Presentation entitled "Hit identification Strategies for Epigenetic Targets" at X-Gen Epigenetics iV, March 5-7th, 2012. Presentation was delivered by Dr Amy Quinn as I had a conflict which prevented my attendance

1. Hit Identification strategies
for Epigenetic Targets
Andy Pope
Platform Technology &
Science, GlaxoSmithKline,
Collegeville PA, USA
X-Gen – Epigenetics IV,
San Diego
March 5-7, 2012

2. 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”

3. 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?

4. Growing literature on applied* epigenetic discovery
* i.e. concerning the discovery and exploitation
of epigenetic modulator compounds

5. 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

6. Histone modifications and complexity
In theory: 2x1030 permutations

7. Components of the Histone Code
- methyl transferases - demethylases - Methyl readers
- acetyl transferases - deacetylases (HDACs) “chromodomains ”
- ubiquitin ligases - deubiquitinases - Acetyl readers
“bromodomains ”
- kinases - phosphatases
18 HDACs, 20 HATs, ~100 HMTs, HDMs, ~100 reader
domains…..

8. A wide range of epigenetic modulator compounds are
now known

9. The changing therapeutic target landscape
Dramatic shift of drug discovery activities into “new biology” post 2005
– Epigenetic targets are a significant component
“classical” targets = GPCR, ion Channel, kinase, protease,
nuclear receptor

10. Highly refined Hit Discovery Engine
- developed for classical drug targets; how well does it work for new epigenetic targets?
Diversity Screening Focused screening
Differential
discovery
Lead/drug
like molecules
Encoded Library Technologies
Integrated
Knowledge-based design discovery
Fragment screening

11. How were current epigenetic modulator compounds
discovered?

12. Selection of therapeutic targets (and target class strategies)
Pope A (2012) The Role of Chemical Biology in
Drug Discovery. Wiley Dictionary of Chemical
Biology; Drug Discovery. Part I; Drug
 Which targets can be linked to disease? Discovery and Development. Submitted
 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?

13. Different approaches to Drug Discovery
Chemical
Genomics
Target Assays & Lead
Validation Reagents Discovery
Specific Drug
Conventional Single Target
Target
Target Assays & Hit Lead
Validation Reagents Discovery Optimization
Phenotypic screening

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

15. 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……..

16. 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 N
molecules => Chemoproteomics N
N
R2
N
-1.4kb Firefly luciferase
R1
Human ApoA1 promoter 5’-UTR 3’-UTR Benzodiazepines
ApoA1 ApoA1

17. Bromodomain Target
Identification
HepG2/THP1 cells
BZD Active compound BZD -ve control
N
N N
N N
N
R2 R2
N N
R1 R1
Series X -ve control
BZD -ve control
Seies X active
BZD Active
BET proteins (Brd2, Brd3, Brd4)

18. High Content screening to measure cellular histone
marks

19. Single Highly Validated Target….many (integrated) hit ID
approaches
Test cpds
High quality Knowledge-based
Target & protein discovery/design ~1-5 x 102
partners crystals
Fragment based-drug
discovery
~1-5 x 103
Biophysical
Protein assays
expression Cross screening ~1-5 x 103
Functional
Enzyme Focused compound sets ~1-5 x 104
assays
Cellular High throughput
Screens
~0.5-2 x 106
assays
Tagged Encoded Library Screens ~1 x 1010
Immobilized
protein

20. Single Target approach example; EZH2
e.g. EZH2 methyl-transferase
• EZH2 5-membered complex
• activity on peptides, histones, multiple
nucleosome types
• H3K27 methylation confirmed – LC/MS
• Screening +/- activating peptide
• 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

21. EZH2 High Throughput Screens
Peptide substrate HTS ~2M compounds tested in screens against both
100
peptide and nucleosome substrates
80
60 HTS successful in identifying validated small
40 molecule inhibitors
20
0
GSK-1: IC50 = 0.8 uM, optimized to IC50 < 5 nM
-20
Selectivity of Enzyme IC50 (nM)
-40
-40 -20 0 20 40 60 80 100
Optimized GSK-X EZH2 13
Response for Rep_2
EZH1 1258
G9a 10000
MMSET 63096
Nucleosome substrate HTS DOT1 >100000
HTS hit GSK-1
100
SUV39H1 >100000
80 SUV39H2 >100000
60
SET7 >100000
SET8 >100000
40
PRMT1 >100000
20 PRMT3 >100000
0
PRMT4 >100000
PRMT5 >100000
-20
PRMT6 >100000
-40 SETMAR >100000
-40 -20 0 20 40 60 80 100
Response for Rep_2 DNMT1 >100000
DNMT3a >100000
DNMT3b 50119
SMYD2 134300
HDAC1-11 >100000

22. Encoded Library Technologies (ELT)
Library size ~1010 compounds
µg target protein
test in biological + µL library pool
assay
affinity-
synthesize based
feature cpds off- selection
DNA
Sequence
DNA tags
Identify chemical
“features”

23. 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

24. 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)

25. Chemical Genomics – e.g. Structural Genomics
Consortium
“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.”
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).

26. 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?

27. Chemical Probe example – UNC0638

28. Rapid scanning for chemical tractability in Encoded
Library Technologies
Pooled ELT libraries
Proteins (~50 uG)
Partially purified
(~109 warheads)
Target
Resin
Simultaneous protein
Purification & selection
-
-
Translate to ELT warheads PCR amplification
DNA sequence
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

29. 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

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