characterization of small molecule ets transcription factor binders nicole m. martinez marius s. pop...

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