Investigating Compound Mechanism of Action

Transcription

1 Investigating Compound Mechanism of Action Using Genetic Interaction Screens Lisa Marshall, Priya Mitty, Michael Ollmann, and Paul Kassner Michael Ollmann, Ph.D. Principal Scientist, Therapeutic Discovery Discovery On Target Boston, MA, October 3-4, 2012

2 The Big Leap: Linking Compound Mechanism of Action with Phenotypes Lee, JMedChem, 2012 Biochemical or ce ell-based assay reado out compound conc.

3 Linking Compound Mechanism of Action with Phenotype correlating biochemical and cell-based assay phenotypes Acetyl CoA Carb boxylase Enzyme Activity Ass say IC50 (um) Angiogenesis Assay IC50 (um) Lee, JMedChem, 2012

4 Linking Compound Mechanism of Action with Phenotype selectivity assays Davis, Nature Biotech, 2011

5 Linking Compound Mechanism of Action with Phenotype genotype-phenotype correlation across cell line panels Genomic alterations in ALK correlate with sensitivity to TAE-684 across 602 cell lines McDermott, Cancer Research, 2008

6 Linking Compound Mechanism of Action with Phenotype resistance mutations Wacker, NatChemBiol, Vol.8, 2012

7 Linking Compound Mechanism of Action with Phenotype drug affinity-based methods Lomenick, ACS Chem Biol, Vol.6, 2011

8 Linking Compound Mechanism of Action with Phenotype chemoinformatics approaches: ligand similarity Adams, PLOS Comp Biol, Vol.5, 2009

9 Linking Compound Mechanism of Action with Phenotype genetic interaction screens Increasing sensitivity to methotrexate DFR1 = dihydrofolate reductase FOL1 & FOL2 = genes involved in folatesynthesis YBT1 = ABC-family transporter Giaver, PNAS, Vol.101, 2004

10 Compound+siRNA Genetic Interaction Screens in Mammalian Cells case study: MDM2:p53 inhibitor MDM2:p53 binding site MDM2 inhibitor AM-8553 Rew, J Med Chem, Vol.55, 2012 Bernard, J Med Chem, Vol.55, 2012 Small Molecule Inhibitor Of MDM2-p53 Interaction p14 ARF MDM2 p53 Transcriptional Activation Cell cycle arrest and apoptosis

11 Anti-proliferative Effects of MDM2 inhibitor AM-8553 are p53-dependent Rew, J Med Chem, 2012

12 Compound + sirna Screening Format A B C D E F G H I J K L M N O P sirnalibrary Plate A B C D E F G H I J K L M N O P A B C D E F G H I J K L M N O P A B C D E F G H I J K L M N O P A B C D E F G H I J K L M N O P A B C D E F G H I J K L M N O P A B C D E F G H I J K L M N O P MDM2 inhibitor treated DMSO treated Screened portion of Amgen whole-genome sirna library 14,000 sirna for 1700 genes arrayed in 384-well format, with control sirna in outer columns Two p53 wild type cell lines screened SJSA1 MDM2 amplified, p53 WT CAL51 p53 WT Cells treated with DMSO, 0.8, or 8uM of MDM2 inhibitor Compound added 24 hours post 10-30nM sirna transfection, followed by 72hr incubation CellTiter-Glo viability assay Data analysis Visualization of paired screening data by scatterplot rank-based p-value calculation for each gene

13 Genetic Interaction Screen with MDM2 Inhibitor in SJSA-1 cells interactions with MDM2 and p53 reveal mechanism of action Cell Viability with MDM2 inhibi Normalized to neutral co itor (8uM) treatment ontrol sirna Cell Viability with DMSO treatment Normalized to neutral control sirna

14 Genetic Interaction Screen with MDM2 Inhibitor in SJSA-1 cells identification of enhancers of sensitivity Cell Viability with MDM2 inhibi Normalized to neutral co itor (8uM) treatment ontrol sirna Cell Viability with DMSO treatment Normalized to neutral control sirna MDM2i sensitizer gene sirna

15 Genetic Interaction Screen with MDM2 Inhibitor in SJSA-1 cells dose-dependent phenotypes Viability with MDM2 ibitor treatment (RLU) 0.2uM MDM2i 1.6uM MDM2i Cell inhi Cell Viability with DMSO treatment (RLU) 5.5uM MDM2i 50uM MDM2i

16 Genetic Interaction Screen with MDM2 Inhibitor in CAL-51 cells RNAi of p53, but not MDM2, alters sensitivity to MDM2 inhibitor in CAL-51 (MDM2 WT) cells Cell Viability with MDM2 inhib bitor treatment (RLU) Cell Viability with DMSO treatment (RLU)

17 Cell Line Selective Effects of MDM2 RNAi differential sensitivity of SJSA-1 (MDM2 amplified) and CAL-51 (MDM2 WT) cells to RNAi of MDM2 SJSA-1 Cells MDM2 Copy # = 68 CAL-51 Cells Wild Type MDM2 MDM2 p value =.01 MDM2 p value =.44 Non-additive interactions between MDM2 inhibitor and MDM2 sirna phenotypes likely account for genetic interaction of MDM2 sirna and MDM2 inhibitor in SJSA-1 cells

18 Linking Compound MOA with Phenotype Using Genetic Interaction Screens Test Agent Drug Mechanism of Action Cell-based Assay Phenotype Rew, J Med Chem, 2012 compound conc. +Inhi ibitor +DMSO

19 Linking Compound MOA with Phenotype Using Genetic Interaction Screens Test Agent Drug Mechanism of Action Cell-based Assay Phenotype Rew, J Med Chem, 2012 compound conc. ibitor +Inhi High-content screening assays +DMSO

20 Questions?

21 Investigating Compound Mechanism of Action Using Genetic Interaction Screens Lisa Marshall, Priya Mitty, Michael Ollmann, and Paul Kassner Abstract Successful drug discovery often requires a clear understanding of the target(s) and mechanism of action that drive both desirable and undesirable drug responses. A variety of methods can be employed to address this challenge, including selectivity assays, correlative approaches, affinity-based methods, and chemoinformatics. Drug targets and mechanism of action can also be revealed by identification of drug-gene interactions using genome-wide RNAi screens. We have undertaken a large-scale RNAi screen to reveal genes that enhance or suppress the activity of a novel MDM2 inhibitor developed at Amgen. The results of this screen clearly reveal the mechanism of action of the compound, while also identifying genetic interactions that may guide patient selection and biomarker development, demonstrating the utility of compound+sirna genetic interaction screens in the drug discovery process. Michael Ollmann, Ph.D. Principal Scientist, Therapeutic Discovery Discovery On Target Boston MA, October 3-4, 2012