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