Unbiased Ligand Discovery for Histone Deacetylases Using Small Molecule Microarrays

Nicole M. Martinez, Summer Research, Chemical Biology Program

HDACs Participate in Regulation of Gene Expression

• Histone Deacetylases HDACs

• Acetylation regulates gene expression

• HAT transcription activation complex

• HDAC transcription repression complex

de Ruijter, A. J.; van Gennip, A. H.; Caron, H. N.; Kemp, S.; van Kuilenburg,A. B. Biochem. J. 2003, 370, 737-749.

HDACs Impact Cancer Biology

• gene expression• oncoprotein stability• cell migration • protein catabolism• cell cycle control

http://targethdac.com

HDACs and Cellular Functions

Minucci, S.; Pelicci, P. G. Nat. Rev. Cancer. 2006, 6, 38-51.

HDAC Inhibitors (HDACi)

• Anti-cancer agents• Few HDACi are isoform selective• Allosteric inhibitors are highly desirable• Tools for assigning function to HDACs

Existing HDACi

Synthetics Natural Products

DMSO stock solutions

protein-small molecule interaction on a microarray

A647 α-GST

GST

HDAC

Small-Molecule Microarrays (SMMs)

fluorescent features revealputative binding interactions

print stocks onto functionalized slides

QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.

Biophysical Assays

Protein *Library Replicates Data Points

HDAC1 DIV 3x 32400

HDAC4 NPC/DIV 3x 97200

HDAC5 NPC/DIV 3x 97200

HDAC6 NPC/DIV 3x 97200

HDAC7 DIV 3x 32400

HDAC8 DIV 3x 32400

HDAC9 NPC/DIV 3x 97200

HDAC10 NPC/DIV 3x 97200

583,200*10,800 compounds per library

DIV: Diversity Oriented Synthesis Small Molecules library

NPC: Natural Products and commercials (Including FDA approved drugs) library

SMM Unbiased Approach

Approach:• Small Molecule Microarrays to determine

unbiased protein-ligand interactions

Benefit:• High-throughput• Quick and cheap• Small amount of protein analyte

From Raw Data to HitsFrom Raw Data to Hits

f = microarray featuremedian pixel intensity

b = local backgroundmedian pixel intensity

xcpd = f - b1.25 3.75 6.25Z*

http://chembank.broad.harvard.edu

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Overlay of GAL File

Z* =cpd - µmock

mock (1+ )cpd0.96

Representative Data: HDAC6

ZScoreB

ZScoreA

ZScoreC

CompositeZ Rounded

DIV library

Structures of Common HDAC Hits

Common HDAC Biased Hits

Structures of HDAC6 Specific Hits

HDAC biased plates 2131-2134

What Happens Next?

• Screen with small molecule microarrays against the“Forma Collection” in order to find allosteric binders

• Surface plasmon resonance (SPR) to evaluate binding kinetics

• in vitro activity assays• Phenotypic assays

ChemBank: A Tool for the CommunityChemBank: A Tool for the Community

DSA-ChemBank:DSA-ChemBank: 796,063 curated compounds, 1,963 assays, 149 projects, 16,942,065 well measurements. ChemBank:ChemBank: 528,062 curated compounds, 529 assays, 45 projects, 5,764,724 well measurements

43,651 users at 8,309 organizations

in 154 countries

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

Acknowledgments

Mentors

Angela Koehler, Ph.D.

Jason Fuller

Collaborators

James Bradner, M.D.

Stephen Haggarty, Ph.D.

Katie Doud

Summer Research Program in

Genomics

Shawna Young (Program

Coordinator)

Bruce Birren, Ph.D.

Common HDAC Hits

1463.A101463.A141463.A201463.C221463.E121463.E242099.F062099.M032100.J132108.B032108.B052108.B112130.L012131.A212131.O212132.G122133.F212133.I092283.N21

HDAC6 Specific

2131.A092131.C232131.D182131.D242131.E232131.G172131.G232131.N222133.D042133.G232133.N242133.P22

HDAC biased plates 2131-2134

HDAC Cross Comparison