Discovery and Molecular Targets

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Discovery and Molecular Targets

• Susan Holbeck, Ph.D.
• National Cancer Institute
• Developmental Therapeutics Program
• Information Technology Branch
• holbecks_at_mail.nih.gov

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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
• RAND
• Compounds, extracts, cell lines, and tumors

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DTP Discovery Resources

• RAND - (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_ind
  ex.html

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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

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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

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60 Cell Screen Endpoints
Adriamycin
50 growth inhibition
Total growth inhibition
50 lethality
GI50
LC50
TGI
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Mean Graphs
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Comparing Mean Graphs
Adriamycin
Daunorubicin
Oxanthrazole
Teniposide
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COMPARE Finds Compounds With Similar 60 Cell
Activity

• Compound PCC
• Adriamycin 1.00
• Daunorubicin 0.950
• Deoxydoxorubicin 0.900
• Teniposide 0.878
• Oxanthrazole 0.864

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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
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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

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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

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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

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Molecular Target Measurements

• Enzyme activity measurements
• e.g., DT-diaphorase, P450s
• 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

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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/targetee
  r.html

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Mean Graphs
Compound
Molecular Target
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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.

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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
• Identification of cellular receptor for AAV5
  virus, Pasquale et al. 2003
• Sensitivity to a glycogen phosphylase inhibitor
  correlates with mRNA levels of GP, Schnier et al.
  2003

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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

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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

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

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Diethyldithiocarbamate (NSC 4857 Et2NCS2H)-An
Inhibitor of NF-kappaB Higher 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

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Interleukin 1 SignalingDaun JM, Fenton MJJ
Interferon Cytokine Res 2000 Oct20(10)843-55
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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

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Cells With Higher Levels of erbB2 are Sensitive
to anti-erbB2 Immunotoxin
erbB2
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COMPARE Results for 1,10-phenanthroline - An MMP
Inhibitor
A matrix metalloproteinase inhibitor
MMP9
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PDGF receptor is the receptor for
adeno-associated virus 5
PDGFRa
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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.

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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
• RAND
• Molecular Targeted Assays
• Compounds, extracts, cell lines, tumors
• Data freely available at
• http//dtp.nci.nih.gov

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Thanks to the Targeteers
Carmen Allegra John Lazo Robert Shoemaker Susan
Bates Dick Leopold Norman Sladek David
Botstein Millennium Barbara Sosnowski Raymond
Budde Anne Monks Patricia Steeg Jay
Chiorini Jeffrey Moscow Sherman Stinson Michael
Dean Novartis Sugen Tito Fojo Patrick
O'Connor Yoshikazu Sugimoto Albert
Fornace Lawrence Panasci Kenneth Tew Steve
Friend Marcus Peter George Vande Woude Stanton
Gerson Garth Powis David Waxman Jean Grem John
Reed John Weinstein David Hyndman Joachim
Schnier Paul Workman Michael Kelley Daominic
Scudiero Guido Kroemer Michael Seiden