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Discovery and Molecular Targets

Discovery and Molecular Targets. Susan Holbeck, Ph.D. National Cancer Institute Developmental Therapeutics Program Information Technology Branch holbecks@mail.nih.gov. NCI-DTP Drug Discovery Resources. NCI panel of 60 human tumor cell lines 2 day 5-dose growth inhibition assay

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Discovery and Molecular Targets

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  1. Discovery and Molecular Targets Susan Holbeck, Ph.D. National Cancer Institute Developmental Therapeutics Program Information Technology Branch holbecks@mail.nih.gov

  2. NCI-DTP Drug Discovery Resources • NCI panel of 60 human tumor cell lines • 2 day 5-dose growth inhibition assay • Molecular targets • 3 cell line high throughput prescreen • In vivo testing • Hollow fiber assay • Xenografts • Molecular targeted assays • R*A*N*D • Compounds, extracts, cell lines, and tumors

  3. DTP Discovery Resources • R*A*N*D - (Rapid Access to NCI Discovery • Resources) - For academics and non-profits. Utilize for • analog or library synthesis, assay development assistance, • microarray technology, early formulation or PK/PD work • in order to select best candidate from multiple early • leads (Requires application and is peer-reviewed) • http://dtp.nci.nih.gov/docs/rand/rand_index.html

  4. Testing Compounds in the 60 Human Tumor Cell Line Screen • Free of charge • Pure compounds and biologicals • Compound structures must be provided • Compounds can be covered by a confidentiality agreement, if desired • All screening data kept confidential for 2 years • Available to academics and companies • On-line submission form at: http://dtp.nci.nih.gov/docs/misc/common_files/submit_compounds.html

  5. Why Test in the 60 Cell Line Screen? • Information on tumor cell types affected by the compound • Information to help choose a lead compound • Qualifying compounds sent for in vivo hollow fiber testing • COMPARE can help suggest possible mechanisms

  6. 60 Cell Screen Endpoints Adriamycin 50% growth inhibition Total growth inhibition 50% lethality GI50 LC50 TGI

  7. Mean Graphs

  8. Comparing Mean Graphs Adriamycin Daunorubicin Oxanthrazole Teniposide

  9. COMPARE Finds Compounds With Similar 60 Cell Activity CompoundPCC Adriamycin 1.00 Daunorubicin 0.950 Deoxydoxorubicin 0.900 Teniposide 0.878 Oxanthrazole 0.864

  10. Diverse Chemical StructuresSimilar Mechanism of ActionSimilar Behavior in 60 Cell Screen Correlation with Colchicine Vincristine sulfate 0.67 Taxol 0.57 Maytansine 0.74 Nocodazole 0.64 Leurosine N-oxide 0.44 Rhizoxin 0.67 Dolastatin 10 0.45 Halichondrin B 0.59

  11. COMPARE Can Help Develop Hypotheses • Start with a compound of known mechanism to find other structures to test for similar mechanism • Start with a compound of unknown mechanism - see if COMPARE can suggest a possible MOA

  12. Compound COMPARE Success Stories • Paullones - a new class of CDK inhibitors • nM PKC ligands that correlate with phorbol esters • Novel inhibitors of the thioredoxin reductase pathway • Numerous novel tubulin binders and Topo interacting agents

  13. DTP Molecular Targets Definition • Measurement of molecular entities in the NCI 60 human tumor cell line panel • Most measurements made by extramural researchers Goals • Correlate patterns of anticancer compound activity with molecular target measurements • Identify cell lines with desired characteristics • Improve understanding of the target

  14. Molecular Target Measurements • Enzyme activity measurements • e.g., DT-diaphorase, P450’s • Gene mutation status • e.g., Ras, p53 • Protein levels • e.g., Thymidylate synthase • mRNA levels • e.g., Microarray • Other • e.g., Phosphorylation status of a protein

  15. Molecular Target Data in the 60 Cell Panel • Data is available from the DTP Web site http://dtp.nci.nih.gov/mtargets/mt_index.html • Searchable by gene name or by text string • Browse the data • Download the datasets • Measure your favorite target • 60 cell line materials/data analysis provided free of charge • data to be made public on DTP web site • apply at:http://dtp.nci.nih.gov/mtargets/targeteer.html

  16. Mean Graphs Compound Molecular Target

  17. COMPARECompounds vs. Molecular Targets • Positive correlations: Cell lines with high levels of the Molecular Target are more sensitive to the compound. • Negative correlations: Cell lines with low levels of the Molecular Target are more sensitive to the compound.

  18. COMPARE Drug-Target Success Stories • CDK4-NSC 680434 (IC50 3.1 microM) Kubo et al. 1999 • EGFR and ErbB2 pathway inhibitors - 14 compounds Wosikowski et al. 1997 • RNR inhibitors more toxic to cells with high BID Krajewska et al. 2002

  19. COMPARE Can Help Find Molecular Targets Important in Drug Sensitivity • Transport of compound • Metabolism of compound • Pathways that may be affected by compound • Possible Molecular Target of compound

  20. COMPARE Can Help Find Molecular Targets Important in Drug Sensitivity • Transport of compound • Metabolism of compound • Pathways that may be affected by compound • Possible Molecular Target of compound

  21. COMPARE Results for DT-Diaphorase Activity(a quinone-metabolizing enzyme)

  22. COMPARE Can Help Find Molecular Targets Important in Drug Sensitivity • Transport of compound • Metabolism of compound • Pathways that may be affected by compound • Possible Molecular Target of compound

  23. Diethyldithiocarbamate (NSC 4857 Et2NCS2H)-An Inhibitor of NF-kappaBHigher levels of these genes correlate with sensitivity 1 0.51 nucleoporin-like protein 1 7 0.41 TRAF family member-associated NFKB activator 11 0.4 acid sphingomyelinase 16 0.39 phospholipase C, gamma 2 38 0.35 interleukin 1 receptor, type II

  24. Interleukin 1 SignalingDaun JM, Fenton MJJ Interferon Cytokine Res 2000 Oct;20(10):843-55

  25. COMPARE Can Help Find Molecular Targets Important in Drug Sensitivity • Transport of compound • Metabolism of compound • Pathways that may be affected by compound • Possible Molecular Target of compound

  26. Cells With Higher Levels of erbB2 are Sensitive to anti-erbB2 Immunotoxin erbB2

  27. COMPARE Results for 1,10-phenanthroline - An MMP Inhibitor A matrix metalloproteinase inhibitor MMP9

  28. COMPARE: Not the Final Answer • COMPARE can provide a starting point for experiments, but is NOT the whole story. • Compounds and Targets with “flat patterns” can give strong correlations. Take a look at the data. • COMPARE at GI50, TGI and LC50 can give different answers. • Correlations with an mRNA target may be misleading if the gene product is post-transcriptionally regulated.

  29. CONCLUSIONS • 60 cell line data for ~40,000 compounds and ~10,000 Molecular Targets • Target data can be correlated with sensitivity to compounds • Other development resources • R*A*N*D • Molecular Targeted Assays • Compounds, extracts, cell lines, tumors • Data freely available at: • http://dtp.nci.nih.gov

  30. Thanks to the Targeteers: Carmen Allegra Patrick O'Connor Susan Bates Parke-Davis David Botstein Garth Powis Pat Brown John Reed Raymond Budde Bob Shoemaker Tito Fojo Norman Sladek Albert Fornace Barbara Sosnowski Steve Friend Patricia Steeg Stanton Gerson Sherman Stinson Jean Grem Sugen, Inc. Michael Kelley Kenneth Tew John Lazo George Vande Woude Millenium Pharmaceuticals David Waxman Anne Monks John Weinstein Jeffrey Moscow Paul Workman Len Neckers

  31. Our next speaker is: Dr. George Johnson Grants & Contracts Operations Branch Developmental Therapeutics Program

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