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. 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(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. 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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.