Upload
andypopeuk
View
382
Download
1
Tags:
Embed Size (px)
DESCRIPTION
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
Citation preview
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