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(12) United States Patent (10) Patent No.: US 7,083,918 B2 Althoff Et Al

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(12) United States Patent (10) Patent No.: US 7,083,918 B2 Althoff Et Al US007083918B2 (12) United States Patent (10) Patent No.: US 7,083,918 B2 Althoff et al. (45) Date of Patent: Aug. 1, 2006 (54) BACTERIAL SMALL-MOLECULE Abstracts, American Chemical Society, 219" ACS National THREE-HYBRD SYSTEM Meeting, San Francisco, CA, p. CHED 401 (Mar. 26-30. 2000). (75) Inventors: Eric A. Althoff, New York, NY (US); Griffith, E.C. et al. “Yeast Three-Hybrid System for Detect Virginia W. Cornish, New York, NY ing Ligand-Receptor Interactions.” Methods in Enzymology, (US) vol. 328, pp. 89-103 (2000). Dove et al., (1997) 'Activation of Prokaryotic Transcription (73) Assignee: The Trustees of Columbia University Through Arbitrary Protein-Protein Contacts' Nature in the City of New York, New York, 386:627-630 ; and. NY (US) Filman et al., (1982) “Crystal Structures of Escherichia coli and Lactobacillus casei Dihydrofolate Reductase Refined at (*) Notice: Subject to any disclaimer, the term of this 1.7. A Resolution”, The Journal of biological Chemistry patent is extended or adjusted under 35 257(22): 13663-13672. U.S.C. 154(b) by 330 days. Amara, J.F. et al. A Versatile Synthetic Dimerizer for the Regulation of Protein-Protein Interactions, Proc. Natl. Acad. (21) Appl. No.: 10/132,039 Sci. USA, 1997, 94, 10618-10623 (Exhibit 1). Austin DJ, et al. Proximity versus allostery: the role of (22) Filed: Apr. 24, 2002 regulated protein dimerization in biology. 1994. Chem Biol. 1(3): 131-6 (Exhibit 13). (65) Prior Publication Data Belshaw PJ, et al. Controlling protein association and US 2003/02O3471 A1 Oct. 30, 2003 Subcellular localization with a synthetic ligand that induces heterodimerization of proteins. 1996. Proc. Natl Acad Sci (51) Int. Cl. USA 93(10):4604-7 (Exhibit 14). CI2O I/68 (2006.01) Belshaw PJ, et al. Controlling programmed cell death with GOIN 33/53 (2006.01) a cyclophilin-cyclosporin-based chemical inducer of GOIN 33/554 (2006.01) dimerization. 1996. Chem. Biol. 3:731-738 (Exhibit 15). GOIN 33/567 (2006.01) Choi J, et al. Structure of the FKBP-12-Rapamycin complex GOIN 33/573 (2006.01) interacting with the binding domain of human FRAP. 1996. (52) U.S. Cl. ............................ 435/6: 435/7.1:435/7.2: Science 273 (5272):239-42 (Exhibit 16). 435/7.32:435/7.4 DeGrado WF, et al. Screening, selection and design: stand (58) Field of Classification Search .................... 435/6, ing at the crossroads in three dimensions. 1997. Current 435/7.1, 7.2, 7.32, 7.4 Opinion in Structural Biology 7:455-456 (Exhibit 17.) See application file for complete search history. (Continued) (56) References Cited Primary Examiner Mark Navarro U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm—John P. White, Esq.; Cooper 5, 194594 A 3, 1993 Khawli et al. & Dunham LLP 5,314,817 A 5, 1994 Schultz 5,468,614 A 11/1995 Fields et al. (57) ABSTRACT 5,736,343 A 4/1998 Landry 5,925,523 A 7, 1999 Dove et al. 5,928,868 A * 7/1999 Liu et al. ....................... 435/6 A transgenic bacterial cell comprising 6,030,785 A 2/2000 Katze et al. (a) a dimeric Small molecule which comprises a first 6,200,759 B1 3, 2001 Dove et al. moiety known to bind a first receptor domain FOREIGN PATENT DOCUMENTS covalently linked to a second moiety known to bind a EP O742O15 11, 1996 second receptor domain; WO 9731113 8, 1997 (b) nucleotide sequences which upon transcription encode WO 98.13353 4f1998 i) a first fusion protein comprising the first receptor WO 99.10508 3, 1999 domain, and WO 99.10510 3, 1999 ii) a second fusion protein comprising the second WO 9630540 10, 1999 receptor domain; and WO O153355 T 2001 WO WOO259272 2, 2003 (c) a reporter gene wherein expression of the reporter WO WOO36OO73 11/2003 gene is conditioned on the proximity of the first fusion WO WO2004O42345 9, 2004 protein to the second fusion protein. The cell is also adapted for use in a method for identifying a molecule OTHER PUBLICATIONS that binds to a known target in a bacterial cell from a Cornish, Virginia W. et al., “Dexamethasone-Methotrexate: pool of candidate molecules, and a method for identi An Efficient Chemical Inducer Of Protein Dimerization. In fying an unknown target receptor to which a molecule Vivo. Abstracts of Papers American Chemical Society, Vol. is capable of binding in a bacterial cell. Also described 219, No. 1-2, p. BIOL 124 (2000). are compounds and kits for carrying out the methods. Cornish, Virginia W. et al., “Synthesis of Dexamethasone Methotrexate CIDS With Variable Linkers.” Book of 42 Claims, 5 Drawing Sheets US 7,083,918 B2 Page 2 OTHER PUBLICATIONS Stockwell BR, et al. TGF-beta-signaliung with small mol Diver SR, et al. Single-step synthesis of cell-permeable ecule FKBP12 antagonists that bind myristoylated FKBP12 protein dimerizers that activate signal transduction and gene TGF-beta type 1 receptor fusion proteins. 1998. Chem Biol. expression. 1997. J. Am. Chem. Soc. 119: 5106-5109 5(7):385-95 (Exhibit 31). (Exhibit 18). Stockwell BR, et al. Probing the role of homomeric and Ho SN, et al. Dimeric ligands define a role for transcriptional heteromeric receptor interactions in TGF-beta signaling activation domains in reinitiation. 1996. Nature. using small molecule dimerizers. 1998. Curr Biol 8(13):761 382(6594):822-6 (Exhibit 19). 70 (Exhibit 32). Holsinger LJ, et al. Signal transduction in T lymphocytes Winkler T., et al. Confocal fluorescence coincidence analy using a conditional allele of Sos. 1995. Proc. Natl. Acad. Sci. sis: An approach to ultra high-throughput screening. 1998 USA 92:9810-9814 (Exhibit 20). Proc. Natl. Acad. Sci. USA 96:1375-1378 (Exhibit 33). Hung DT, et al. Understanding and controlling the cell cycle Yang, J., et al. Small-molecule control of insulin and PDGF with natural products. 1996. Chem. Biol. 3:623-639 (Exhibit receptor signaling and the role of membrane attachment. 21). 1997 Curr. Biol. 8:11-18 (Exhibit 34). Klemm JD, et al. Dimerization as a regulatory mechanism in Zlokarnik G. et al. Quatitation of tranmscription and clonal signal transduction. 1998. Annu. Rev. Immunol. 16:569-92 selection of single living cells with beta-lactamase as (Exhibit 22). reporter. 1998, Science 279(5347):84-8 (Exhibit 35). Liberles SD, et al. Inducilble gene expression and protein Search Report dated May 14, 2001 corresponding PCT translocation using nontoxic ligands identified by a mam International Application No. PCT/US01/02285 (Exhibit malian three-hybrid screen. 1997. Proc. Natl. Acad. Sci. 36). USA 94(15):7825–7830 (Exhibit 23). Lin, H., et al. Dexamethasone-Methotrexate: An Efficient Licitra EJ, et al. A three-hybrid system for detecting small Chemical Inducer of Protein Dimerization. In Vivo. 2000. ligand-protein receptor interactions. 1996. Proc. Natl. Acad. J.Am...Chem.Soc. 122:4247-4248 (Exhibit 37). Sci. USA 98: 12817-12821 (Exhibit 24). Kopytek, S.J., et al. Chemically Induced Dimerization of Pedersen H, et al. A method for directed evolution and Dihydrofolate Reductase by a Homobifunctional Dimer of functional cloning of enzymes. 1998. Proc. Natl. Acad. Sci. Methotrexate. 2000. Chem Biol 7:313-321 (Exhibit 38). USA95:10523-10528 (Exhibit 25). Firestine, S.M., et al. Using an AraC-Based three-hybrid Pruschy MN, et al. Mechanistic studies of a signaling system to detect biocatalysts in vivo. 2000. Nature pathway activated by the organic dimerizer FK1012. 1994. Biotechnology. 18, 544-547 (Exhibit 40). Cbem. Biol. 1:163–172 (Exhibit 26). Pelletier, J.N., et al. Oligomerization domain-directed reas Schreiber SL. Chemical genetics resulting from a passion sembly of active dihydrofolate reductase from rationally for synthetic organic chemistry. 1998. Bioorganic & Medici designed fragments. 1998. Proc. Natl. Acad.Sci. USA. 95, nal Chemistry 6:1127-1152 (Exhibit 27). 12141-12146 (Exhibit 41). Spencer DM, et al. Controlling signal transduction with Ladant, D., Karimova, G. Genetic systems for analyzing synthetic ligands. 1993. Science 262(5136):1019-1024 protein-protein interactions in bacteria. 2000. Res. (Exhibit 28). Microbiol. 151, 711-720 (Exhibit 42). Spencer DM, et al. Functional analysis of Fas signaling in Pollack et al., (2002) "Dimerizer-regulated gene expression' vivo using synthetic inducers of dimerization. 1996. Curr Current Opinion in Biotechnology 13:459-467. Biol. 6(7):839-47 (Exhibit 29). Spencer DM, et al. A general Strategy for producing condi U.S. Appl. No. 10/056,874, filed Jan. 24, 2002. tional alleles of Src-like tyrosine kinases. 1995. Proc. Natl. U.S. Appl. No. 10/084,388, filed Feb. 25, 2002. Acad. Sci. 92:9805-9809 (Exhibit 30). * cited by examiner U.S. Patent Aug. 1, 2006 Sheet 1 of 5 US 7,083,918 B2 Figure 1. /.../2.S.B. Alex). U.S. Patent Aug. 1, 2006 Sheet 2 of 5 US 7,083,918 B2 Figure 2 OMe O SS NH NH H H S - Ole 2Y-2. S-1 />-1s- su() . O - H NSN1 Mitx-SLF O O Oalso N -NH, N HNNN-S-N- sy1 NH C o CoatBu 3. O U.S. Patent Aug. 1, 2006 Sheet 3 of 5 US 7,083,918 B2 Figure 3 U.S. Patent Aug. 1, 2006 Sheet 4 of 5 US 7,083,918 B2 Figure 4 M Mtx-SLF M. Mitx-Slf No S.M. aA? Mix-SLF Gaw Mix-SLF O M Mix 10, MSF-Ofsts 350 250 200 SO OO 50 2 3 4 5 12345 1234s 1 2 3 4 5 1 2 3 4 5 U.S. Patent Aug. 1, 2006 Sheet S of 5 US 7,083,918 B2 Figure 5 3OO 2SO 2G 0 ci-GalAfonTD-Goil SO a ci-FKBP2 fond-DHFR a c-FKBPfein) CO O.
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