Characterization of Small Molecule ETS Transcription Factor Binders Nicole M. Martinez Marius S. Pop and Levi A. Garraway Cancer Biology Program

Targeted Therapy in Cancer “Druggable” targets –Obvious active site Kinases Other enzymes “Undruggable” targets –No obvious pocket DOI: /nrd2275

Many “Driver” Cancer Proteins are Currently “Undruggable” Example: Oncogenic Transcription Factors –ETS Transcription Factors Translocated in >50% of prostate cancers ETV1 ERG Otis Brawley, National Cancer Institute Prostate Tumor

ETS Transcription Factor Role in Prostate Cancer /ETV1 doi: /nm

Can we develop a therapeutic?

DMSO stock solutions protein-small molecule interaction on a microarray aMouse-IgG-Cy5 ETV1 fluorescent features reveal putative binding interactions Small-Molecule Microarrays (SMMs) ha.11 Lysates expressing target protein αHA MITF ETV1 ERG truncated ERG full

f = microarray feature median pixel intensity b = local background median pixel intensity x cpd = f - b Z* Overlay of GAL File Z* =  cpd - µ mock  mock ( 1+1+ )  cpd 0.96 From Raw Data to Hits

ETV1 Selection of Hits ZScoreA ZScoreB ZScoreC CompositeZ

ChemBank: Tool for Filtering Compounds

List of ETS “Hit” Compounds *LibraryHEK-293T cell lysate MITFETV1ERGtERG NPC PDI NPC: Natural Products and commercials (Including FDA approved drugs) library PDI: Psychiatric Disease Initiative compounds *10,800 compounds per library

Assess Inhibitory Capabilities by Luciferase Assays ETSLuciferase ERG rep rep + ERG rep + ERG + compound Fold

Lead Compounds Cpd1 Cpd2

Assess Inhibitory Capabilities by Luciferase Assays ETSLuciferase ERG rep rep + ERG rep + ERG + compound Fold

Dud Compounds Ctrl DMSO ERG cpds A B C D MITFETV1 Ctrl ERG cpds Ctrl tERG cpds A B C D MITF ETV1

Conclusions SMM allow us to find binders Luciferase assays allow us to determine inhibitory capabilities Future work –Surface Plasmon Resonance –Screen w/ more compounds

Acknowledgements Mentors Marius Pop, PhD Levi Garraway, MD, PhD Collaborators Angela Koehler, PhD Jason Fuller Summer Research Program in Genomics Shawna Young Lucia Vielma Bruce Birren, PhD

ETS Fusion Products Exon 1 of TMPRSS2 with the beginning of exon 4 of ETV1 Exons 1 and 2 of TMPRSS2 with the beginning of exon 4 of ETV1 Exon 1 of TMPRSS2 with the beginning of exon 4 of ERG