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(12) United States Patent (10) Patent No.: US 6,333,155 B1 L0ckhart Et Al

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(12) United States Patent (10) Patent No.: US 6,333,155 B1 L0ckhart Et Al USOO6333155B1 (12) United States Patent (10) Patent No.: US 6,333,155 B1 L0ckhart et al. (45) Date of Patent: Dec. 25, 2001 (54) EXPLOITING GENOMICS IN THE SEARCH Sedlak et al. “Gene Chip Technology Ready to Impact FOR NEW DRUGS Diagnostic Markets' Genetic Engineering News, Dec. 1997, p. 1. (75) Inventors: David J. Lockhart, Del Mar; Lisa Lockhart et al. “Expression monitoring by hybridization to Wodicka, San Diego; Ming Hsiu Ho, high-density oligonucleotide arrays' Nature Biotechnology, San Jose, all of CA (US) vol. 14, Dec. 1996, pp. 1674–1680. Schow et al. “Synthesis and Activity of 2,6,9–Trisubstituted (73) Assignee: Affymetrix, Inc., Santa Clara, CA (US) Purines’ Bioorganic & Medicinal Chemistry Letters, vol. 7, No. 21, pp. 2697–2702 1997. (*) Notice: Subject to any disclaimer, the term of this Konig et al. “The novel cyclin-dependent kinase inhibitor patent is extended or adjusted under 35 flavopiridol downregulates Bcl-2 and induces growth arrest U.S.C. 154(b) by 0 days. and apoptosis in chronic B-cell leukemia lines' Blood vol. 90, No. 11, 1997 pp. 4307-4312. (21) Appl. No.: 09/215,207 De Azevedo et al. “Inhibition of cyclin-dependnet kinase by (22) Filed: Dec. 18, 1998 purine analogues Crystal Structure of human cdk2 com plexed with roscovitine” European Journal of biochemistry, Related U.S. Application Data vol. 243, No. 1/02, 1997 pp. 518–526. (60) Provisional application No. 60/068,289, filed on Dec. 19, Norman et al. “A Structure-based library approach to kinase 1997. inhibitors' The Journal of the American Chemical Society, (51) Int. Cl. .............................. C12Q 1/68; C12P 19/34; vol. 118, 1996 pp. 7430–7431. C07H 21/02; CO7H 21/04 Brooks et al. “CVT 313, a specific and potent inhibitor of (52) U.S. Cl. ............................ 435/6; 435/912; 536/23.1; CDK2 that prevents neointimal proliferation” Journal of 536/24.3; 536/24.31; 536/24.33; 935/6; Biological Chemistry, vol. 272, No. 46, Nov. 14, 1997 pp. 935/18 292O7. 2921.1. (58) Field of Search ....................... 435/6, 91.2, 536/23.1 Gray "Exploiting chemical libraries, Structure, and genom ics in the search for kinase inhibitors' Science, vol. 281, (56) References Cited 1998 pp. 533–538. U.S. PATENT DOCUMENTS * cited by examiner 5,700,637 A 12/1997 Southern ........................... 435/6 Primary Examiner Jeffrey Fredman FOREIGN PATENT DOCUMENTS ASSistant Examiner Arun Kr. Chakrabarti WO 89/10977 11/1989 (74) Attorney, Agent, or Firm-Banner & Witcoff WO 97/10365 3/1997 (57) ABSTRACT WO 97/27317 7/1997 WO 97/29091 8/1997 The cellular effects of potentially therapeutic compounds are WO 98/O5335 2/1998 characterized in mammalian cells and yeast. In the latter OTHER PUBLICATIONS case the effects can be characterized on a genome-wide Scale by monitoring changes in messenger RNA levels in treated Ecker et al., “Estimation of the chemosensitizing activity of cells with high-density oligonucleotide probe arrayS. modulators of multi-drug resistance via combined Simulta neous analysis of Sigmoidal-dose response curves', J. Phar 14 Claims, 6 Drawing Sheets macy and Pharmacology, vol. 49, pp. 305-309, Jan. 1997.* Stephen Fodor “Massively parallel genomics' Science vol. 277, Jul 18, 1997 pp. 393-394. (3 of 6 Drawing Sheet(s) Filed in Color) U.S. Patent Dec. 25, 2001 Sheet 2 of 6 US 6,333,155 B1 A/G 24 U.S. Patent Dec. 25, 2001 Sheet 3 of 6 US 6,333,155 B1 U.S. Patent Dec. 25, 2001 Sheet 4 of 6 US 6,333,155 B1 f 30 ; gorgot w r : 8 : ya- ::: Y s 8:33:8: 88: '. &e:8x. 3:38 .: --- c-8-8-8 :- :- ---------------------- ; : 3 S & 3 U.S. Patent Dec. 25, 2001 Sheet S of 6 US 6,333,155 B1 Observed for flavopirido bers (see caption U.S. Patent Dec. 25, 2001 Sheet 6 of 6 US 6,333,155 B1 *8 was assssssssssssss 888cripts at ci::ge excisivey for 52 x saw spiricii : xic:8: 8: al 383 3.33333333338.3:: 3:38 . 3838;. ; 3:33.33.; 8 3333333 *:::::::::8xig 38: 8388x8 is:x: x8:::::::: US 6,333,155 B1 1 2 EXPLOITING GENOMICS IN THE SEARCH chain contacts are indicated by lines connecting the respec FOR NEW DRUGS tive residue box and interactions to main chain atoms are shown as lines to the Specific main chain atoms. Van der This Application benefit of provisional No. 60/068,289 Waals contacts are indicated by thin dotted lines, and H filed Dec. 19, 1997. bonds by dashed lines. For H bonds the distances between the nonhydrogen atom are indicated in angstroms. W, water. BACKGROUND OF THE INVENTION FIG. 3. Representative transcripts observed to change more than twofold for triplicate hybridizations for each of Many biological functions are accomplished by altering two independent experiments (except for cdc28-4, which the expression of various genes through transcriptional (e.g. represents triplicate hybridizations of RNA from a single through control of initiation, provision of RNA precursors, experiment). (FIG. 3A) Names of the genes whose mRNA RNA processing, etc.) and/or translational control. For levels change in common to 52 and flavopiridol (none of example, fundamental biological processes Such as cell these transcripts changed significantly in the 52Me profile): cycle, cell differentiation and cell death, are often charac terized by the variations in the expression levels of groups YBR214w (similar to Schizosaccharomyces pombe protein 15 moc1 involved in meiosis and mitosis); YGR108W (CLB1, of genes. G-M phase cyclin); YBL003c (HTA2, histone); YBL002w Changes in gene expression also are associated with (HTB2, histone); YNL327W(EGT2, involved in timing of pathogenesis. For example, the lack of Sufficient expression cell separation); YLR286C (CTS1, endochitinase); of functional tumor Suppressor genes and/or the over expres YJL157C* (FAR1, inhibitor of Cdc28p/Cln1,2p Sion of oncogene/protoncogenes could lead to tumorgenesis complexes); YPR119W* (CLB2, G-M phase cyclin); (Marshall, Cell, 64; 313–326 (1991); Weinberg, Science, YHR096C (HXT5, homologous to hexose transporters); 254: 1138-1146 (1991), incorporated herein by reference for YAL061W (unknown, similar to alcohol or sorbitol all purposes). Thus, changes in the expression levels of dehydrogenase); YKR097W (PCK1, phosphoenolpyruvate particular genes (e.g. oncogenes or tumor Suppressors) serve carboxykinase); Y GR043 C (similar to Tallp, a as Signposts for the presence and progression of various 25 transaldolase); YMR105C (PGM2, phosphoglucomutase); diseases. YBR169c (SSE2, heat shock protein of HSP70 family); Often drugs are Screened and prescreened for the ability YBR072W (HSP26, heat shock protein induced by to interact with a major target without regard to other effects osmostress); YLL026W (HSP104); YCR021c (HSP30); the drugs have on cells. Often Such other effects cause YPL240C (HSP82, chaperonin homologous to Escherichia toxicity in the whole animal, which prevent the development coli HtpG); YDR171W (HSP42, involved in restoration of and use of the potential drug. Therefore, there is a need in cytoskeleton during mild stress); YOR328W (PDR10, mem the art to develop a Systematic approach to test and develop ber of the ATP binding cassette Superfamily); YDR406w new drugs for their effects on cellular metabolism without (PDR15); YDL223c (unknown); YER 150w (similar to relying on groSS morphologic and phenotypic effects. Sedlp an abundant cell surface glycoprotein); YGR032W 35 (GSC2, component of -1,3-glucan synthase); YGL179C* SUMMARY OF THE INVENTION (Serine-threonine kinase similar to Elm1p and Kin82p); This invention provides methods and compositions for YLR178C (TFS1, Cdc25-dependent nutrient and ammonia Studying the complex relationships among drugs and genes. response cell cycle regulator); YNR009W (unknown); In Some of its Specific applications, this invention provides YFLO31W (HAC1, basic leucine zipper protein, activates methods and compositions for detecting alternate targets for 40 unfolded-protein response pathway); and YHR 143W drug Screening and development by monitoring the expres (unknown). (FIG. 3B) Transcript changes that may result from Pho85p kinase inhibition observed in either the 52 or Sion of genes affected by a drug or mutation. flavopiridol profiles: YOLOO1W (PHO80, a cyclin that asso BRIEF DESCRIPTION OF THE DRAWINGS ciates with Pho85p); YGR233C (PHO81, inhibitory protein 45 that associates with Pho80p or Pho85p);YFLO14W (HSP12, The file of this patent contains at least one drawing heat shock protein); YHR071W (PCL5, cyclinlike and asso executed in color. Copies of this patent with color drawings ciates with Pho85p); YGR088W (CTT1, cytosolic catalase are provided to the Patent and Trademark Office with T); YBR093c (PHO5, secreted acid phosphatase); YLL039c payment of the necessary fee. (UBI4, ubiquitin); YCL009c (PHO84, phosphate FIG. 1. (FIG. 1A) Scheme for the combinatorial synthesis 50 transporter); YML116W(PHO8, vacuolar alkaline of 2,6,9-trisubstituted purines from a 2-, 6-, or 9-linked phosphatase); YBR296c (homologous to a phosphate purine Scaffold with amination and alkylation chemistries. repressible permease). (FIG. 3C) Transcripts that change for Chemical structures of CDK inhibitors (FIG. 1B) flavopiri cdc28-4, cdc28-4 and 52, cdc28-4 and flavopiridol, and 52: dol (FIG. 1C) olomoucine and roscovitine, and (FIG. 1D) YBR 147W (unknown, has 7 potential transmembrane purvalanol A and B and (FIG. 1E) Compound 40 and (FIG. 55 domains); YOL155C (unknown, similar to glucan 1,4- 1F) Compounds 100 and 101. glucosidase); YJR 127C (ZMS1, similar to Arp1p, an FIG. 2. (FIG. 2A) Purvalanol B bound to CDK2 (black N-acetyltransferase); YKL109W (HAP4, transcriptional Sticks, principal conformation only) is compared with bound activator protein involved in activation of CCAAT box (1) olomoucine (white Sticks) and bound roScovitine (orange containing genes); YBL015w (ACH1, acetyl-coenyme A Sticks), (2) bound flavopiridol (green Sticks), and (3) bound 60 hydrolase); YPR160W (GPH1, glycogen phosphorylase); ATP (yellow sticks). The comparisons are based on Super YAL039C (CYC3, cytochrome cheme lyase); YML116W position of the C atoms of CDK2.
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