Characterization of Small Molecule ETS Transcription Factor Binders

Nicole M. MartinezMarius 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:10.1038/nrd2275

Many “Driver” Cancer Proteins are Currently “Undruggable”

• Example: Oncogenic Transcription Factors– ETS Transcription FactorsTranslocated in >50% of

prostate cancersETV1

ERG

Otis Brawley, National Cancer Institute

Prostate Tumor

ETS Transcription Factor Role in Prostate Cancer

/ETV1

doi:10.1038/nm0106-14

Can we develop a therapeutic?

DMSO stock solutions

protein-small molecule interaction on a microarray

aMouse-IgG-Cy5ETV1

fluorescent features revealputative binding interactions

Small-Molecule Microarrays (SMMs)

ha.11

Lysates expressing target protein

αHA

MIT

FE

TV1

ER

G tr

unca

ted

ER

G fu

ll

f = microarray featuremedian pixel intensity

b = local backgroundmedian pixel intensity

xcpd = f - b1.25 3.75 6.25Z*

Overlay of GAL File

Z* =cpd - µmock

mock (1+ )cpd0.96

From Raw Data to HitsFrom Raw Data to Hits

ETV1 Selection of Hits

ZScoreA

ZScoreB

ZScoreC

CompositeZ

CompositeZ

ChemBank: Tool for Filtering ChemBank: Tool for Filtering CompoundsCompounds

http://chembank.broad.harvard.eduhttp://chembank.broad.harvard.edu

List of ETS “Hit” Compounds

*Library

HEK-293T cell

lysate

MITF ETV1 ERG tERG

NPC 2 2 87 31 30

PDI 6 3 15 8 1

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

ETS Luciferase

ERG

rep

rep +

ERG

rep + ERG

+ compound

Fold

Lead Compounds

MMP1/ETV1 in 501 mel

0

1

2

3

4

5F

old

rep rep+ETV1 rep+cpd rep+ETV1+cpd

Cpd1 Cpd2

Assess Inhibitory Capabilities by Luciferase Assays

ETS Luciferase

ERG

rep

rep +

ERG

rep + ERG

+ compound

Fold

Dud CompoundsAP20T/ERG with cpds (501mel)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

Fo

ld in

du

ctio

n

AP20T AP20T+ERG AP20T+cpd AP20T+ERG+cpd

CtrlDMSO ERG cpds

A B C D

MITF ETV1

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Ctrl ERG cpds

AP20T/tERG + cpds (501mel)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Fo

ld i

nd

uct

ion

reporter reporter+tERG

CtrltERG cpds

A B C DMITF 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 ResearchProgram 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