Andy Pope Platform Technology &

Science, GlaxoSmithKline, Collegeville PA, USA

X-Gen – Epigenetics IV,

San Diego March 5-7, 2012

Hit Identification strategies

for Epigenetic Targets

Epigenetic Drug Discovery

“….epigenetics is emerging not just as a discipline with a solid

theoretical and mechanistic foundation, but as a highly promising if

still confusing source of new drug targets”

Epigenetic Drug Discovery

“….epigenetics is emerging not just as a discipline with a solid

theoretical and mechanistic foundation, but as a highly promising if

still confusing source of new drug targets”

• How were existing epigenetic modulating molecules discovered?

• How is this being currently approached?

• How does this relate to drug discovery against “traditional” target classes?

• How might current approaches change our views and/or accelerate progress in epigenetic drug discovery?

Growing literature on applied* epigenetic discovery

* i.e. concerning the discovery and exploitation

of epigenetic modulator compounds

DNA Methylation & DNMT Inhibitors

• Oldest class of epigenetic

modulators

• Hypomethylation via

cytidine analogs (eg. AZA/DAC) - covalent DNMT complex triggers

proteasome mediated DNMT

removal

• Discovered prior to

understanding mechanisms

• Selective & reversible DNMT

inhibitors currently being

sought

Histone modifications and complexity

In theory: 2x1030 permutations

Components of the Histone Code

18 HDACs, 20 HATs, ~100 HMTs, HDMs, ~100 reader domains…..

- methyl transferases

- acetyl transferases

- ubiquitin ligases

- kinases

- demethylases

- d eacetylases (HDACs)

- deubiquitinases

- phosphatases

- Methyl readers

“ chromodomains ”

- Acetyl readers

“ bromodomains ”

A wide range of epigenetic modulator compounds are now known

The changing therapeutic target landscape

“classical” targets = GPCR, ion Channel, kinase, protease,

nuclear receptor

Dramatic shift of drug discovery activities into “new biology” post 2005

– Epigenetic targets are a significant component

Integrated

discovery

Lead/drug

like molecules

Differential

discovery

Highly refined Hit Discovery Engine - developed for classical drug targets; how well does it work for new epigenetic targets?

Encoded Library Technologies

Diversity Screening

Fragment screening

Focused screening

Knowledge-based design

How were current epigenetic modulator compounds discovered?

Selection of therapeutic targets (and target class strategies)

Which targets can be linked to disease?

How safe will it be to perturb epigenetic systems?

Which targets are chemically tractable?

What is the best way to discover new Leads?

Can whole classes of target be exploited?

What selectivity and specificity is required?

Can probes be generated which open up new biology?

Pope A (2012) The Role of Chemical Biology in Drug Discovery. Wiley Dictionary of Chemical Biology; Drug Discovery. Part I; Drug Discovery and Development. Submitted

Different approaches to Drug Discovery

Lead Optimization

Assays &

Reagents

Target Validation

Chemical Genomics

Conventional Single Target

Phenotypic screening

TargetValidation

Assays &Reagents

LeadDiscovery

Spe

cific Dru

g Target

Hit

Discovery

Phenotypic approaches played a significant role in first in class drug discovery

Phenotypic approach; epigenetics examples

HDAC inhibitors – discovered and optimized as inhibitors of proliferation

before mechanism was identified

Bromodomain Inhibitors – phenotypic screen for Apo-A1 inducers

Historically, phenotypic approaches have pre-dated key target discoveries - Currently being re-emphasized……..

Bromodomain Inhibitor discovery via “black box” screening

• Apo-A1 expression linked to the Nuclear Receptor LXR – target for dislipidemia

• In 2001 GSK ran a reporter gene HTS coupling the ApoA1 promotor to luciferase

(~500K compounds)

• Hits were triaged for direct interactions with LXR

• One series (BZD) gave consistent ApoA1 induction, but did not act via LXR directly

• Medicinal chemistry successfully optimized without knowledge of the molecular

target.

• Profiling of compounds against numerous assays did not identify target for these

molecules => Chemoproteomics N

N

R2

N

R1

N

Benzodiazepines 5’-UTR ApoA1

3’-UTR ApoA1

-1.4kb

Human ApoA1 promoter

Firefly luciferase

Bromodomain Target Identification

HepG2/THP1 cells

N

N

R2

N

R1

N

N

N

R2

N

R1

N

BZD Active compound BZD -ve control

BZ

D A

cti

ve

BZ

D -

ve

c

on

tro

l

Se

rie

s X

-v

e c

on

tro

l

Se

ies

X a

cti

ve

BET proteins (Brd2, Brd3, Brd4)

High Content screening to measure cellular histone marks

Single Highly Validated Target….many (integrated) hit ID approaches

Target & partners

Protein expression

Functional Enzyme assays

Focused compound sets

Fragment based-drug discovery Biophysical

assays

High throughput Screens

Cross screening

Encoded Library Screens

Knowledge-based discovery/design

Tagged Immobilized

protein

High quality protein crystals

Cellular assays

Test cpds

~1-5 x 103

~1-5 x 102

~1-5 x 104

~1-5 x 103

~0.5-2 x 106

~1 x 1010

Single Target approach example; EZH2

e.g. EZH2 methyl-transferase

• Over-expressed in tumors (prostate, breast, lung)

• Activating mutations are pro-oncogenic

• knockdown in prostate & breast cancer lines, result in

↓proliferation ↓ anchorage independent growth

↓ invasion/migration ↓ tumor formation in mice

• EZH2 5-membered complex

• activity on peptides, histones, multiple nucleosome types

• H3K27 methylation confirmed – LC/MS

• Screening +/- activating peptide

EZH2 High Throughput Screens

Response for Rep_2

-40 -20 0 20 40 60 80 100

-40

-20

0

20

40

60

80

100

Response for Rep_2

-40 -20 0 20 40 60 80 100

-40

-20

0

20

40

60

80

100

~2M compounds tested in screens against both peptide and nucleosome substrates HTS successful in identifying validated small molecule inhibitors

GSK-1: IC50 = 0.8 uM, optimized to IC50 < 5 nM

Enzyme IC50 (nM) EZH2 13

EZH1 1258

G9a 10000

MMSET 63096

DOT1 >100000

SUV39H1 >100000

SUV39H2 >100000

SET7 >100000

SET8 >100000

PRMT1 >100000

PRMT3 >100000

PRMT4 >100000

PRMT5 >100000

PRMT6 >100000

SETMAR >100000

DNMT1 >100000

DNMT3a >100000

DNMT3b 50119

SMYD2 134300

HDAC1-11 >100000

Peptide substrate HTS

Nucleosome substrate HTS HTS hit GSK-1

Selectivity of Optimized GSK-X

Encoded Library Technologies (ELT)

Library size ~1010 compounds

synthesize feature cpds off-

DNA

affinity-

based selection

Sequence DNA tags

Identify chemical “features”

test in biological assay

µg target protein + µL library pool

Can chemical connectivity drive epigenetic lead discovery?

Focused libraries based upon emerging templates, substrate elements Cross-screening members of the same protein class

Increasing number of crystal structures > knowledge-based design

Enzymes versus protein:protein interactions

• Bias against protein:protein interactions as too difficult c.f. enzymes

- tight binding, de-localized

• Reader: Histone mark interactions appear to be chemically tractable

• Perhaps also other opportunities (e.g. methyltransferase complexes)

Chemical Genomics – e.g. Structural Genomics Consortium

Structural Genomics Consortium (SGC), also includes GlaxoSmithKline, Novartis, Pfizer, Eli Lilly,

NCGC Bethesda, Center for Integrative Chemical Biology and Drug Discovery at the University of

North Carolina at Chapel Hill, the Departments of Chemistry and Biochemistry at the University of

Oxford and the Department of Chemistry at Umeå University (Sweden).

“SGC aims to develop "chemical probes", small molecules that can selectively stimulate or block the activity of

a protein, specifically designed to affect the activity of proteins involved in epigenetic control. They will

complement genetic knockouts and RNAi approaches to understand the cellular role of these proteins. The

probes need to be selective for their target protein, and suitable for use in cellular settings. It is hoped that

some probes may be a starting point for drug discovery.”

Chemical Probes

Potent and selective enough to probe target biology

Demonstrate target chemical tractability

HTS as major hit discovery method so far

Methods to increase success and throughput of probe discovery?

Chemical Probe example – UNC0638

Rapid scanning for chemical tractability in Encoded Library Technologies

-

-

Res

in

Targ

et

Gross J (2011) Parallel Small‐Scale Expression and ELT Screening of Drug Targets to Explore Druggability and Generate Chemical Probes. SBS Conference Orlando, March 28-31

Par

tial

ly p

uri

fie

d

Pro

tein

s (

~50

uG

)

Po

ole

d E

LT li

bra

rie

s (~

10

9 w

arh

ead

s)

DNA sequence

PCR amplification

Simultaneous protein Purification & selection

Tran

slat

e to

ELT

war

he

ads

Conclusions

• “Applied” epigenetic discovery is a active field

• Rapid discovery of probes/leads against many of the players in histone modification

• Similar methods are being applied as for “classical” drug targets, with apparently similar success rates

• Chemical probe/rapid tractability methods are opening up new target

classes for exploration

• Tool molecules will likely play a key role in decoding epigenetic signaling and open up new ways to modify disease

• Tools should allow key questions about where and how epigenetic mechanisms can be safely modified to treat disease

Acknowledgements

Chun-Wa Chung Deepak Bandyophyay Martin Brandt Murray Brown Elizabeth Davenport Lorena Kallal Alan Graves Enoch Gao Tony Jurewicz Glenn Hoffman Bob Hertzberg Mike Hann Tom Heightman Roy Katso Quinn Lu Carl Machutta Bill Miller Gordon McIntrye Barry Morgan Mehul Patel Simon Semus Sharon Sweitzer Peter Tumino Sara Thrall Amy Quinn Zining Wu Jess Schneck

…..plus the numerous other authors whose work was cited