, as used
herein, the phrase optionally substituted indicates the designated hydrocarbon group may be unsubstituted (e.g., substituted with H) or substituted with, for example one or more independently chosen common substituents. Typically, substituted hydrocarbons are hydrocarbons with a hydrogen atom removed and replaced by a substituent (e.g., a common substituent). It is understood by one of ordinary skill in the chemistry art that substitution at a given atom is limited by valency. The use of a substituent (radical) prefix names such as alkyl without the modifier optionally substituted or substituted is understood to mean that the particular substituent is optionally substittued. Additionally, the use of haloalkyl without the modifier optionally substituted or substituted is still understood to mean an alkyl group, in which at least one hydrogen atom is replaced by halo. Compounds and agents provided herein (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agents 1-30) can have one or more asymmetric carbon atoms and can exist in the form of optically pure enantiomers, mixtures of enantiomers such as racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates. The optically active forms can be obtained for example by resolution of the racemates, by asymmetric synthesis or asymmetric chromatography (chromatography with a chiral adsorbent or eluant). That is, certain of the disclosed compounds may exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not superimposable, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center. “Enantiomer” means one of a pair of molecules that are mirror images of each other and are not superimposable. Diastereomers are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms and represent the conFIGuration of substituents around one or more chiral carbon atoms. Enantiomers of a compound can be prepared, for example, by separating an enantiomer from a racemate using one or more well-known techniques and methods, such as chiral chromatography and separation methods based thereon. The appropriate technique and/or method for separating an enantiomer of a compound described herein from a racemic mixture can be readily determined by those of skill in the art. “Racemate” or “racemic mixture” means a mixture containing two enantiomers, wherein such mixtures exhibit no optical activity; i.e., they do not rotate the plane of polarized light. “Geometric isomer” means isomers that differ in the orientation of substituent atoms (e.g., to a carbon-carbon double bond, to a cycloalkyl ring, to a bridged bicyclic system, etc.). Atoms (other than H) on each side of a carbon- carbon double bond may be in an E (substituents are on
opposite sides of the carbon- carbon double bond) or Z (substituents are oriented on the same side) conFIGuration. “R,” “S,” “S*,” “R*,” “E,” “Z,” “cis,” and “trans,” indicate configurations relative to the core molecule. Certain of the disclosed compounds may exist in atropisomeric forms. Atropisomers are stereoisomers resulting from hindered rotation about single bonds where the steric strain barrier to rotation is high enough to allow for the isolation of the conformers. The compounds disclosed herein may be prepared as individual isomers by either isomer-specific synthesis or resolved from an isomeric mixture. Conventional resolution techniques include forming the salt of a free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming the salt of the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each of the isomers of an isomeric pair using an optically pure acid, amine or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of either a starting material or a final product using various well known chromatographic methods. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9%) by weight relative to the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight optically pure. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight pure. Percent optical purity is the ratio of the weight of the enantiomer or over the weight of the enantiomer plus the weight of its optical isomer. Diastereomeric purity by weight is the ratio of the weight of one diastereomer or over the weight of all the diastereomers. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction pure relative to the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction pure. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by mole fraction pure. Percent purity by mole fraction is the ratio of the moles of the enantiomer or over the moles of the enantiomer plus the moles of its optical isomer. Similarly, percent purity by moles fraction is the ratio of the moles of the diastereomer or over the moles of the diastereomer plus the moles of its isomer. When a disclosed compound is named or depicted by structure without indicating the stereochemistry, and the compound has at least one chiral center, it is to be understood that the name or structure encompasses either enantiomer of the compound free from the corresponding optical isomer, a racemic mixture of the compound or mixtures enriched in one enantiomer relative to its
corresponding optical isomer. When a disclosed compound is named or depicted by structure without indicating the stereochemistry and has two or more chiral centers, it is to be understood that the name or structure encompasses a diastereomer free of other diastereomers, a number of diastereomers free from other diastereomeric pairs, mixtures of diastereomers, mixtures of diastereomeric pairs, mixtures of diastereomers in which one diastereomer is enriched relative to the other diastereomer(s) or mixtures of diastereomers in which one or more diastereomer is enriched relative to the other diastereomers. The disclosure embraces all of these forms. It will be understood that the description of compounds herein is limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding with regard to valencies, etc., and to give compounds which are not inherently unstable. For example, any carbon atom will be bonded to two, three, or four other atoms, consistent with the four valence electrons of carbon. Additionally, when a structure has less than the required number of functional groups indicated, those carbon atoms without an indicated functional group are bonded to the requisite number of hydrogen atoms to satisfy the valency of that carbon. The term “pharmaceutical composition,” as used herein, represents a composition containing a compound described herein (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agents 1-30) formulated with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gel cap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other formulation described herein (see below). The pharmaceutical composition may comprise, for example, from 0.1% to 25% of the compounds of the present disclosure by weight of the composition. Useful pharmaceutical carriers for the preparation of the compositions hereof, can be solids, liquids, or gases. Thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g., binding on ion-exchange resins or packaging in lipid-protein vesicles), sustained release formulations, solutions, suspensions, elixirs, and aerosols. The carrier can be selected from the various oils including those of petroleum, animal,
vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, and sesame oil. Water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly (when isotonic with the blood) for injectable solutions. For example, formulations for intravenous administration comprise sterile aqueous solutions of the active ingredient(s) which are prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution and rendering the solution sterile. Suitable pharmaceutical excipients include starch, cellulose, chitosan, talc, glucose, lactose, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. The compositions may be subjected to conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, and buffers. Suitable pharmaceutical carriers and their formulation are described in Remington’s Pharmaceutical Sciences by E. W. Martin, which is hereby incorporated by reference in its entirety. Such compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for administration to the recipient. As used herein, the term “pharmaceutically acceptable salt” refers to salts of any of the compounds described herein that within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008, each of which are hereby incorporated by reference in their entirety. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, dichloroacetate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glutamate, glycerophosphate, hemisulfate, heptonate, hexanoate, hippurate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, isethionate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, mucate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts. Representative basic salts include alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, aluminum salts, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to
ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, caffeine, and ethylamine. Transcription factors typically refer to a DNA-binding protein that regulates transcription of DNA into RNA, for example, by activation or repression of transcription. Some transcription factors effect regulation of transcription alone, while others act in concert with other proteins. Some transcription factors can both activate and repress transcription under certain conditions. In general, transcription factors bind a specific target sequence or sequences highly similar to a specific consensus sequence in a regulatory region of a target gene. Transcription factors may regulate transcription of a target gene alone or in a complex with other molecules. Examples of transcription factors include, but are not limited to, Sp1, NF1, CCAAT, GATA, HNF, PIT-1, MyoD, Myf5, Hox, Winged Helix, SREBP, p53, CREB, AP-1, Mef2, STAT, R-SMAD, NF-κB, Notch, TUBBY, and NFAT. Transcription factors generally bind DNA in a sequence-specific manner and either activate or repress transcription initiation. In some embodiments, the transcription factor is a tumor suppressor. For example, the transcription factor may be SMAD4, RFX7, REST, FOXP3, FOXP1, p53, FOXO3A, or IRF3. At least three types of separate domains have been identified within transcription factors. One is essential for sequence-specific DNA recognition, one for the activation/repression of transcriptional initiation, and one for the formation of protein-protein interactions (such as dimerization). Studies indicate that many plant transcription factors can be grouped into distinct classes based on their conserved DNA binding domains (Katagiri F and Chua N H, 1992, Trends Genet.8:22-27; Menkens A E, Schindler U and Cashmore A R, 1995, Trends in Biochem Sci. 13:506-510; Martin C and Paz-Ares J, 1997, Trends Genet.13:67-73). Each member of these families interacts and binds with distinct DNA sequence motifs that are often found in multiple gene promoters controlled by different regulatory signals. As used herein, the term “subject” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. In most embodiments, the subject is a human. Other subjects may include mammals such as mice, rats, rabbits, cats, dogs, non-human primates. The subject may be domesticated animals (e.g., cows, calves, sheep, goat, lambs, horses, poultry, foals, pigs, piglets, etc.), or animals in the family Muridae (e.g., rats, mice, etc.), or animals in the family Felidae. A subject may seek or be in need of treatment, require treatment, be receiving treatment, may be receiving treatment in the future, or a human or animal that is under care by a trained professional for a particular disease or condition (e.g., cancer, etc.). Physiological conditions typically refer to a set of conditions including temperature, salt concentration, pH that mimic those conditions of a living subject. A host of physiologically relevant
conditions for use in in vitro assays and chemical syntheses have been established. Generally, a physiological buffer contains a physiological concentration of salt and at adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. A variety of physiological buffers may be used to produce physiological conditions in, for example, reaction medium, including phosphate buffered saline (PBS). Physiologically relevant temperature ranges from 25° C. to 40° C or from 30° C. to 38° C. Typically, a proliferative disease refers to the physiological condition in a subject characterized by unregulated cell growth such as cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer (“NSCLC”), vulval cancer, thyroid cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. In yet other embodiments, the cancer is at least one selected from the group consisting of ALL, T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma, B- cell ALL, Philadelphia chromosome positive ALL, Philadelphia chromosome positive CML, lymphoma, leukemia, multiple myeloma, myeloproliferative diseases, large B cell lymphoma, and B cell Lymphoma. The term “unit dosage form” refers to a physically discrete unit suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active material (e.g., a compound of the present disclosure) calculated to produce the desired therapeutic effect, in association with any suitable pharmaceutical excipient or excipients. Exemplary, non- limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip, gel cap, and syrup. BRIEF DESCRIPTION OF DRAWINGS FIG.1 (FIGs.1A-1B) provide information regarding the TF-DUBTAC platform. FIG.1A is a schematic diagram of the TF-DUBTAC platform. The BCN-linked OTUB1 binder (DUBL-X-BCN, X=(CH2)n, n=2-11) was conjugated onto an azide-modified DNA oligomer (N3-ODN) via a copper- free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, resulting in a TF-DUBTAC that recruits the OTUB1 deubiquitinase to remove polyubiquitin chain(s) from the targeted transcription
factor. FIG.1B illustrates a general synthetic scheme for the TF-DUBTACs of the present disclosure. FIG.2 is an NMR of the DUBL-X-BCN #1 compound which may be used to form DUBTACs. FIG.3 is an NMR of the DUBL-X-BCN #2 compound which may be used to form DUBTACs. FIG.4 is an NMR of the DUBL-X-BCN #3 compound which may be used to form DUBTACs. FIG.5 is an NMR of the DUBL-X-BCN #4 compound which may be used to form DUBTACs. FIG.6 (FIGs.6A-6C) provide measurements of DUBL-X-BCN #5 compound which may be used to form DUBTACs including NMR (FIGs.6A-6B) and mass spectrometry data (FIG.6C). FIG.7 (FIGs.7A-7C) provide measurements of DUBL-X-BCN #6 compound which may be used to form DUBTACs including NMR (FIGs.7A-6B) and mass spectrometry data (FIG.7C). FIG.8 (FIGs.8A-8C) provide measurements of DUBL-X-BCN #7 compound which may be used to form DUBTACs including NMR (FIGs.8A-8B) and mass spectrometry data (FIG.6C). FIG.9 is an NMR of the DUBL-X-BCN #8 compound which may be used to form DUBTACs. FIG.10 is an NMR of the DUBL-X-BCN #9 compound which may be used to form DUBTACs. FIG.11 is an NMR of the DUBL-X-BCN #10 compound which may be used to form DUBTACs. FIG.12 (FIGs 12A-12V) illustrate how FOXO-DUBTACs, and particularly FOXO- DUBTAC#6, stabilize FOXO3A in an OTUB1-dependent manner. FIG.12A is a schematic diagram for the DNA binding motifs of FOXO family members (top) and a diagram for the FOXO3A motif and FOXO-ODN (bottom). FIG.12B illustrates that FOXO-ODN binds both FOXO3A and its close family member FOXO1 and the binding can be antagonized by free FOXO-ODN. Cell lysates with Flag-FOXO3A or HA-FOXO1 protein were extracted from HEK293T cells that expressed indicated constructs for 48 hours. FIGs.12C-D show that Biotin-FOXO-ODN binds FOXO3A (C) and
FOXO1 (D) in a streptavidin-biotin pulldown assay. The cell lysates derived from HEK293T cells that expressed Flag-FOXO3A (C) or HA-FOXO1 (D) construct were incubated with biotin or Biotin- FOXO-ODN at 4 ^C for 3 hours, followed by another 1-hour incubation with Streptavidin agarose beads and further analyzed with immunoblotting. FIG.12E illustrates how FOXO-ODN binds both FOXO3A and FOXO1, which can be antagonized by free FOXO-ODN. Repeat assay for FIG.12B. FIG.12F shows that linkers with 6-8 methylene groups are optimal for the in vitro SPAAC reactions between BCN-modified OTUB1 ligands and N3-FOXO-ODN to form FOXO-DUBTACs. The SPAAC reaction products were separated by 20% native PAGE. The arrow indicates the click reaction products, FOXO-DUBTACs, and the arrowhead indicates N3-FOXO-ODN. FIG.12G provices the chemical structures of BCN-linked OTUB1 binders, DUBL-X-BCN #5 and #6. FIG. 12H provides the chemical structure of of FOXO-DUBTAC #6. FIG.12I illustrates how FOXO- DUBTAC #6 stabilizes FOXO3A protein in HeLa cells. HeLa cells were treated with the indicated concentrations of FOXO-ODN, FOXO-DUBTAC #5, or #6 for 24 hours, followed by immunoblot analysis. FIGs.12J-K illustrate how FOXO-DUBTAC #6 stabilizes FOXO3A protein in HeLa cells. Repeat assay for FIG.12I. FIG.12L illustrates that the OTUB1 ligands, DUBL-X-BCN #5, #6, and #7 did not affect the basal expressions of FOXO3A or p53. FIG.12M shows that FOXO-DUBTAC #6 mediates the binding between OTUB1 and FOXO3A. Flag-OTUB1 protein was purified by Flag- bead immunoprecipitation and further used for pulldown of FOXO3A with or without adding FOXO3-DUBTAC #6. FIG.12N shows that FOXO-DUBTAC #6 treatment increases the expression of p27 and BIM. *, p<0.05; **, p<0.01. FIG.12O shows how FOXO-DUBTAC #6 treatment changes the proteome profile of HeLa cells. FIG.12P provides a diagram of the enrichment of MYC- related protein network in FOXO-DUBTAC #6-treated cells. Those in the dotted box are increased proteins, and and those outside the box are decreased proteins. FIG.12Q is a schematic diagram to showing that FOXO3A represses MYC expression and function, resulting in increase of NDRG1. FIG.12R illustrates that FOXO-DUBTAC #6 suppresses the tumorigenesis of HeLa cells. HeLa cells were treated with 1 ^g/mL of FOXO-DUBTAC #6, followed by assessment in a colony formation assay. *, p<0.05. FIG.12S shows that FOXO-DUBTAC #6 increases FOXO3A protein abundance in OTUB1+/+ cells, but not in OTUB1-/- cells. HeLa cells were infected with sgControl or sgOTUB1 virus to knock out endogenous OTUB1 and selected with puromycin for 72 hours, followed by the treatment with FOXO-DUBTAC #6 for 24 hours and further analyzed with immunoblotting. FIG. 12T shows that FOXO-DUBTAC #6 increases FOXO3A protein abundance in an OTUB1-dependent manner. Repeat assay for FIG.12Q. FIG.12U illustrates FOXO-DUBTAC #6 treatment increases the expression of p27 and BIM in a OTUB1-dependent manner. *, p<0.05; **, p<0.01. FIG.12V illustrates how FOXO-DUBTAC #6 treatment represses colony formation in a OTUB1-dependent manner. ***, p<0.001.
FIG.13 (FIGs 13A-13L) illustrate how p53-DUBTACs such as p53-DUBTAC #6 and p53- DUBTAC #7 stabilize p53 in an OTUB1-dependent manner. These figures illustrate that in vitro SPAAC reactions between p53-ODN and DUBL-X-BCNs lead to the formation of p53-DUBTACs. FIG.13A is a schematic diagram for the p53 motif, p53-ODN, biotin-modified p53-ODN (biotin- p53-ODN) and azide-modified p53-ODN (N3-p53-ODN). FIG.13B shows that biotin-p53-ODN binds Flag-tagged p53. The cell lysates with p53 protein were extracted from HEK293T cells that expressed the Flag-p53 construct for 48 hours. FIG.13C provides the native PAGE analysis of the in vitro SPAAC reaction products between BCN-linked OTUB1 binders (DUBL-X-BCNs 1-9) and N3- p53-ODN. The arrow indicates the click reaction products, p53-DUBTACs, and the arrowhead indicates p53-ODN. FIG.13D provides the chemical structure of p53-DUBTAC #6 (top), p53- DUBTAC #5 (middle) and p53-DUBTAC #7 (bottom). FIG.13E shows that p53-DUBTACs #5, #6 and #7 (1 µg/mL, 24 hours) increase the p53 protein level in HeLa cells. FIG.13F shows that p53- DUBTACs #5, #6 and #7 (0.1, 0.2 and 0.4 µg/mL, 24 hours) increase the p53 protein level in HeLa cells. Vehicle stands for the treatment with transfection reagents and control stands for the treatment without transfection reagents. FIG.13G provides the increase the p53 protein level in HeLa cells. FIG.13H provides that p53-DUBTACs #5, #6 and #7 increase the p53 protein level in HeLa cells. Repeat assay for FIG 13F. FIG 13I shows that p53-DUBTACs #6 and #7 mediate the binding between OTUB1 and p53. Flag-OTUB1 protein was purified by Flag-bead immunoprecipitation and further used for pulldown of p53 with or without adding of p53-DUBTACs. FIG.13J shows that p53-DUBTAC #6 treatment changes the proteome profile of HeLa cells. FIG.13K illustrates Enrichment of AURKA-related protein network in p53-DUBTAC #6-treated cells. Within the dotted box indicates increased proteins, and outside the box indicates decreased proteins. FIG.13L shows that p53-DUBTACs #6 and #7 (1 µg/mL, 24 hours) increase the p53 protein level in an OTUB1- dependent manner in HeLa cells. HeLa cells infected with either sgControl or sgOTUB1 virus and selected with puromycin, followed by treatment with indicated compound for 24 hours and immunoblotting analysis of p53 protein abundance. FIG.14 (FIGs.14A-14H) shows that IRF-DUBTACs, including IRF-DUBTAC #7, stabilizes IRF3 in vitro (e.g., in HeLa cells). FIG.14A is a schematic diagram for the motifs of IRF family members. FIG.14B is a schematic diagram for the IRF3 motif, IRF-ODN, biotin-modified IRF- ODN (biotin-IRF-ODN) and azide-modified IRF-ODN (N3-IRF-ODN). FIG.14C shows that Biotin-IRF-ODN binds all IRF family members, except IRF9. The cell lysates were extracted from HEK293T cells that overexpressed indicated Flag-IRF constructs. FIG.14D illustrates that Biotin- IRF-ODN binds IRF2 and IRF3 in a streptavidin-biotin pulldown assay. The cell lysates derived from HEK293T cells that expressed Flag-IRF2 or Flag-IRF3 were incubated with biotin or Biotin- IRF-ODN at 4 ^C for 3 hours, followed by another 1-hour incubation with Streptavidin agarose beads
and further analyzed with immunoblotting analysis. FIG.14E illustrates that DUBL-X-BCNs #5-7 are most effective in forming IRF-DUBTACs via in vitro SPAAC reactions with N3-IRF-ODN. The SPAAC reaction products were separated by 20% native PAGE. The arrow indicates the click reaction products, IRF-DUBTACs, and the arrowhead indicates IRF-ODN. FIG.14F shows that IRF-DUBTAC #7 (2 µg/mL, 24 hours) is most effective in increasing the IRF3 protein level in HeLa cells. Vehicle stands for the treatment with transfection reagents. FIG.14G provides the chemical structure of IRF-DUBTAC #7. FIG.14H illustrates that IRF-DUBTAC #7 is most effective in increasing the IRF3 protein level in HeLa cells as compared to the other IRF-DUBTACs measured. Repeat assay for Figure 14F. DETAILED DESCRIPTION The invention provides compositions and methods relating to Deubiquitinase-Targeting Chimera (DUBTACS) and their use for stabilizing proteins of interest. PROTACs typically adopts small molecule inhibitor (SMIs) as ligand for protein of interest (POI), but inhibitor cannot be used for DUBTAC, due to a high possibility of residual inhibitory effect on POI from these engineered DUBTACs. Prior to the experiments and disclosure provided herein, it was unknown whether the OTUB1 ligand could be used for stabilizing tumor suppressive proteins. PROTACS using transcription factor binding modalities have been reported such as in Liu et al, JACS 143.23 (2021): 8902-8910 and WO 2022251614, each of which are hereby incorporated by reference in their entirety and particularly in relation to transcription factor conjugation strategies, however these degrade oncogenic transcription factors (TFs). Without wishing to be bound by theory, the TF-DUBTAC platform described herein provides targeted stabilization of TFs with tumor suppressive function leading to therapeutic efficacy. The present disclosure includes a generalizable platform termed TF-DUBTAC to selectively stabilize tumor suppressive transcription factors. Through a series of alkyl linkers to bridge OTUB1 ligand EN523 with BCN-1, a panel of clickable DUB ligands (referred to herein as DUBL1-10) were further conjugated onto the 5’-terminal of azide-modified DNA oligomers (N3-ODN) via a copper- free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, to generate the TF-DUBTACs for stabilizing tumor suppressor transcription factors in cells. Briefly, TF-DUBTAC is composed of the OTUB1 ligand (e.g., EN523) and oligonucleotide corresponding to specific TF, linked by a series of linkers. DUBLs were chemically clicked on to the Azide-modifed oligonucleotide (N3-ODN) corresponding to the DNA binding motif of respective TF, in vitro using a copper free strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. Three series of TF-DUBTACs for FOXO3A, p53 and IRF3 were screened and validated, and identified agents such as compounds that efficiently stabilize FOXO3A, p53 and IRF3,
respectively, in cells. This TF-DUBTAC is a generalizable platform for selectively stabilizing transcription factors with tumor suppressive function for the first time. In certain embodiments, the TF-DUBTAC may have an affinity for its Transcription Factor or binding motif thereof (Kd) of less than 1 mM (e.g. from 500 μM to 1 mM, etc.) or less than 500 μM (e.g., less than 450 μM, less than 400 μM, less than 350 μM, less than 300 μM, less than 250 μM, less than 200 μM, less than 150 μM, less than 100 μM, less than 100 μM, less than 50 μM, less than 10 μM, less than 1 μM, less than 500 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, etc.). In some implementations, the TF-DUBTAC does not inhibit or has minimal inhibitory activity on the Transcpactor Factor of interest, such as, for example, having an IC50 of greater than (or up to 1M) 1μM or greater than 10 μM or greater than 100 μM or greater than 1000 μM. The compounds, agents, methods and compositions described herein include the use of N- oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the disclosure, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether, etc.) or alcohol (e.g., ethanol, etc.) solvates, acetates and the like. In certain embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In other embodiments, the compounds described herein exist in unsolvated form. In certain embodiments, the compounds and agents of the disclosure may exist as tautomers or mixtures of tautomers, racemates, mixtures of racemates, stereoisomers, mixtures of stereoisomers, or combinations thereof. All tautomers are included within the scope of the compounds presented herein. The DUBTACs of the present disclosure (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) typically include a moiety (ODN) which is capable of binding to a transcription factor (e.g., tumor suppressing transcription factor). The target protein may be any class of protein, for example, any protein found in a cell (e.g., a mammalian cell, a plant cell, a fungal cell, an insect cell, a bacterial cell) or a viral particle. In some embodiments, the protein is a soluble protein or a membrane protein. In some embodiments, the protein is a soluble protein. In some embodiments, the protein is a membrane protein. The target protein may comprise a post-translational modification, e.g., a sugar moiety, acyl moiety, lipid
moiety. In some embodiments, the target protein is glycosylated, e.g., at an asparagine, serine, threonine, tyrosine, or tryptophan residue. In some embodiments, the target protein is modified with a ubiquitin or a ubiquitin-like protein (collectively referred to herein as “Ubls”). In some embodiments, the Ubl is ubiquitin. In some embodiments, the Ubl is SUMO, NEDD8, or Agp12. In some embodiments, the target protein is monoubiquitinated or polyubiquitinated. The target protein may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Ubl chains, e.g., on a lysine amino acid residue. The target protein may comprise polyubiquitin chains linked in any manner, for example, K48-linked polyubiquitin chains, K63- linked polyubiquitin linked chains, K29-linked polyubiquitin chains, or K33-linked polyubiquitin chains. In some embodiments, the target protein comprises a plurality of polyubiquitin chains. In some embodiments, the target protein comprising a Ubl is capable of binding to a protein comprising a Ubl-binding domain (e.g., a ubiquitin binding domain). The target protein may comprise a feature that increases its instability or impairs its activity, e.g., relative to the wild-type target protein. For example, the target protein may be mutated or misfolded. In some embodiments, the target protein has a reduced capacity for binding to a binding partner, e.g., by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% relative to the wild type target protein. In some embodiments, the target protein is less active than the wild type target protein, e.g., by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%. In some embodiments, the target protein is more active than the wild type target protein, e.g., by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%. This ODN moietiy is conjugated, directly or indirectly to a moiety capable of binding to a deubiquitinase (DUB). Deubiquitinases comprise a large family of proteases responsible for hydrolyzing Ubl-Ubl bonds or Ubl-target protein bonds and play a role in numerous cellular processes. Deubiquitinases serve several functions, including generating free ubiquitin monomers from polyubiquitin chains, modulating the size of polyubiquitin chains, and reversing ubiquitin signaling by removal of a from a ubiquitinated target protein. Misregulation of deubiquitinase function is associated with many diseases, including cancer, metabolic diseases, genetic disorders, haploinsufficiency targets, and neurological diseases. Roughly 80 different functional deubiquitinases have been identified in human cells to date. The presently disclosed DUBTACs (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) comprise a DUB Recruiter capable
of binding to a deubiquitinase. The deubiquitinase may be any deubiquitinase, e.g., in a cell, including cysteine protease deubiquitinases and metalloprotease deubiquitinases. In some embodiments, the deubiquitinase is a cysteine protease, e.g., comprising a catalytic site cysteine amino acid residue. The deubiquitinase may be a full-length protein or a fragment thereof. In some embodiments, the deubiquitinase comprises a single active site. In other embodiments, the deubiquitinase is one function of a multifunctional protein. Exemplary deubiquitinases include BAP1, CYLD, OTUB1, OTUB2, OTUD3, OTUD5, OTUD7A, OTUD7B, TNFAIP3, UCHL1, UCHL3, UCHL5, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP17L1, USP17L2, USP17L24, USP17L3, USP17L5, USP18, USP19, USP2, USP20, USP21, USP22, USP24, USP25, USP26, USP27X, USP28, USP3, USP30, USP31, USP33, USP34, USP35, USP36, USP37, USP38, USP4, USP40, USP41, USP42, USP43, USP44, USP45, USP46, USP47, USP48, USP49, USP5, USP50, USP51, USP54, USP7, USP8, USP9X, VCPIP1, WDR48, YOD1, ZRANB1, and ZUP1, or a fragment or variant thereof. In some embodiments, the deubiquitinase is selected from the group consisting of WDR48, YOD1, OYUD3, OTUB1, USP8, USP5, USP16, UCHL3, UCHL1, and USP14, or a fragment thereof. In some embodiments, the deubiquitinase is selected from the group consisting of WDR48, YOD1, OYUD3, OTUB1, OTUD5, USP8, USP5, USP14, USP15, USP16, UCHL3, and UCHL1, or a fragment thereof. In some embodiments, the deubiquitinase comprises OTUB1 or a fragment or variant thereof. In some embodiments, the deubiquitinase comprises OTUD5 or a fragment or variant thereof. In some embodiments, the deubiquitinase comprises USP15 or a fragment or variant thereof. The DUBTACs of the present disclosure (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) may bind to a deubiquitinase in a covalent or non-covalent manner. In some embodiments, the DUBTAC (e.g., via the DUB moiety) binds to a site other than a catalytic site within the deubiquitinase. In some embodiments, the DUBTAC (e.g., via the DUB moiety) binds to an allosteric site within the deubiquitinase. In some embodiments, binding of the DUBTAC (e.g., DUB) to the deubiquitinase does not modulate the activity of the deubiquitinase more than 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%, relative to the activity of the deubiquitinase in the absence of the bifunctional compound. In some embodiments, binding of the bifunctional compound (e.g., DUB) to the deubiquitinase does not modulate the activity of the deubiquitinase more than 0.1-50%, 1-50%, 1-25%, 1-10%, 0.1-10%, 1-5%, or 0.1-2%, relative to the activity of the deubiquitinase in the absence of the bifunctional compound. In some embodiments,
the binding of the bifunctional compound (e.g., DUB) to the deubiquitinase does not substantially modulate (e.g., inhibit) the activity (e.g., deubiquitinase activity) of the deubiquitinase. The DUBTACs of the present disclosure (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) may be capable of binding to a cysteine amino acid residue (e.g., a thiol moiety), e.g., within the deubiquitinase. In some embodiments, the cysteine amino acid residue is an allosteric cysteine amino acid residue. In some embodiments, the cysteine amino acid residue is present on a surface of the deubiquitinase. In some embodiments, the cysteine amino acid residue is present on or in the interior of the deubiquitinase. In some embodiments, the cysteine amino acid residue is not a catalytic cysteine amino acid residue. In some embodiments, the bifunctional compound preferentially binds to an allosteric cysteine amino acid residue over a catalytic cysteine amino acid residue. In some embodiments, the bifunctional compound does not substantially bind to a cysteine amino acid residue in the catalytic site of the deubiquitinase (e.g., a catalytic cysteine). In some embodiments, the DUBTAC (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) comprises a functional group selected from the group consisting of an amide, heterocyclyl, cycloalkyl, heterocyclyl, cycloalkyl, carbonyl, ester, alkyl, alkenyl, alkynyl, acyl, or acrylamide. In some embodiments, the DUB moiety comprises a heterocyclyl (e.g., a piperazinonyl). In some embodiments, DUB comprises an acrylamide moiety. In some embodiments, DUB comprises a heteroaryl (e.g., a furan moiety). Pharmaceutical Compositions The disclosure includes a pharmaceutical composition comprising a DUBTAC described herein (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) and at least one pharmaceutically acceptable carrier, diluent, or diluent. In certain embodiments, the composition is formulated for an administration route such as oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical,
intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. The pharmaceutical composition may comprise a compound disclosed herein (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) and one or more pharmaceutically acceptable salts, carriers, or diluents. In specific embodiments, the compound is formulated as a topical composition (e.g., ointment, gel, etc.). In some embodiments, the composition comprises from 0.1%-90% (e.g., 0.1%-50%, 0.1%-20%, 0.1%- 10%, etc.) of the compound by weight of the composition. The pharmaceutical composition may be formulated to allow for or enhance transfection of the compounds of the present disclosure (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) into a cell. Transfection may occur, for example, in vivo, ex vivo, or in vitro. The compounds of the present disclosure can be introduced by suitable transfection mechanisms such as electroporation, lipofection, particle gun acceleration. In some examples, the method is a chemical method (e.g., calcium-phosphate transfection), physical method (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), or receptor-mediated endocytosis. In some embodiments, a pharmaceutical composition may comprise one or more lipids with the compounds of the presente disclosure selected from cationic lipids, ionizable lipids, anionic lipids, sterols, pegylated lipids, and any combination of the foregoing. In some embodiments, the pharmaceutical composition containing a translatable compound comprises a cationic lipid, a phospholipid, cholesterol, and a pegylated lipid. In certain embodiments, a pharmaceutical composition can be substantially free of liposomes. In some embodiments, the pharmaceutical composition comprises liposomes. In some embodiments, the pharmaceutical composition comprises lyophilized liposomes. In further embodiments, a pharmaceutical composition can include nanoparticles. Lipid-based formulations have been increasingly recognized as one of the most promising delivery systems due to their biocompatibility with certain DNA and RNA and their ease of large-scale production. Cationic lipids, for example, have been widely studied as synthetic materials for compound. After mixing together, nucleic acids may be condensed by cationic lipids to form lipid/nucleic acid complexes known as lipoplexes.
These lipid complexes may be able to protect compounds of the present disclosure from the action of nucleases and to deliver it into cells by interacting with the negatively charged cell membrane. Lipoplexes can be prepared by directly mixing positively charged lipids at physiological pH with negatively charged nucleic acids. Conventional liposomes typically consist of a lipid bilayer that can be composed of cationic, anionic, or neutral (phospho)lipids and cholesterol, which encloses an aqueous core. Both the lipid bilayer and the aqueous space can incorporate hydrophobic or hydrophilic compounds, respectively. Liposome characteristics and behaviour in vivo can be modified by addition of a hydrophilic polymer coating, e.g. polyethylene glycol (PEG), to the liposome surface to confer steric stabilization. Furthermore, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (Front Pharmacol.2015 Dec.1; 6:286, which is hereby incorporated by reference in its entirety). Pharmaceutical compostions of the present disclosure comprising liposomes may be colloidal lipid-based and surfactant-based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment. These liposomes may be present as spherical vesicles and can range in size from 20 nm to a few microns. Cationic lipid-based liposomes are able to complex with negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required for in vivo clinical applications. Liposomes can fuse with the plasma membrane for uptake; once inside the cell, the liposomes are processed via the endocytic pathway and the genetic material is then released from the endosome/carrier into the cytoplasm. Cationic liposomes have been traditionally the most commonly used non-viral delivery systems for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA/small hairpin R A-shRNA). Cationic lipids, such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids to form nanoparticles by electrostatic interaction, providing high transfection efficiency. Furthermore, neutral lipid-based nanoliposomes for compound delivery as e.g. neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomes may be well suited for delivery of the compounds of the present disclosure (Adv Drug Deliv Rev.2014 February; 66: 110-116, which is hereby incorporated by reference in its entirety). According to some embodiments, the compounds described herein are lipid formulated. The lipid formulation is preferably selected from, but not limited to, liposomes, lipoplexes, copolymers, such as PLGA, and lipid nanoparticles. Lipid nanoparticles may include a cationic lipid suitable for forming a lipid nanoparticle. The cationic lipid may be, for example, N,N-dioleyl-N,N-
dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-Dioleyloxy-3- trimethylaminopropane chloride salt), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-dilinoleylcarbamoyloxy-3- dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin- DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy- 3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1,2- dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2- propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DM A), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]- dioxolane (DLin-K-DMA), (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12- dienyl)tetrahydro- -3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)ami- no)ethyl)piperazin-1- yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]- dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K- DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,2831-tetraen-19-yl 4-(dimethylamino) butanoate (DLin- M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethy- lpropan-1-amine (MC3 Ether), 4-((6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N- dimethylbutan-1-amine (MC4 Ether), or any combination of any of the foregoing. Other cationic lipids include, but are not limited to, N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P- (N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(1-(2,3-dioleyloxy)propyl)-N- 2-(sperminecarboxamido)ethyl)-N,N-dimethyl- ammoniumtrifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(1,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), and 2,2-Dilinoleyl-4-dimethylaminoethyl- [1,3]-dioxolane (XTC). Additionally, commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and Lipofectamine (comprising DOSPA and DOPE, available from GIBCO/BRL).
Other suitable cationic lipids are disclosed in International Publication Nos. WO 09/086558, WO 09/127060, WO 10/048536, WO 10/054406, WO 10/088537, WO 10/129709, and WO 2011/153493; U.S. Patent Publication Nos.2011/0256175, 2012/0128760, and 2012/0027803; U.S. Pat. No.8,158,601; and Love et al, PNAS, 107.5 (2010): 1864-69, each of which are hereby incorporated by reference in their entirety. Other suitable amino lipids useful in the compositions of the present disclosure include those having alternative fatty acid groups and other dialkylamino groups, including those, in which the alkyl substituents are different (e.g., N-ethyl-N-methylamino-, and N-propyl-N-ethylamino-). In general, amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below 0.3 microns, for purposes of filter sterilization. Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of C14 to C22 may be used. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid. In various implementations, amino or cationic lipids used in lipid formulations of the present disclosure may have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded from use in the invention. In certain embodiments, the protonatable lipids have a pKa of the protonatable group in the range of 4 to 11, e.g., a pKa of 5 to 7. The cationic lipid can comprise from 20 mol % to 70 or 75 mol % or from 45 to 65 mol % or 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, or 65-70 mol % of the total lipid present in the particle. In another embodiment, the lipid nanoparticles include from 25% to 75% on a molar basis of cationic lipid, e.g., from 20 to 70%, from 35 to 65%, from 45 to 65%, on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle). In certain embodiments, the ratio of cationic lipid to nucleic acid is from 3 to 15, such as from 5 to 13 or from 7 to 11. The lipid-based formulations may als contain a non-cationic lipid. The non-cationic lipid can be a neutral lipid, an anionic lipid, or an amphipathic lipid. Neutral lipids, when present, can be any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH. Such neutral lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for use in the particles described herein is generally guided by consideration of, e.g., lipid particle size and stability of the lipid particle in the
bloodstream. Preferably, the neutral lipid is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine). In one embodiment, the neutral lipids contain saturated fatty acids with carbon chain lengths in the range of C10 to C20. In another embodiment, neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of C10 to C20 are used. Additionally, neutral lipids having mixtures of saturated and unsaturated fatty acid chains can be used. Suitable neutral lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dimyristoyl phosphatidylcholine (D PC), distearoyl-phosphatidyl-ethanolamine (DSPE), SM, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. Anionic lipids suitable for use in lipid particles of the invention include, but are not limited to, phosphatidyl lycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids. The non-cationic lipid can be from 5 mol % to 90 mol %, 5 mol % to 10 mol %, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, or 85-90 mol % of the total lipid present in the particle. In one embodiment, the lipid nanoparticles include from less than (or from 0.1% to) 15% or less than 45% on a molar basis of neutral lipid, e.g., from 3 to 12% or from 5 to 10% (based upon 100% total moles of lipid in the lipid nanoparticle). The lipid-based formulations may also contain a sterol such as cholesterol. The sterol can be from 10 mol % to 60 mol % or from 25 mol % to 40 mol % of the lipid particle. In one embodiment, the sterol is 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, or 55-60 mol % of the total lipid present in the lipid particle. In another embodiment, the lipid nanoparticles include from 5% to 50% on a molar basis of the sterol (based upon 100% total moles of lipid in the lipid nanoparticle). When formulated as a nanoparticle, the lipid nanoparticles may have the structure of a liposome. A liposome is typically a structure having lipid-containing membranes enclosing an aqueous interior. Liposomes preferably have one or more lipid membranes. In certain embodiments, liposomes can be single-layered, referred to as unilamellar, or multi-layered, referred to as
multilamellar. When complexed with nucleic acids (e.g., RNA, DNA, the ODN moiety of the compounds of the present disclosure), lipid particles may also be lipoplexes, which are preferably composed of cationic lipid bilayers sandwiched between nucleic acid layers. Liposomes can further be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. In certain embodiments, liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low (e.g. an acidic) or a high (e.g. a basic) pH in order to improve the delivery of the pharmaceutical formulations. As a non-limiting example, liposomes such as synthetic membrane vesicles may be prepared by the methods, apparatus and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372, each of which are herein incorporated by reference in their entirety. In certain implementations, ODN moiety or entire compound may be encapsulated by the liposome, and/or it may be contained in an aqueous core, which may then be encapsulated by the liposome as described in International Pub. Nos. WO2012031046, WO2012031043, WO2012030901 and WO2012006378 and US Patent Publication No. US20130189351, US20130195969 and US20130202684, each of which are herein incorporated by reference in their entirety). Examples of cancers that can be treated or prevented by the present disclosure include but are not limited to: squamous cell cancer, lung cancer including small cell lung cancer, non-small cell lung cancer, vulval cancer, thyroid cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer. In certain embodiments, the cancer is at least one selected from the group consisting of ALL, T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma, B- cell ALL, Philadelphia chromosome positive ALL, Philadelphia chromosome positive CML, lymphoma, leukemia, multiple myeloma, myeloproliferative diseases, large B cell lymphoma, and B
cell Lymphoma. In certain embodiments, the proliferative disease is melanoma, leukemia, lymphoma, or retinal blastoma. The methods of the disclosure may comprise administering to the subject a therapeutically effective amount of at least one compound of the disclosure, which is optionally formulated in a pharmaceutical composition. In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent that treats or prevents cancer. The compound may be a compound for the treatment of a proliferative disease. In certain embodiments, the compound may be a compound for the manufacture of a medicament for the treatment of a proliferative disease. The method for the treatment of a proliferative disease may comprise the administration of a compound or pharmaceutical composition as disclosed herein. In certain embodiments, administering the compound of the disclosure to the subject allows for administering a lower dose of the additional therapeutic agent as compared to the dose of the additional therapeutic agent alone that is required to achieve similar results in treating or preventing a cancer in the subject. For example, in certain embodiments, the compounds disclosed herein may enhance the anti-cancer activity of the additional therapeutic compound, thereby allowing for a lower dose of the additional therapeutic compound to provide the same effect. In certain embodiments, the compounds and the therapeutic agent are co-administered to the subject. In other embodiments, the compound of the disclosure and the therapeutic agent are coformulated and co-administered to the subject. In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human. The therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of a cancer in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors. For example, the therapeutically effective amount may be determined based on the amount of DUBTAC (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30) required to treat a proliferative disease of interest. For example, the therapeutically effective amount may be based on more than 20% or more than 30% or more than 40% or more than 50% or more than 60% or more than 70% or more than 80% or more than 90% or more than 95% or more than 95% or more than 99% or 100% being decaged following endocytosis f the compounds described herein by weight of the composition.
For example, a suitable dose of a compound of the present disclosure may be in the range of from 0.01 mg to 5,000 mg per day, such as from 0.1 mg to 1,000 mg, for example, from 1 mg to 500 mg, such as 5 mg to 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with, for example, a 12-hour interval between doses. It is understood that the amount of compound dosed per day may be administered, in non- limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of the inhibitor of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time. The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Once improvement of the patient’s conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection. The compounds for use in the method of the disclosure may be formulated in unit dosage form. Typically, unit dosage forms are physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Many applications of the compositions of the present disclosure may involve cell surface localization or targeting. However, the general methods of this invention may also be extended to include internalization of the compound in cells. In these embodiments, constructs may incorporate
an internalization or targeting moiety that causes the cell to internalize a DUBTAC (e.g., agents having the structure of formula (I), (IIA), (IIB), (IIC), (IID), (III), (IVa), (IVb), (IVc), (IVd), (IVf), (IVg), (IVh), (IVi), (IVj), (IVk), (IVl), (IVm), (IVn), (IVo), (IVp), (IVq), (IVr), (IVs), (IVt), (IVa1), (IVb1), (IVc1), (IVd1), (IVf1), (IVg1), (IVh1), (IVi1), (IVj1), (IVk1), (IVl1), (IVm1), (IVn1), (IVo1), (IVp1), (IVq1), (IVr1), (IVs1), (IVt1), (V), (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), Agent 1-30). Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized. It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. Unless otherwise apparent from context, the sum of all weight percentages should not exceed 100% and all percentages in relation to components refer to percentages by weight of the composition. EXAMPLES The following examples illustrate specific aspects of the instant description. The examples should not be construed as limiting, as the example merely provides specific understanding and practice of the embodiments and its various aspects. Three series of VHL-based TF-DUBTACs lead compounds, FOXO3a-DUBTAC, p53- DUBTAC and IRF3-DUBTAC, were synthesized which efficiently stabilize FOXO3a, p53 and IRF3 proteins, respectively, in cells. We are in the process of using this TF-DUBTAC technology to target other tumor suppressive TFs such as SMAD4, this design provides a generalizable platform of TF- DUBTACs to achieve selective stabilizaton of TFs in cells. In vivo animal models including on zebra fish or mice will evaluate the efficacy of these DUBTACs in tissues. More importantly, this technology can also be applied to conventional targeted chemotherapy reagents as listed below. Many transcriptional factors are characterized as tumor suppressors such as p53, FOXO3a or IRF3, however, targeted therapies for these tumor suppressors are not available and TF-DUBTACs provide a generalizable technology to target any tumor suppressive TFs with known
DNA binding motif. To this end, we can design TF-DUBTAC to target other tumor suppressive TFs including SMAD4, RFX7, REST, FOXP3, and FOXP1. This will create a TF-directed targeted chemotherapy platform to achieve better efficacy and target many previous untargetable tumor suppressive transcriptional factors for stabilization, which can present tremendous opportunity to expand our arsenal for anti-cancer therapies. TF-DUBTACs selectively stabilize tumor suppressor transcription factors. Through a series of alkylene linkers to bridge the OTUB1 ligand EN523 and bicyclononyne (BCN), a panel of clickable DUB binders (DUBL-X-BCN 1-10) were synthesized for creation of the TF-DUBTACs. These BCN-linked OTUB1 binders were conjugated onto the 5’-terminal of azide-modified DNA oligomers (N3-ODN) via a copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction (FIGs.1A and 1B), resulting in TF-DUBTACs for stabilizing tumor suppressor transcription factors in cells (FIG.1A). Three tumor suppressor transcription factors, FOXO3A, p53 and IRF3, with well-defined DNA binding motifs were used to experimentally test the feasibility of the TF-DUBTAC approach. The OTUB ligand EN523, is described in the following reference, Henning et al., Nat. Chem. Biol.2022, 18:412-421, which is incorporated herein in its entirety particularly in relation to EN523, its use, and synthetic methods thereof. It will be understood that in the event of any inconsistency in chemical structure between the EN523 OTUB ligand in the present application and in Henning et al., Nat. Chem. Biol.2022, 18:412-421, both compounds will be considered embraced by the present disclosure. Example 1: DUBTAC Synthesis A general synthetic route for agents of the present disclosure is:
((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (2-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)ethyl)carbamate (DUBL-X-BCN #1).
Compound 1 was prepared as shown in Henning, N. J. et al. Nat Chem Biol 18.4 (2022): 412- 421, which is hereby incorporated by reference in its entirety and particularly in relation to synthesis of Compound 1:
Compound 1 Briefly, to a solution of compound 1 (29.2 mg, 0.1 mmol) in DMSO (1 mL) were added commercially available tert-butyl (2-aminoethyl)carbamate (2, 16 mg, 0.1 mmol, 1.0 equiv), 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDCI, 28.8 mg, 0.015 mmol, 1.5 equiv), 1-hydroxy-7- azabenzo-triazole (HOAt, 20.4 mg, 0.015 mmol, 1.5 equiv), and N-methylmorpholine (NMM, 30.3 mg, 0.3 mmol, 3.0 equiv). After being stirred for 3 hours at rt, the resulting mixture was purified by preparative HPLC (10%-100% acetonitrile / 0.1% TFA in H2O) to afford white solid (19.7 mg, yield 45%). The solid was dissolved in DCM (1 mL) and TFA (1 mL) and the reaction was stirred at rt for 10 min. Then, the solvent was evaporated, and the resulting compound 12 was used for the next step without purification. To a solution of compound 12 and ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (2,5- dioxopyrrolidin-1-yl) carbonate (22, 13.2 mg, 0.045 mmol, 1.0 equiv) in DMF (1 mL) was added triethylamine (TEA, 13.6 mg, 0.135 mmol, 3.0 equiv). After being stirred at rt for 30 min, the resulting mixture was purified by preparative HPLC (10%-100% acetonitrile / 0.08% NH4HCO3 in H2O). The collected fractions were extracted with ethyl acetate (EA, 3 x 10 mL). The combined organic layers were washed with water (10 mL) and brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated to afford DUBL-X-BCN #1 as white solid (16.9 mg, yield 33% for two steps).1H NMR (600 MHz, CDCl3) is shown in FIG.2 with δ in ppm of 6.46 (s, 1H), 6.33 (dd, J = 16.8, 1.8 Hz, 1H), 6.28 (s, 1H), 6.11 (d, J = 3.3 Hz, 1H), 5.98 (d, J = 3.2 Hz, 1H), 5.75 (d, J = 10.4 Hz, 1H), 5.23 (s, 1H), 4.44 – 4.26 (m, 2H), 4.06 (d, J = 8.0 Hz, 2H), 3.89 (d, J = 40.9 Hz, 2H), 3.75 (s, 2H), 3.25 (q, J = 5.7 Hz, 2H), 3.15 (q, J = 5.8 Hz, 2H), 2.86 (dd, J = 14.9, 7.6 Hz, 2H), 2.42 (t, J = 7.3 Hz, 2H), 2.26 – 2.09 (m, 6H), 1.54 – 1.43 (m, 2H), 1.33 – 1.22 (m, 1H), 0.93 – 0.78 (m, 2H). HRMS (m/z) for C27H35N4O6+ [M + H]+: calculated 511.2551, found 511.2567.
((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (3-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)propyl)carbamate (DUBL-X-BCN #2).
DUBL-X-BCN #2 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (3- aminopropyl)carbamate (3, 17.4 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #2 was obtained as white solid (19.6 mg, yield 37% for three steps).1H NMR (600 MHz, CDCl3) is shown in FIG.3 with δ in ppm of 6.46 (s, 1H), 6.33 (dd, J = 16.8, 1.8 Hz, 1H), 6.21 (s, 1H), 6.13 (d, J = 3.3 Hz, 1H), 5.98 (d, J = 3.2 Hz, 1H), 5.74 (d, J = 10.5 Hz, 1H), 5.17 (s, 1H), 4.41 – 4.25 (m, 2H), 4.05 (d, J = 8.1 Hz, 2H), 3.96 – 3.81 (m, 2H), 3.77 (s, 2H), 3.17 (q, J = 6.2 Hz, 2H), 3.04 (t, J = 6.7 Hz, 2H), 2.91 – 2.82 (m, 2H), 2.42 (t, J = 7.4 Hz, 2H), 2.25 – 2.06 (m, 6H), 1.56 – 1.44 (m, 4H), 1.32 – 1.22 (m, 1H), 0.92 – 0.81 (m, 2H). HRMS (m/z) for C28H37N4O6 + [M + H]+: calculated 525.2708, found 525.2717. (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (4-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)butyl)carbamate (DUBL-X-BCN #3).
DUBL-X-BCN #3 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (4- aminobutyl)carbamate (4, 18.8 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #3 was obtained as white solid (7.9 mg, yield 15% for three steps).1
NMR is shown in FIG.4 (600 MHz, CDCl3) with δ in ppm of 6.46 (s, 1H), 6.33 (dd, J = 16.8, 1.8 Hz, 1H), 6.13 (d, J = 3.3 Hz, 1H), 5.98 (d, J = 3.2 Hz, 1H), 5.74 (d, J = 10.5 Hz, 2H), 4.82 (s, 1H), 4.42 – 4.26 (m, 2H), 4.06 (d, J = 8.1 Hz, 2H), 3.99 – 3.79 (m, 2H), 3.76 (s, 2H), 3.14 (d, J = 5.9 Hz, 2H), 3.07 (d, J = 6.3 Hz, 2H), 2.86 (t, J = 7.3 Hz, 2H), 2.40 (t, J = 7.3 Hz, 2H), 2.25 – 2.08 (m, 6H), 1.56 – 1.43 (m, 2H), 1.39 (dd, J = 7.5, 4.1 Hz, 4H), 1.31 – 1.22 (m, 1H), 0.86 (t, J = 9.8 Hz, 2H). HRMS (m/z) for C29H39N4O6 + [M + H]+: calculated 539.2864, found 539.2851.
((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (5-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)pentyl)carbamate (DUBL-X-BCN #4).
DUBL-X-BCN #4 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (5- aminopentyl)carbamate (5, 20.2 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #4 was obtained as white solid (16.3 mg, yield 30% for three steps).1H NMR (600 MHz, CDCl3) is shown in FIG.5 with δ in ppm of 6.46 (s, 1H), 6.33 (dd, J = 16.7, 1.8 Hz, 1H), 6.14 (s, 1H), 5.97 (s, 1H), 5.74 (d, J = 10.5 Hz, 2H), 4.75 (s, 1H), 4.34 (d, J = 31.1 Hz, 2H), 4.05 (d, J = 8.1 Hz, 2H), 3.97 – 3.80 (m, 2H), 3.77 (s, 2H), 3.20 – 3.01 (m, 4H), 2.86 (t, J = 7.4 Hz, 2H), 2.40 (t, J = 7.4 Hz, 2H), 2.28 – 2.06 (m, 6H), 1.55 – 1.45 (m, 2H), 1.45 – 1.34 (m, 4H), 1.31 – 1.17 (m, 3H), 0.90 – 0.81 (m, 2H). HRMS (m/z) for C30H41N4O6+ [M + H]+: calculated 553.3021, found 553.3039. ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (6-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)hexyl)carbamate (DUBL-X-BCN #5).
DUBL-X-BCN #5 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (6- aminohexyl)carbamate (6, 21.6 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #5 was obtained as white solid (15.5 mg, yield 27% for three steps).1H NMR (600 MHz, CDCl3) is shown in FIGs.6A and 6B with δ in ppm of 6.46 (s, 1H), 6.33 (dd, J = 16.7, 1.8 Hz, 1H), 6.15 (d, J = 3.3 Hz, 1H), 5.97 (d, J = 3.3 Hz, 1H), 5.80 – 5.68 (m, 1H), 5.63 (s, 1H), 4.70 (d, J = 6.2 Hz, 1H), 4.42 – 4.24 (m, 2H), 4.06 (d, J = 8.1 Hz, 2H), 3.97 – 3.72 (m, 4H), 3.19 – 2.99 (m, 4H), 2.86 (t, J = 7.4 Hz, 2H), 2.39 (t, J = 7.4 Hz, 2H), 2.26 – 2.08 (m, 6H), 1.56 – 1.45 (m, 2H), 1.45 – 1.33 (m, 4H), 1.31 – 1.15 (m, 5H), 0.86 (t, J = 10.0 Hz, 2H).13C NMR (101 MHz, CD3OD) δ 173.06, 165.84, 165.35, 157.92, 149.97, 144.80, 128.31, 126.77, 120.68, 106.66, 100.75, 98.12, 62.12, 46.54, 42.37, 40.18, 38.88, 33.92, 29.45, 28.91, 28.77 (2C), 26.15, 26.03, 23.70, 20.52 (3C), 19.98 (2C), 17.58. HRMS (m/z) for C31H43N4O6+ [M + H]+: calculated 567.3177, found 567.3153 as shown in FIG.6C.
((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (7-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)heptyl)carbamate (DUBL-X-BCN #6).
DUBL-X-BCN #6 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediates 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (7- aminoheptyl)carbamate (7, 23 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #6 was obtained as white solid (12.7 mg, yield 22% for three steps).1H NMR (600 MHz, CDCl3) is provided in FIGs.7A and 7B with δ in ppm of 6.44 (d, J = 18.9 Hz, 1H), 6.33 (dd, J = 16.7, 1.8 Hz, 1H), 6.15 (d, J = 3.3 Hz, 1H), 5.97 (d, J = 3.2 Hz, 1H), 5.74 (d, J = 10.5 Hz, 1H), 5.56 (s, 1H), 4.67 (s, 1H), 4.39 – 4.22 (m, 2H), 4.06 (d, J = 8.1 Hz, 2H), 3.95 – 3.80 (m, 2H), 3.77 (s, 2H), 3.17 – 3.01 (m, 4H), 2.86 (t, J = 7.4 Hz, 2H), 2.39 (t, J = 7.4 Hz, 2H), 2.25 – 2.09 (m, 6H), 1.56 – 1.45 (m, 2H), 1.44 – 1.32 (m, 4H), 1.29 – 1.11 (m, 7H), 0.86 (t, J = 9.9 Hz, 2H).13C NMR (151 MHz, CD3OD) δ 173.03, 165.82, 157.91, 149.96, 144.80, 128.31, 126.77, 120.41, 106.65, 100.74, 98.13, 82.97, 62.12, 46.23, 42.38, 40.28, 38.96, 33.93, 29.47, 28.91, 28.77 (2C), 28.63, 26.46, 26.33, 23.71, 20.54 (3C), 19.98 (2C), 17.59. HRMS (m/z) for C32H45N4O6 + [M + H]+: calculated 581.3334, found 581.3322 as shown in FIG.7C. ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (8-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)octyl)carbamate (DUBL-X-BCN #7).
DUBL-X-BCN #7 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (8- aminooctyl)carbamate (8, 24.4 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #7 was obtained as white solid (12.7 mg, yield 21% for three steps).1H NMR (600 MHz, CDCl3) is shown in FIGs.8A and 8B δ 6.46 (d, J = 14.7 Hz, 1H), 6.33 (dd, J = 16.7, 1.8 Hz, 1H), 6.15 (d, J = 3.3 Hz, 1H), 5.97 (d, J = 3.3 Hz, 1H), 5.79 – 5.69 (m, 1H), 5.54 (s, 1H), 4.64 (s, 1H), 4.40 – 4.26 (m, 2H), 4.06 (d, J = 8.1 Hz, 2H), 3.98 – 3.71 (m, 4H), 3.18 – 3.03 (m, 4H), 2.86 (t, J = 7.4 Hz, 2H), 2.39 (t, J = 7.4 Hz, 2H), 2.26 – 2.08 (m, 6H), 1.52 (dd, J = 17.7, 7.3 Hz, 2H), 1.45 – 1.31 (m, 4H), 1.31 – 1.12 (m, 9H), 0.91 – 0.79 (m, 2H).13C NMR (151 MHz, CD3OD) δ 173.02, 165.81, 165.26, 157.90, 149.95, 144.83, 128.32, 126.95, 126.77, 106.64, 100.69, 98.14, 62.11, 48.84, 46.23, 42.38, 40.32, 39.00, 33.93, 29.53, 28.97,
28.92 (2C), 28.77, 26.47, 26.36, 23.71, 20.55 (3C), 19.99 (2C), 17.60. HRMS (m/z) for C33H47N4O6+ [M + H]+: calculated 595.3490, found 595.3495 as shown in FIG.8C. ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (9-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2- yl)propanamido)nonyl)carbamate (DUBL-X-BCN #8).
DUBL-X-BCN #8 was synthesized following the standard procedure for preparing DUBL-X- BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (9- aminononyl)carbamate (9, 25.8 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #8 was obtained as white solid (13.8 mg, yield 23% for three steps).1H NMR (600 MHz, CDCl3) is shown in FIG.9 with δ in ppm of 6.45 (s, 1H), 6.33 (dd, J = 16.8, 1.8 Hz, 1H), 6.15 (d, J = 3.3 Hz, 1H), 5.97 (d, J = 3.2 Hz, 1H), 5.74 (d, J = 10.6 Hz, 1H), 5.52 (s, 1H), 4.64 (s, 1H), 4.41 – 4.27 (m, 2H), 4.06 (d, J = 8.1 Hz, 2H), 3.84 (d, J = 89.0 Hz, 4H), 3.15 – 3.00 (m, 4H), 2.86 (t, J = 7.5 Hz, 2H), 2.39 (t, J = 7.5 Hz, 2H), 2.25 – 2.08 (m, 6H), 1.57 – 1.44 (m, 2H), 1.44 – 1.31 (m, 3H), 1.30 – 1.13 (m, 12H), 0.86 (t, J = 9.9 Hz, 2H). HRMS (m/z) for C34H49N4O6 + [M + H]+: calculated 609.3647, found 609.3636. ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (10-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan- 2-yl)propanamido)decyl)carbamate (DUBL-X-BCN #9).
DUBL-X-BCN #9 was synthesized following the standard procedure for preparing DUBL-X- BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (10- aminodecyl)carbamate (10, 27.2 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #9 was obtained as white solid (8.4 mg, yield 14% for three steps).1H NMR (600 MHz, CDCl3) is shown in FIG. 10 with δ in ppm of 6.45 (s, 1H), 6.33 (dd, J = 16.8, 1.8 Hz, 1H), 6.15 (d, J = 3.2 Hz, 1H), 5.97 (d, J = 3.2 Hz, 1H), 5.74 (d, J = 10.4 Hz, 1H), 5.49 (s, 1H), 4.62 (s, 1H), 4.43 – 4.24 (m, 2H), 4.06 (d, J = 8.1 Hz, 2H), 3.96 – 3.72 (m, 4H), 3.19 – 3.03 (m, 4H), 2.86 (t, J = 7.5 Hz, 2H), 2.39 (t, J = 7.5 Hz, 2H), 2.27 – 2.08 (m, 6H), 1.55 – 1.45 (m, 2H), 1.44 – 1.31 (m, 3H), 1.29 – 1.12 (m, 14H), 0.86 (t, J = 10.0 Hz, 2H). HRMS (m/z) for C35H51N4O6+ [M + H]+: calculated 623.3803, found 623.3812.
((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (11-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan- 2-yl)propanamido)undecyl)carbamate (DUBL-X-BCN #10).
DUBL-X-BCN #10 was synthesized following the standard procedure for preparing DUBL- X-BCN #1 from intermediate 1 (29.2 mg, 0.01 mmol) and commercially available tert-butyl (11- aminoundecyl)carbamate (11, 28.6 mg, 0.01 mmol, 1.0 equiv). DUBL-X-BCN #10 was obtained as white solid (14.2 mg, yield 22% for three steps).1H NMR (600 MHz, CDCl3) is shown in FIG.11 with δ in ppm of 6.44 (d, J = 20.0 Hz, 1H), 6.33 (dd, J = 16.8, 1.8 Hz, 1H), 6.15 (d, J = 3.2 Hz, 1H), 5.97 (d, J = 3.3 Hz, 1H), 5.74 (d, J = 10.4 Hz, 1H), 5.49 (s, 1H), 4.61 (s, 1H), 4.40 – 4.23 (m, 2H), 4.06 (d, J = 8.1 Hz, 2H), 3.94 – 3.71 (m, 4H), 3.17 – 3.00 (m, 4H), 2.86 (t, J = 7.5 Hz, 2H), 2.39 (t, J = 7.5 Hz, 2H), 2.25 – 2.08 (m, 6H), 1.57 – 1.45 (m, 2H), 1.45 – 1.31 (m, 4H), 1.25 – 1.14 (m, 15H), 0.86 (t, J = 10.0 Hz, 2H). HRMS (m/z) for C36H53N4O6+ [M + H]+: calculated 637.3960, found 637.3974. Example 2:FOXO-DUBTACs FOXO3A is a member of FOXO family transcription factors, all of which have similar DNA binding motifs, also known as forkhead consensus binding sites (FIG.12A) and described in Brunet A. et al., Cell 96.6 (1999): 857-868, Tsai, K. et al. Nucleic Acids Res 35.20 (2007): 6984-6994, Pierrou S, et al. EMBO 13.20 (1994): 5002-5012 each of which are incorporated by reference in their entirety and particularly in relation to binding motifs and forkhead consensus binding sites. As a downstream mediator of several oncogenic pathways, such as Akt and I ^B, FOXO3A plays a tumor suppressive role. A single strand DNA oligonucleotide (5’-CTATGTAAACAACTTTGTTGTTTACATAG-3’, FOXO-ODN) that contains a FOXO consensus binding sequence (G/TAAAC/TA) and forms double strand hairpin structure via intra-dimerization (FIG.12A). Moreover, the biotin-modified FOXO- ODN (Biotin-FOXO-ODN, FIG.12A) was also synthesized to test the binding and specificity of FOXO-ODN to FOXO family members. As expected, Biotin-FOXO-ODN bound both FOXO3A and FOXO1 to a similar extent, and this binding could be blocked by an excess amount of free FOXO- ODN (FIGs.12B-E). The azide-modified FOXO-ODN (N3-FOXO-ODN) was conjugated to a DUB ligand via a SPAAC reaction (FIG.1A and 12A). A series of compounds were synthesized through attaching bicyclo[6.1.0]nonyne (BCN) to EN523, a covalent ligand of OTUB1, with various alkylene linkers (herein termed DUBL-X-BCN, X = (CH2)n, n = 2-11, FIGs.1A-B). In vitro SPAAC reactions between N3-FOXO-ODN and these DUBL-X-BCNs (#1-10) were performed using an excess
amount of DUBL-X-BCNs (10-fold) under physiological conditions (PBS, 37 ^C), to ensure a relatively high yield of TF-DUBTACs (FIG.12F). DUBL-X-BCNs #5-7, which contain 6-8 methylene groups as the linkers, are optimal for the SPAAC reaction to form FOXO-DUBTACs. However, the compounds with a longer linker (such as DUBL-X-BCNs #9 and #10) displayed drastically reduced efficiency in the click reaction to generate FOXO-DUBTACs, while shorter linkers (in DUBL-X-BCNs #1-4) mildly disrupted the intra-molecular dimerization, potentially reducing the binding of these FOXO-DUBTACs to FOXO3A. FOXO-DUBTAC #5 and #6 were used for further experiments in cells (FIG.12F-G). FOXO-DUBTAC #6 (FIG.12H) was effective in stabilizing FOXO3A in a concentration- dependent manner in HeLa cells (0.2-1 µg/mL, FIGs.12I-K). Furthermore, the protein level of FOXO1, a close family member of FOXO3A, was not significantly elevated by the treatment with FOXO-DUBTAC #6 even at 1 µg/mL (FIG.12I), although FOXO-ODN displayed comparable binding to both FOXO3A and FOXO1 (FIGs.12B-E), indicating that FOXO-DUBTAC #6 preferentially stabilizes FOXO3A. This selectivity might be due to that: (1) the potential difference in FOXO3A and FOXO1 protein structures outside of the DNA binding pocket may impact the formation of OTUB1-DUBTAC-FOXO1 ternary complex, and/or (2) the distance between OTUB1 and polyubiquitin chain(s) on FOXO1 may not be optimal for its removal. In contrast, the OTUB1 ligands have no effect on the stability of FOXO3A (FIG.12L). In keeping with stabilization results, FOXO-DUBTAC #6 was capable of inducing the interaction between OTUB1 and FOXO3A (FIG. 12M). The expression level of known downstream targets of FOXO3A, including p27 and BIM, both of which were elevated by FOXO-DUBTAC #6 in a dose-dependent manner (FIG.12N). To determine the efficiency and specificity of the FOXO-DUBTAC, we further performed the unbiased mass spectrum analysis to quantify the proteomics of FOXO-DUBTAC-treated and control cells. Notably, we found 106 elevated and 36 decreased proteins among the total of 6,333 detected proteins (FIG.12O). Although the level of FOXO3A itself was under the detective limit, an enrichment of MYC-related protein networks was identified, with a reduction in MYC expression and an increase of NDRG1, one of the most import MYC-downstream genes (FIGs.12O-P). MYC is a well-defined FOXO3A downstream protein, and FOXO3A suppresses MYC in transcriptional, translational and post-translational level, and FOXO3A also antagonizes the function of MYC through directly binding to the promoters of MYC-target genes. WDR5 recruits MYC onto the chromatin and is essential for the oncogenic function of MYC. MYC forms a complex with TOP1 on chromatin to favor transcriptions. Thus, the reduction of WRD5 and TOP1 might be an indirect effect through MYC. Moreover, MYC suppresses the transcription of a series of genes, including DNRG1, a cancer metastasis repressor, and FOXO-DUBTAC treatment led to an increase in NDRG1 level, which
could also be due to the reduction of MYC. These results indicated that FOXO-DUBTAC stabilizes FOXO3A, which suppress the transcription and function of MYC, unleashing the repression of NDRG1 by MYC (FIGs.12P-Q). In line with its effect on increasing the FOXO3A protein level and repressing MYC-driven transcription, FOXO-DUBTAC #6 suppressed tumorigenesis of HeLa cells in vitro in a colony formation assay (FIG.12R). Endogenous OTUB1 was further depleted using CRISPR/Cas9 in HeLa cells to determine the dependence of DUBTAC on OTUB1. FOXO-DUBTAC #6 increased the protein level of FOXO3A in OTUB1+/+ cells, but not in OTUB1-/- cells (FIGs.12S-T). Similarly, FOXO-DUBTAC #6 increased the transcription level of p27 and BIM only in OTUB1+/+ cells but not in OTUB1-/- cells (FIG.12U). Consistent with these findings, FOXO-DUBTAC #6 suppressed tumorigenesis of wild type HeLa cells but not OTUB1-KO cells (FIG.12V). These results indicated that the FOXO3A stabilization induced by FOXO-DUBTAC #6 is dependent on OTUB1. Example 3: p53-DUBTACs To explore whether other tumor suppressors with known DNA binding motifs can be targeted by this TF-DUBTAC approach, TF-DUBTACs that bind to p53, one of the most important tumor suppressors, were synthesized an analyzed. The tumor suppressive role of p53 largely depends on its DNA binding and subsequent transcription activation of downstream targets that suppress cell proliferation. Tumor-driven p53 mutations usually locate in its DNA binding domain. Wild type p53 protein binds a DNA consensus sequence consisting of two tandem repeats of 10 base-pair motif 5'- PuPuPuC(A/T)(T/A)GPyPyPy-3' (Pu stands for purine and Py for pyrimidine) separated by 0-13 base-pairs as shown in Wang, Y. et al. Mol Cell Biol 15.4 (1995): 2157-2165 and El-Deiry, et al. Nat Genet 1.1 (1999): 45-49, which are hereby incorporated by reference in their entirety and particularly in relation to binding sites of p53. A double strand p53-ODN, with a sense strand of 5’- AGACATGCCTAGACATGCCT-3’ was synthesized. An azide-modified oligomer N3-p53-ODN by attaching an azide group onto the 5’ end of the sense strand as well (FIG.13A). A streptavidin pulldown study showed p53 binding to the biotin-p53-ODN (FIG.13B), illustrating that the designed p53-ODN can bind p53. Furthermore, in vitro SPAAC reactions between N3-p53-ODN and a series of BCN-containing compounds DUBL-X-BCNs 1-9 resulted in the formation of p53-DUBTACs, as evident by a shift in native PAGE gel (FIG.13C). Three p53- DUBTACs (#5, #6 and #7, FIG.13D) for further evaluation of stabilizing and increasing the p53 protein level. p53-DUBTAC #6 and #7 significantly increased the p53 protein level in HeLa cells (over 3-fold of increase, 1 ^g/mL, 24 hours, FIGs.13E-H), while p53-DUBTAC #5 had a weaker effect. On the other hand, DUBL-X-BCNs #5-#7 or p53-ODN had minimal effects on the p53 protein level (FIGs.13E-F), thereby excluding the possibility of an indirect effect from ligands or
oligomer. Moreover, the OTUB1 ligands have no effect on the stability of p53 (FIG.12L). Furthermore, p53-DUBTAC #6 and #7 were capable of guiding the interaction between OTUB1 and p53 (FIG.13I). Through an unbiased MS-based proteomic analysis, 76 elevated and 33 decreased proteins were identified among the 6,333 quantified proteins in p53-DUBTAC-treated HeLa cell in comparison to control cells (FIG.13J). Similar to FOXO3A, p53 was also below the detection limit. Several ARUKA-related proteins were found among the proteins with reduced expression, while the level of several other p53 related proteins were increased, such as KAT5 (FIGs 13J-K). ARUKA has been determined as a p53 partner, and p53 suppresses AURKA function, by transcription regulation, direct inhibition through protein-protein interaction, as well as posttranslational regulation of ARUKA stability. KAT5, also known as Tip60, is another p53-binding protein that promotes p53 acetylation, and is essential for p53 signaling through direct association on the promoters of target genes. These results indicate that p53-DUBTAC has a preference to affect p53-related proteins, partially through suppressing AURKA. Furthermore, p53-DUBTAC were incapable of increasing the p53 protein levels in OTUB1-knockout cells (FIG.13L), indicating that the p53 stabilization induced by these p53-DUBTACs is OTUB1-dependent. Example 4: IRF-DUBTACs The generalizability of this TF-DUBTAC strategy was demonstrated by targeting IRF transcription factor family members, which have a conserved DNA binding motif (FIG.14). IRF transcription factor family members are responsible for the transcription of genes involving in inflammation, anti-bacteria and anti-virus protective pathways, such as interferons and interleukins. All IRF family members bind DNA with a consensus motif of AANNGAAA (N stands for any nucleotide, FIG.14A) as described in Panne, D. et al. Cell 129.6 (2007): 1111-1123 and Panne D. et al, EMBO 23.22 (2004): 4384-4393, which are hereby incorporated by reference in their entirety and particularly in relation to IRF binding motifs. A single strand oligomer with two GAAA core sequences (5’-GAAACTGAAACTTTTAGTTTCAGTTTC-3’, IRF-ODN) was synthesized as were corresponding biotin-IRF-ODN and N3-IRF-ODN compounds (FIG.14B). We found that biotin- IRF-ODN binds all IRF family members except IRF9 (FIGs.14C-D). In vitro click reactions between N3-IRF3-ODN and DUBL-X-BCNs #1-10 showed a similar trend as N3-FOXO-ODN, with DUBL-X-BCNs #5, 6 and 7 exhibiting the best click efficiency in forming IRF-DUBTACs (FIG. 14E). Subsequent cellular experiments validated that IRF-DUBTAC #7 was most effective in stabilizing IRF3, while other IRF-DUBTACs had some effect (FIG.14F-H) 4D, S4C, D). Taken together, these results suggest that TF-DUBTACs could be a general platform for stabilizing TFs with known DNA binding motifs.
In summary, proof-of-concept TF-DUBTACs that target and stabilize FOXO3A, p53 and IRF3 in cells were synthesized and characterized. In particular, FOXO-DUBTAC #6, p53-DUBTAC #6, #7 and IRF-DUBTAC #7 effectively stabilized FOXO3A, p53 and IRF3 tumor suppressor transcription factors, respectively, in an OTUB1-dependent manner. Collectively, these suggest that this TF-DUBTAC technology provides a universal strategy for targeting TFs with a tumor suppressor role, most of which are still “undruggable” due to the lack of small-molecule activators. EXPERIMENTAL METHODS General chemistry methods All commercially available solvents and reagents were used as received without further purification. Final compounds for biological evaluation were purified with preparative high- performance liquid chromatography (HPLC) on an Agilent Prep 1200 series with the UV detector set to 254/220 nm with solvent A (0.08% ammonium bicarbonate in water) and solvent B (acetonitrile) as eluents with a flow rate of 40 mL/min at rt. Purities of the final compounds were determined by high-performance liquid chromatography (HPLC) and were greater than 95%. HPLC conditions to evaluate purity were as follows: Agilent 1200 series system, 2.1 mm x 150 mm Zorbax 300SB-C185 ^m column 1-99% gradient of 0.1% trifluoroacetic acid in water, and 0.1% trifluoroacetic acid in acetonitrile at a flow rate of 0.4 mL/min; High-resolution mass spectra (HRMS) data was acquired on an Agilent G1969A API-TOF with an electrospray ionization (ESI) source. Nuclear magnetic resonance (NMR) spectra were recorded on either AVANCE NEO 600 MHz or 500 MHz spectrometers. Proton and carbon nuclear magnetic resonance spectra are reported in parts per million (ppm) on the ^ scale. Oligomer synthesis The single strand oligonucleotides containing the FOXO3A binding motif, namely FOXO- ODN, was synthesized based on the reported FOXO3A DNA binding consensus. The sequence of 29-mer FOXO-ODN is 5’- CTATGTAAACAACTTTGTTGTTTACATAG -3’, in which the core consensus is underlined. The single strand IRF-ODN was synthesized based on the reported IRF3 DNA binding consensus with two repeat of core consensus, and the sequence is 5’- GAAACTGAAACTTTTAGTTTCAGTTTC-3’. The double stain p53-ODN was synthesized based on the well-defined p53-DNA binding consensus with two repeats of 10 base-pair motif. The sense stand of p53-ODN is 5’-AGACATGCCTAGACATGCCT-3’, and the antisense stand is 5’- AGGCATGTCTAGGCATGTCT-3’. Moreover, the biotin modification of ODNs were synthesized by adding a biotin to the 5’ end of single strand ODN or the 5’ end of sense stand for double strand ODN using a spacer of NH2-(CH2)2-O-(CH2)2-OH. The azide modification of ODNs were
synthesized by incorporating an azide group on the same site as biotin through the 5’ amino modifier C6. All oligomers were synthesized by Integrated DNA Technologies, Inc. The unmodified oligomers were purified by standard desalting, while both biotin- and azide-modified oligomers were purified by HPLC. The single FOXO-ODN and IRF-ODN were annealed by heating to 95 ^C for 5 min, followed by cooled down to room temperature in 5 ^C per min. Similarly, the sense and antisense p53 oligomers were mixed at 1:1 ratio and annealed before use. In vitro copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction For incorporation of OTUB1 ligands onto the 5’ end of oligomers, BCN-modified OTUB1 ligands (DUBL-X-BCN) were incubated with either azide-modified oligomer at 37 ^C for 24 hours. The reaction mixtures were further purified by the Nucleotide Removal Kit (QIAGEN) to remove extra DUB ligands and salts in the reaction mixture, followed by annealing as described above. Native DNA polyacrylamide gel electrophoresis (PAGE) Oligomers and SPAAC reaction products were separated by 20% native polyacrylamide gel electrophoresis (PAGE) as described in Liu, J. et al., JACS 143.23 (2021): 8902-8910, which is hereby incorporated by reference in its entirety and particularly in relation to TF-PROTACs and separations of SPAAC products therefrom. Briefly, 0.5 ug oligomers were separated by nageive PAGE at 100 V for 1 hour, followed by incubated in 0.2% EtBr solution in 1 x Tris-boric acid-EDTA (TBE, pH 8.3) buffer that consisted of 89 mM Tris, 89 mM boric acid, 2 mM EDTA. The native gels contain 20% acrylamide and 2.5% glycerol, 0.075 ammonium persulfate (APS), 0.05% tetramethylethylenediamine (TEMED) in 0.5 x TBE buffer. Finally, the gels were imaged with UV illumination with the ChemiDoc™ Touch Imaging System (Bio-Rad). Cell culture and treatment Human embryonic kidney 293T (HEK293T) and HeLa cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 Units/mL of penicillin and 100 µg/mL streptomycin. For ectopic expression of transcription factors, Flag- FOXO3A, HA-FOXO1, Flag-p53, Flag-IRF2, 3, 4, 5, 6, 8 and 9 were transfected into HEK293T cells, and harvested for lysis 48 hours after transfection. For TF-DUBTACs treatment, HeLa cells in 6-well plate were transfected with individual TF-DUBTAC for 24 hours, followed by harvest for western blot analysis. For depletion of endogenous OTUB1, the sgRNA for OTUB1 (5’- TATCAACAGAAGATCAAGGT-3’) was synthesized and inserted into lenti-CRISPR-V2 construct as described in Sanjan, N. Nat Methods 11.8 (2014): 783-784, which is hereby incorporated by reference in its entirety. The sgOTUB1 lentivirus were generated in HEK293T cells as described in Liu, J. et al. Sci. Adv.6.8 (2020): eaay5154 and Liu, J. et al. JACS 143.19 (2021): 7380-7387, which
are hereby incorporated by reference in their entirety, for infection of HeLa cells overnight, followed by selection with puromycin for 72 h. Streptavidin-biotin pulldown assay Streptavidin pulldown assay for biotin-ODN with respective transcription factor was performed as described in Liu, J. et al., JACS 143.23 (2021): 8902-8910, which is hereby incorporated by reference in its entirety and particularly in relation to streptavidin pulldown assays. HEK293T cells that ectopically expressed transcription factors were lysed by EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Pierce) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations of the lysates were measured using the Bio-Rad protein assay reagent on a Beckman Coulter DU-800 spectrophotometer. The cell lysates (1 mg) were further incubated with biotin-ODN together with/without excess free ODN (3 or 10 folds) at 4 ^C for 3 hours, followed by adding 10 ^µL of Streptavidin agarose beads (Thermo Fisher) for another 1 hour. The beads were washed 4 times with NETN buffer (100 mM NaCl, 20 mM Tris-Cl, pH 8.0, 0.5 mM EDTA, 0.5 % NP-40), boiled in SDS loading buffer and further separated by 10% SDS-PAGE and blotted with individual antibody. Western blot assay Cells were lysed in EBC buffer supplemented with protease inhibitors cocktail and phosphatase inhibitors, and the protein concentrations were measured as described in Liu, J. et al., JACS 143.23 (2021): 8902-8910, which is hereby incorporated by reference in its entirety and particularly in relation to Western blot assays. The lysates (60 µg protein) were then resolved by 10% SDS-PAGE at 130 V for 80 min and immunoblotted with indicated antibodies at 4°C overnight, washed 4 times with Tris-buffered saline with 0.1% Tween-20 (TBST), incubated with secondary antibody for 1 hour at room temperature, and then washed 4 times with TBST buffer. Anti-FOXO3A (#12829, 1:1,000), FOXO1 (#2880, 1:1000), OTUB1 (#3783, 1:1000) and IRF3(#11904, 1:1000) antibodies were purchased from Cell Signaling Technologies. Anti-HA antibody (clone 16B12, #901513, 1:1,000) was purchased from Biolegend. Anti-Flag (F3165, 1:5,000), anti-vinculin antibody (V-4505, 1:50,000), peroxidase-conjugated anti-mouse secondary antibody (A-4416, 1:3,000) and peroxidase-conjugated anti-rabbit secondary antibody (A-4914, 1:3,000) were purchased from Sigma. Anti-p53 antibody (clone DO-1, sc-126, 1:1000) was purchased from Santa Cruz. All primary antibodies were diluted in 1% bovine serum albumin (BSA) in TBST buffer, and secondary antibodies were diluted in 5% non-fat milk in TBST buffer. All western blot assays were repeated at least twice. The protein quantification in immunoblot assay was performed with Quantity One software (Bio-Rad).
RT-qPCR Total RNAs were extracted using Qiagen RNeasy mini kit (Qiagen. #74106) and reversed transcripted into cDNA using iScript™ Reverse Transcription Supermix (Bio-Rad, # 1708841). RT- qPCR was performed with SYBR Select Master Mix (Thermo Fisher, #4472908) using primers as below: p27-forward: AACGTGCGAGTGTCTAACGG, p27-reverse: CCCTCTAGGGGTTTGTGATTCT, BIM-forward: TAAGTTCTGAGTGTGACCGAGA, BIM- reverse: GCTCTGTCTGTAGGGAGGTAGG, GAPDH-forward: TTGAGGTCAATGAAGGGGTC, GAPDH-reverse: GAAGGTGAAGGTCGGAGTCA. Proteomics sample preparation The cell pellets were resuspended in 8 M Urea, 50 mM Tris-HCl pH 8.0, reduced with dithiothreitol (5 mM final) for 30 min at room temperature, and alkylated with iodoacetamide (15 mM final) for 45 min in the dark at room temperature. Samples were diluted 4-fold with 25 mM Tris-HCl pH 8.0, 1 mM CaCl2 and digested with trypsin at 1:100 (w/w, trypsin:protein) ratio overnight at room temperature. There were two biological samples at each condition and total 8 samples. Peptides were cleaned by homemade C18 stage tips and the concentration was determined (Peptide assay, Thermo 23275).25 µg each was used for labeling with isobaric stable tandem mass tags (TMT10-126, 127C, 128N, 129N, 129C, 130N, 130C, 131, Thermo Fisher Scientific, San Jose, CA) following manufacture instruction. The mixture of labeled peptides was fractionated into 22 fractions on C18 stage tip with buffer 10 mM Trimethylammonium bicarbonate (TMAB), pH 8.5 containing 5 to 50% acetonitrile. Mass spectrometry analysis Dried peptides were dissolved in 0.1% formic acid, 2% acetonitrile.0.5 ^g of peptides of each fraction was analyzed on a Q-Exactive HF-X coupled with an Easy nanoLC 1200 (Thermo Fisher Scientific, San Jose, CA). Peptides were loaded on to a nanoEase MZ HSS T3 Column (100Å, 1.8 µm, 75 µm × 250 mm, Waters). Analytical separation of all peptides was achieved with 110-min gradient. A linear gradient of 5 to 10% buffer B over 5 min, 10% to 31% buffer B over 70 min, 31% to 75% buffer B over 15 min was executed at a 250 nL/min flow rate followed a ramp to 100%B in 1 min and 19-min wash with 100%B, where buffer A was aqueous 0.1% formic acid, and buffer B was 80% acetonitrile and 0.1% formic acid. MS experiments were also carried out in a data-dependent mode with full MS (externally calibrated to a resolution of 60,000 at m/z 200) followed by high energy collision-activated dissociation-MS/MS of the top 10 most intense ions with a resolution of 45,000 at m/z 200. High energy collision-activated dissociation-MS/MS was used to dissociate peptides at a normalized collision energy of 32 eV in the presence of nitrogen bath gas atoms. Dynamic exclusion was 45 seconds.
Raw proteomics data processing and analysis Peptide identification and quantification with TMT reporter ions were performed using the MaxQuant software version 1.6.10.43 (Max Planck Institute, Germany). Protein database searches were performed against the UniProt human protein sequence database (UP000005640). A false discovery rate (FDR) for both peptide-spectrum match (PSM) and protein assignment was set at 1%. Search parameters included up to two missed cleavages at Lys/Arg on the sequence, oxidation of methionine, and protein N-terminal acetylation as a dynamic modification. Carbamidomethylation of cysteine residues was considered as a static modification. Peptide identifications are reported by filtering of reverse and contaminant entries and assigning to their leading razor protein. Data processing and statistical analysis were performed on Perseus (Version 1.6.10.50). Protein quantitation was performed on biological replicates and a two-sample t-test statistics was used with a p-value of 5% to report statistically significant protein abundance fold-changes.
Claims
CLAIMS What is claimed is: 1. An agent comprising an oligonucleotide that hybridizes to a transcription factor of interest, wherein the oligonucleotide is directly or indirectly bound to a ligand of a deubiquitinase. 2. The agent of claim 1, wherein the agent has the structure of formula (IIA):
wherein ODN is the oligonucleotide; L1 is absent or absent; Ring A is a cycloalkyl, heterocyclyl, aryl, or heteroaryl (and conjugation to L1 may occur through a substitution group) which may have 1-10 points of optional substitution which may be independently substituted with, for example, alkyl, haloalkyl, heteroalkyl, halo; R8 is hydrogen, alkyl, or an electrophilic moiety; n may be 0, 1,
2, 3, 4, 5, or 6; R9 is alkyl, haloalkyl, heteroalkyl, halo; or a pharmaceutically acceptable salt thereof.
4. The agent of any one of claims 1-3, wherein the deubiquitinase is OTUB1.
5. The agent of claim 1, wherein the oligonucleotide is covalently bound to the ligand via a linker.
6. The agent of claim 5, wherein the linker comprises an azide/alkyne cycloaddition reaction product.
7. The agent of claim 5 or 6, wherein the linker comprises a strained click chemistry reaction product.
8. The agent of any one of claims 1-7, wherein the oligonucleotide has a double-strand hairpin structure formed via intra-dimerization.
9. The agent of any one of claims 1-8, wherein the oligonucleotide is a double-stranded DNA comprising a sense chain and an anti-sense chain.
10. The agent of any one of claims 1-9, wherein the transcription factor is a tumor suppressor.
11. The agent of claim 10, wherein the transcription factor is p53, FOXO3A, or IRF3.
12. A pharmaceutical composition comprising the agent of any one of claims 1-11 and one or more pharmaceutically acceptable salts, carriers, or diluents.
13. The pharmaceutical composition according to claim 12 wherein said compound is formulated in a liposome.
14. A method for reducing the proliferation or survival of a neoplastic cell or a virus, the method comprising contacting the cell with the agent according to any one of claims 1-11 to stabilize a transciption factor of interest or subunits thereof in the cell.
15. A method for the treatment or prophylaxis of a proliferative disease in a subject in need thereof comprising administering the agent according to any one of claims 1-11, or the pharmaceutical composition according to claim 12 or 13 to said subject.
16. A method of forming the agent according to any one of claims 1-11 comprising reacting a DNA oligomer protein comprising a transcription factor binding motif that binds to a transcription factor or subunit thereof; where said DNA oligomer protein is conjugated to an azide.
17. A compound having the structure of formula (IV): Y2–Y3–Y4–Y5–Y6–Y7–Y8–Y9–DUB (VI) wherein DUB is a deubiquitinase binding moiety; and
wherein Y2 is optionally substituted alkynyl, optionally substituted monocyclic or bicyclic cycloalkynyl, optionally substituted heteroalkynyl, or optionally substituted monocyclic or bicyclic heterocycloalkynyl; Y3-Y9 are independently selected from absent, an optionally substituted heteroarylene group optionally fused to an mono or bicyclic cycloalkyl, –C(O)–, –O–, –OC(O)–, –NRa–, –N(Ra)C(O)–, –(C(Ra)(Ra))1-12, –(C(Ra)(Ra)C(Ra)(Ra)O)1-12–, and –S–S–; and Ra is independently selected at each occurrence from hydrogen and optionally substitued alkyl.
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Title |
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DATABASE PUBCHEM COMPOUND 24 February 2022 (2022-02-24), ANONYMOUS : "NJH-2-057", XP093128303, retrieved from PUBCHEM Database accession no. 461446677 * |
LIU JING, CHEN HE, KANISKAN H. ÜMIT, XIE LING, CHEN XIAN, JIN JIAN, WEI WENYI: "TF-PROTACs Enable Targeted Degradation of Transcription Factors", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 143, no. 23, 16 June 2021 (2021-06-16), pages 8902 - 8910, XP093070826, ISSN: 0002-7863, DOI: 10.1021/jacs.1c03852 * |
LIU JING, YU XUFEN, CHEN HE, KANISKAN H. ÜMIT, XIE LING, CHEN XIAN, JIN JIAN, WEI WENYI: "TF-DUBTACs Stabilize Tumor Suppressor Transcription Factors", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 144, no. 28, 20 July 2022 (2022-07-20), pages 12934 - 12941, XP093128300, ISSN: 0002-7863, DOI: 10.1021/jacs.2c04824 * |
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