USOO6617114B1 (12) United States Patent (10) Patent No.: US 6,617,114 B1 Fowlkes et al. (45) Date of Patent: Sep. 9, 2003 (54) IDENTIFICATION OF DRUG OTHER PUBLICATIONS COMPLEMENTARYLIBRARIES COMBINATORIAL Bottger, et al., Identification- Y - of novel mam2 binding peptides by phage display, Oncogene, vol. 13, pp. 2141-2147, 1996. (75) Inventors: Dana M. Fowlkes, Chapel Hill, NC Lu, et al., Expression of Thioredoxin Random Peptide (US); Brian K. Kay, Madison, WI Libraries On the Escherichia coli Cell Surface as Functional (US); Jeffrey A. Frelinger, Chapel Hill, Fusions to Flagellin. A System Designed for Exploring NC (US); Robin Parish Protein-Protein Interactions, Biotechnology, vol. 13, pp. Hyde-Deruyscher, Chapel Hill, NC 366-372, Apr. 1995. (US) Valadon, et al., Peptide Libraries Define the Fine Specificity of Antipolysaccharide Antibodies to Cryptococcus neofor mans, J. Mol. Biol., vol. 261, pp. 11-22, 1996. (73) Assignee: Karo Bio AB, Huddinge (SE) Giebel, L. et al., “Screening of Cyclic Peptide Phage Librar ies Identifies Ligands that Bind Streptavidin with High (*) Notice: Subject to any disclaimer, the term of this Affinities”, Biochemistry 34:15430–15435 (1995). patent is extended or adjusted under 35 Kay, Brian K., et al., “An M13 phage library displaying U.S.C. 154(b) by 0 days. random 38-amino-acid peptides as a Source of novel sequences with affinity to seleted targets', Gene 128:59-65 (21) Appl. No.: 09/069,827 (1993). Lam, Kit S. et al., “A new type of Synthetic peptide library for identifying ligand-binding activity' Nature 354:82-84 (22) Filed: Apr. 30, 1998 (1991). O O Smith, George P. et al., “A ribonuclease S-peptide antago Related U.S. Application Data nist discovered with a bacteriophage display library, Gene 63) Continuation-in-part of application No. 09/050,359, filed on 128:37-42 (1993). (63) Mar. 31, 1998, WSh is FENSE of application Healy, Judith M. et al., “Peptide ligands for Integrin avB3 No. PCT/US97/19638, filed on Oct. 31, 1997, which is a Selected from random phage display libraries”, Biochemis continuation-in-part of application No. 08/740,671, filed on try 34:3948–3955 (1995). Oct. 31, 1996, now abandoned. Koivunen, Erkki et al., “Isolation of a highly Specific ligand 7 for the C5B1 integrin from a phage display library”, J. Cell (51) Int. C. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - GO1N 33/53 Biology 124:373-380 (1994). Houghten, Richard A. et al., “Generation and use of Syn (52) U.S. Cl. ................................ 435/7.1; 435/6; 435/5; thetic peptide combinatorial libraries for basic research and 435/4, 435/DIG.2, 435/DIG 9, 435/DIG. 14; drug discovery”, Nature 354:84-86 (1991). 435/DIG. 27: 530/350, 530/324; 530/325; 530/330 (List continued on next page.) Primary Examiner T. D. Wessendorf (58) Field of Search ............................. 405/7.1, 5, 6, 4, (74) Attorney, Agent, Or Firm-ver P. Cooper 405/DIG. 2, DIG. 9, DIG. 14, DIG. 22; 530/324, 325, 350, 330 (57) ABSTRACT (56) References Cited The present invention is directed to the identification of compounds in a compound library which can mediate the U.S. PATENT DOCUMENTS biological activity of a target receptor protein, even when the 4461619 A 7/1984 Hendry et al ligands which mediate that activity through binding to that 4664,989 A 5/1987 Johnson receptor are not already known. 5,133,866 A 7/1992 Kauvar ....................... 210/635 5,223,409 A 6/1993 Ladner et al. The method of the invention includes the following three 5,288,514 A 2/1994 Ellman Steps: 5.449,754 A 9/1995 Nishioka (1) Screen a first combinatorial library for members 5,506,337 A 4/1996 Summerton et al. binding to the target protein (TP) and hence capable of 5. A E. R al. use as Surrogates for the unknown ligand in Steps (2) 5549.974. A s/1996 Holmes and (3). 5,556,762 A 9/1996 Pinilla et al. (2) Screen a second library, for compounds which inhibit 5,892,014 A * 4/1999 Coughlin et al. .......... 536/23.5 the binding of one or more Surrogates from Step (1) to 6.255,059 B1 * 7/2001 Klein et al. ................ 435/7.31 TP, and, optionally. FOREIGN PATENT DOCUMENTS (3) Determine whether the inhibitory compound mediates the biological activity of the said TP. WO 94.01.102 1/1994 WO 9519359 7/1995 The first library is composed of peptides, peptoids and/or W WO GR 3. nucleic acids, and the Second is not. WO WO 9729372 8/1997 WO WO974OO65 10/1997 10 Claims, 19 Drawing Sheets US 6,617,114 B1 Page 2 OTHER PUBLICATIONS Eichler, et al., Peptide, Peptidomimetic, and Organic Syn Marks, James D. et al., “By-passing Immunization Human thetic Combinatorial Libraries, Medical Research Reviews, Antibodies from V-gene Libraries Displayed on Phage”, J. vol. 15, No. 6, pp. 481–496, 1995. Mol. Biol. 222:581–597 (1991). Ellington, et al., In vitro Selection of RNA molecules that Colas, Pierre et al., “Genetic Selection of peptide aptamers bind specific ligands, Nature, vol. 346, pp. 818-822, Aug. that recognize and inhibit cyclin-dependent kinase 2', 30, 1990. Nature 380:548–550 (1996). JacobSSon, et al., Cloning of Ligand-Binding Domains of Smith, “Filamentous fusion phage: Novel expression vectors Bacterial Receptors by Phage Display, Biotechniques, vol. that display cloned antigens on the virion Surfaces”, Science 18, No. 5, pp. 878-885, 1995. 128:1315-1317 (1985). Schmitz, Rita et al., “Catalytic Specificity of Phosphotry Saggio, et al., Biotin binderS Selected from a random peptide oSine Kinases Blk, Lyn, c-Src and Syk as ASSessed by Phage library expressed on phage, Biochem J., vol. 293 (Pt3), pp. Display”, J. Mol. Biol. 260:664–677 (1996). 613-616, 1993, (Abstract). Hong and Boulanger, “Protein ligands of the human aden Sepetov, et al., Library of libraries. Approach to synthetic Ovirus type 2 outer capsid idnetified by biopanning of a combinatorial library design and Screening of "pharma phage-displayed peptide library on Separate domains of cophore” motifs, Proc. Natl. Acad. Sci. USA, Vol. 92, pp. wild-type and mutant penton capsomers”, EMBO.J. 14 No. 5426–5430, Jun. 1995. 19:4714-4727 (1995). Simon et al., “Peptoids: A moldular approach to drug dis Siani, et al., Chuckles. A Method for Representing and covery”, Proc. Natl. Acad. Sci. USA89:9367–9371 (1992). Searching Peptide and Peptoid Sequences on Both Mono Bunin et al., “The combinatorial Synthesis and chemical and mer and Atomic Levels, J. Chem. Inf. Comput. Sci., vol. 34, biological evaluation of a 1,4-benzodiazepine library', pp. 588-593, 1994. Proc. Natl. Acad. Sci. USA 91:4708–4712 (1994). Sparks, et al., Cloning of ligand targets. Systematic isolation DeWitt et al., “DiverSomers: An approach to nonpeptide, of SH3 domain-containing proteins, Nature Biotechnology, nonoligomeric chemical diversity', Proc. Natl. Acad. Sci. vol. 14, pp. 741-744, Jun. 1996. USA 90:6909-6913 (1993). Chen et al., “Analogous Organic Synthesis of Small-Com Bunin BA, et al., (1994) The combinatorial synthesis and pound Libraries: Validation of Combinatorial Chemistry in chemical and biological evaluation of a 1,4-benzodiazepine Small-Molecule Synthesis”, J. Am. Chem. Soc. library. Proc. Natl. Acad.Sci.U.S.A. 91: 4708–4712, 1994.* 116:2661-2662 (1994). Oliphant et al, Methods in Enzymology, 155, 1987, Bunin et al., “A General and Expedient Method for the 568-582. Solid-Phase Synthesis of 1,4-Benzodiazepine Derivatives”, J. Am. Chem. Society 114:10997–10998 (1992). * cited by examiner U.S. Patent Sep. 9, 2003 Sheet 1 of 19 US 6,617,114 B1 NHB poc C Sr. O -- OC Sn Mes R R 4. Fmoc HN COOH -a-resse O R4 p R2 R R2 ACETIC AC D iN DMF R4 R O N s R2 R5 x --- N R4 R1 R3 N SR2O N R4 U.S. Patent Sep. 9, 2003 Sheet 2 of 19 US 6,617,114 B1 A / G 2 pFLAG. ATS-BAP U.S. Patent Sep. 9, 2003 Sheet 3 of 19 US 6,617,114 B1 s O C) d O O s CO t CN O Ng33d U.S. Patent Sep. 9, 2003 Sheet 4 of 19 US 6,617,114 B1 s O c O o O d d O O V N O CO (O V- CN O 350 W. N3O3 d U.S. Patent Sep. 9, 2003 Sheet 5 of 19 US 6,617,114 B1 F. G. 5 ENRCHMENT OF PHAGE BINDING TO CMV UL44. 8 Q $o32 © ---- I z |- [TT= L. BRARY |---- F. G. 6 NDVDUAL PAGE ESAS FOR CMV UL44. 2 2 1 U.S. Patent Sep. 9, 2003 Sheet 7 of 19 US 6,617,114 B1 A/G 5 Streptavidin GST-U 44 GST GS-Src SH3 A / G 9 5 ' - O 1 V -A O.4 er -1 O O O.5 2 3 4. T ME (hours) U.S. Patent Sep. 9, 2003 Sheet 8 of 19 US 6,617,114 B1 O 4. O O. 1 O2 0.3 O.4 o5 BOKEY CONCENTRATION (M) O 1O 2O 3O 4O 5O (COMPETITOR), IM U.S. Patent Sep. 9, 2003 Sheet 9 of 19 US 6,617,114 B1 A/G 72 1.8 1.6 4. .2 O.O.O. OO O. 1 O 1 OO OF PHAGE -- 20 - H2 ------- 20 - A11 -- 25 D7 -x- 23 - F7 ------ 23 - E9 -o- 23 - DO ----- 19 - C 2O - D9 23 - F7 ------- 20 - C8 -- 9 - D4 U.S. Patent Sep. 9, 2003 Sheet 10 0f 19 US 6,617,114 B1 E O V G O 2 C O ? C A / G.