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WO 2013/137920 Al 19 September 2013 (19.09.2013) P O P C T

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WO 2013/137920 Al 19 September 2013 (19.09.2013) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2013/137920 Al 19 September 2013 (19.09.2013) P O P C T (51) International Patent Classification: PHILLIPS, Aaron, H. [US/US]; 1 DNA Way, Ms49, C07K 14/00 (2006.01) A61K 38/16 (2006.01) South San Francisco, CA 94080-4990 (US). G01N 33/68 (2006.01) A61P 35/00 (2006.01) (74) Agents: AMII, Lisa, A. et al; Morrison & Foerster LLP, (21) International Application Number: 755 Page Mill Road, Palo Alto, CA 94304-1018 (US). PCT/US20 12/043 177 (81) Designated States (unless otherwise indicated, for every (22) International Filing Date: kind of national protection available): AE, AG, AL, AM, 19 June 2012 (19.06.2012) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (25) Filing Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (26) Publication Language: English HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (30) Priority Data: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 61/612,228 16 March 2012 (16.03.2012) US OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, (71) Applicant (for all designated States except US): GEN- SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, ENTECH, INC. [US/US]; 1 DNA Way, Ms49, South San TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. Francisco, CA 94080-4990 (US). (84) Designated States (unless otherwise indicated, for every (72) Inventors; and kind of regional protection available): ARIPO (BW, GH, (75) Inventors/Applicants (for US only): CORN, Jacob, E. GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, [US/US]; 1 DNA Way, Ms49, South San Francisco, CA UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 94080-4990 (US). ZHANG, Yingnan [US/US]; 1 DNA TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Way, Ms49, South San Francisco, CA 94080-4990 (US). EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, [Continued on nextpage] (54) Title: ENGINEERED CONFORMATIONALLY-STABILIZED PROTEINS (57) Abstract: Provided herein are conformationally stabilized ubiquitin pro teins and methods for using the same to identify agents that bind to the stabil ized ubiquitin protein or that bind to a protein that interacts with or processes 5 the stabilized form of the ubiquitin protein. Also provided herein are methods for screening for conformationally stabilized proteins having increased bind ing affinity to a binding partner in comparison to the binding affinity of the wildtype form of the protein to the binding partner. FIG. 7A TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Published: ML, MR, NE, SN, TD, TG). ENGINEERED CONFORMATIONALLY-STABILIZED PROTEINS CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Patent Application No. 61/612,228 filed on March 16, 2012, the disclosure of which is incorporated by reference herein in its entirety. TECHNICAL FIELD [0002] Provided herein are methods for screening for and using conformationally stabilized forms of a conformationally dynamic protein, such as a conformationally stabilized ubiquitin protein, as well as compositions comprising the same. BACKGROUND [0003] Proteins are remarkably dynamic macromolecules, with conformational motions that play roles in diverse processes such as generating mechanical work, carrying out enzymatic reactions, and mediating signal transduction. Since the various states of a molecule may potentiate different functions, there is considerable interest in the ability to generate reagents that specifically recognize discrete conformational states. 1'2 [0004] Ubiquitin is a small regulatory protein that is found in almost all tissues (ubiquitously) in eukaryotic organisms. Protein ubiquitination mediates numerous cellular processes, such as cell cycle control, apoptosis, epigenetics, and transcriptional regulation.4 However, ubiquitin is perhaps best known for the role it plays in the labeling of proteins that are to be destroyed and recycled by a cell's proteolytic machinery. When a protein is covalently "tagged" by ubiquitin, the ubiquitin molecule directs the protein to the proteasome, which is a large multi-component protein complex in the cell that degrades and recycles proteins tagged for destruction. The ubiquitin protein itself consists of 76 amino acids with a molecular mass of about 8.5 kDa. Key features include its C-terminal tail and seven lysine residues.6 The amino acid sequence of ubiquitin is highly conserved among eukaryotic species, with human and yeast ubiquitin sharing approximately 96% sequence identity. In mammals, ubiquitin is encoded by four separate genes. The genes UBA52 and RPS27A encode a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively, whereas the UBB and UBC genes encode the polyubiquitin precursor proteins. [0005] Ubiquitination is an enzymatic, post-translational modification process in which the terminal glycine from the ubiquitin C-terminal di-glycine motif in activated ubiquitin forms an amide bond to the epsilon amine of a lysine residue in a modified protein. Ubiquitin is activated in a two-step reaction by an El ubiquitin-activating enzyme in a process requiring ATP as an energy source. This involves production of an ubiquitin-adenylate intermediate followed by the transference of ubiquitin to the El active site cysteine residue, resulting in a thioester linkage between the C-terminal carboxyl group of ubiquitin and the El cysteine sulfhydryl group. Next, ubiquitin is transferred from the Elenzyme to the active site cysteine of an E2 ubiquitin-conjugating enzyme via a trans(thio)esterification reaction. The final step of the ubiqutination enzyme cascade creates an isopeptide bond between a lysine in the target protein and the C-terminal glycine of ubiquitin. In general, this step requires the activity of one of the hundreds of known E3 ubiquitin-protein ligases (often termed simply "ubiquitin ligase").5 E3 enzymes function as the substrate recognition modules of the system and are capable of interaction with both E2 and the modified protein substrate. [0006] Following the addition of a single ubiquitin to a protein (monoubiquitination), further ubiquitin proteins can be added to the first ubiquitin molecule on one or more of its seven lysine residues, yielding a polyubiquitin chain. In addition, some substrates are modified by the addition of ubiquitin molecules to multiple lysine residues in a process termed multiubiquitination. The most studied polyubiquitin chains - lysine48-linked - target proteins for proteolysis in the proteosome. The condemned protein must be modified by at least four ubiquitin molecules in order for it to be recognized by the cell's proteolytic machinery. Ubiquitin molecules are cleaved off the protein immediately prior to destruction and are recycled for further use by enzymes belonging to the ubiquitin C-terminal hydrolase (UCH) family of deubiquitinases. [0007] Deubiquitinases (DUBs) are a class of specialized proteases that regulate ubiquitin-mediated signaling by disassembling ubiquitin chains or removing monoubiquitination from substrates. DUBs are also commonly referred to as deubiquitinating peptidases, deubiquitinating isopeptidases, deubiquitinases, ubiquitin proteases, ubiquitin hydrolyases, ubiquitin isopeptidases, or DUbs. The human genome encodes nearly 100 DUBs with specificity for ubiquitin in five gene families. DUBs may act as negative and positive regulators of the ubiquitin system. In addition to ubiquitin recycling, they are involved in the initial processing of ubiquitin precursors, in the proofreading of protein ubiquitination, and in disassembly of inhibitory ubiquitin chains. Additionally, DUBs such as the ubiquitin specific protease (USP) family of DUBs reverse the ubiquitination or ubiquitin-like modification of target proteins while DUBs such as members of the UCH family of DUBs are responsible for the regeneration of monoubiquitin from unanchored polyubiquitin, i.e., free polyubiquitin that is synthesized de novo by the conjugating machinery or that has been released from target proteins by other DUBs. [0008] The majority of DUBs have yet to be extensively characterized. One exception is USP7 (HAUSP), which has a well-established role in tumorigenesis. A critical function of USP7 is to regulate cell survival by deubiquitinating and stabilizing the oncoprotein Mdm2, thereby downregulating the p53 tumor suppressor.8'9 Since USP7 indirectly destabilizes p53 and regulates additional tumor suppressors, including FOX04 and PTEN, inhibition of USP7 is an attractive therapeutic strategy.8'9 Another example is USP14, which is thought to play a role in degradation of proteins involved in amyloidogenic neurodegeneration. 32 [0009] All patents, patent applications, publications, documents, nucleotide and protein sequence database accession numbers, the sequences to which they refer, and articles cited herein are all incorporated herein by reference in their entireties. SUMMARY [0010] The invention provided herein discloses, inter alia, compositions comprising conformationally stabilized ubiquitin proteins and methods for using the same to inhibit the action of one or more enzymes which bind to the stabilized form of these proteins as well as methods for identifying agents which bind to these stabilized proteins. Also provided herein are methods for screening for a conformationally stabilized form of a protein. [0011] The present application in one aspect provides a conformationally stabilized ubiquitin protein comprising one or more amino acid substitutions relative to a wild-type ubiquitin protein. In some embodiments, said one or more amino acid substitutions stabilize(s) the β1/β2 loop of the conformationally stabilized ubiquitin protein such that the β1/ β2 loop is confined to a region within about 1.6 A root mean square deviation of Protein Data Bank code 3NHE chain B.
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