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Integrated Solution for Epigenetic Drug Discovery and Development
Elizabeth Quinn, PhDDirector, LeadHunter Discovery Services
[email protected] September 2013
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DiscoveRx Platform Technologies
Product & Service Solution Portfolio
LeadHunter Services
Quantitative Binding
Enzyme Complementation
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Assay Development
Kits and Cell Lines
Primary Human Cell Profiling
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>80% Coverage of Druggable Targets + Primary Cell Phenotypic Assays
HDAC/HMT
Dep
th of C
overage
100%
0%284
394 21 23
34
GPCRs Kinases NHRs Pathways Bromodomain
1,118 assays covering 751 druggable targets• All major drug target classes covered• Multiple assays for single target
Primary Cell Profiling
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Challenges: • Limited HTP compound screening options• Cellular toxicity and adverse effects have resulted in low uptake of epigenetic therapies in the clinic
Opportunities: • Proven therapeutic potential for epigenetic targets
• Innovative approaches combined with “lessons‐learned” can help guide therapeutic development in the epigenetic space
Targeting Epigenetics For Therapeutic DevelopmentHDAC, Demethylases and Bromodomain Target Classes
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Integrated Strategy for Therapeutic Development
CellularActivity
Primary Cell Model Systems
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“…knowing a target is not the same as knowing what the target does, let alone knowing the effects of a chemical inhibitor in diverse disease settings.” Nature Biotech 22, 1253‐1259 (2004).
HTSSAR
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Integrated Strategy for Therapeutic DevelopmentKey Questions
• Hit identification• Is my compound potent and selective?
• What is the phenotypic impact of my compound in the context of disease?
• What efficacy and safety biomarkers are detected?
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• Does my compound bind the target in a cellular environment?
Relevant assays + optimized tool compounds Increased probability of success
CellularActivity
Primary Cell Model Systems
HTSSAR
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Integrated Strategy for Therapeutic DevelopmentTarget Biochemistry: SAR
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Quantitative Binding
• Hit identification• Is my compound potent and selective?
CellularActivity
Primary Cell Model Systems
HTSSAR
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Epigenetic Proteins
Modification Write Erase Read
Acetyl HAT HDAC Bromo
Methyl HMT HDM Chromo, PHD, Tudor, MBT
Enzyme Enzyme Binding8
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KINOMEscan® Technology ‐ The Solution
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KINOMEscanapplied to BRDs
World’s Largest Kinase Panel• Cover >80% human kinome• Now 456 assays – lipid, TK, mutants• Enables new drug discovery paradigms
Our Goal for Bromodomains• Leverage our expertise • Build a first in class panel of bromodomain binding assays
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BROMOscan Technology: How it Works
Measure amount of bromodomain bound to immobilized ligand in the presence and absence of test compound
Competition No Competition
‐ Test Compound + Test Compound
Ultrasensitive qPCR readout (6‐log range)
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Bromodomain Assay Validation
• Small Molecules‐ Measure Kds consistent with known ITC values (when available)
‐ Inhibitors: • JQ1 (active enantiomer)• I‐BET (active and inactive enantiomers)• Other known BRD inhibitors
• Acetylated Histone Tail Peptide Ligands‐ Measure Kds consistent with published values (when available)
‐ Demonstrate selectivity for acetylated over non‐acetylated peptides
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Bromodomain Menu
Bromodomain Targets
ATAD2A BRD3(1,2) BRDT(2) PBRM1(5)ATAD2B BRD4(1) BRDT(1,2) PCAFBAZ2A BRD4(2) BRPF1 SMARCA2BAZ2B BRD4(1,2) BRPF3 TAF1(2)
BRD1BRD4(full‐length, short‐iso) CECR2 TAF1L(2)
BRD2(1) BRD7 CREBBP TRIM24(Bromo.)BRD2(2) BRD8(1) EP300 TRIM24(PHD,Bromo.)BRD2(1,2) BRD8(2) FALZ TRIM33(PHD,Bromo.)BRD3(1) BRD9 GCN5L2 WDR9(2)BRD3(2) BRDT(1) PBRM1(2)
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Roman numerals indicate SGC‐defined families
Human Bromodomain Dendrogram
•39 bromodomain assays •Almost 60% coverage of the entire family •Multiple putative therapeutic targets: BET, ATAD2, CECR2, and TRIM24(PHD,Bromo.)
Current BROMOscan assays
New assays available October 2013
* Tandem BRD2, BRD3, BRDT and full‐length, short‐iso BRD4 assay also available.
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Bromosporine Kd (nM)
T mSh
ift (1
00 u
M B
rom
ospo
rine)
Comparison of BROMOscan and SGC Tm Shift Data for the Promiscuous Inhibitor Bromosporine
• 24/25 BROMOscan assays analyzed• Each square represents a unique bromodomain • Outstanding agreement between BROMOscanand Tm Shift data− Cross‐validates the formats− Further quantifies Bromosporine promiscuity
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Striking agreement between two very different assay readouts
Global Validation of BROMOscan Panel
SGC Tm shift data provided by S. Knapp, PhD in collaboration with DiscoverX
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BET Family Assay Validation JQ1 Kds Consistent with Reported Values
Data Comparison• Similar Kd values measured with BROMOscan & ITC
‐ Domain affinity rank order identical• Optimized conditions ensure true thermodynamic Kd
measurements *Nature (2010) 408: 1067
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BET Family ValidationI‐BET
Nature (2010) 468: 1119.
BROMOscan data consistent with published ITC data• Potency• Rank order• Lack of potent activity for inactive I‐BET enantiomer
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Bromodomain Inhibitor Profiles
I‐BET Bromosporine
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BET‐Selective Promiscuous
PFI‐1
Semi‐BET‐Selective
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72 known kinase inhbitors tested (6% hit rate)• Potent (Kd = 0.01‐ 1 uM) & diverse BRD profiles• Inhibitors that target TKs, STKs and lipid kinases have BRD activity
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Kinase & Bromodomain Polypharmacology
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BROMOscan Services Portfolio
Service Number of Assays Description
24Set of 24 human bromodomainsvalidated for single point testing
1-24 Personalized or custom panels
1-34 Quantitative binding constants
34 Kd profile of entire BROMOscanmenu
1-24Compound library screening against a single target or against the full panelLIBRARY
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14 additional assays available in October 2013
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BROMOscan ‐ Drive Your Epigenetic Programs Forward
Your Collection of Drug Candidates
Phenotypic Screens
Chemoproteomics
Biophysical assays
Crystallography
Single Target HTSFollow upKd Determinations
Selectivity Profiling
Jumpstart Your Program – Only 2 Weeks to Results!19
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BROMOscan Summary
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• BROMOscan is a robust and highly validated panel of 39 bromodomain assays
• Quantitative binding information• Rigorously validated, state‐of‐the‐art reference inhibitors• Accurate, precise & reproducible
• BROMOscan can be used to rapidly generate inhibitor profiles across the entire bromodomain family
• Identify lead compounds • Optimize potency & selectivity
• BROMOscan can be used as a discovery tool to screen existing chemical assets
• Gain starting points for new programs• Uncover off‐target liabilities
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Integrated Strategy for Therapeutic DevelopmentCellular Assays
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Quantitative Binding
• Is my compound potent and selective?
• Does my compound bind the target in a cellular environment?
Enzyme Complementation
CellularActivity
Primary Cell Model Systems
HTSSAR
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• Bromodomains are not enzymatic • Cell‐based assays are typically difficult and
low‐throughput• FRAP assays commonplace• Limited quantitation, impractical for dose
response analysis
Dey et al. MBC 2009
Is my compound cell‐permeable? Does it interact specifically with the target? How does the biochemical potency relate to
the cellular potency?
Challenges of Targeting BromodomainsUsing Cell‐Based Assays
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InCELL Hunter™ – Ligand Induced Stabilization
‐ In vitro compound binding enhances protein stability
Protein PurificationStructural Biology Thermal Shift Assays
TargetConcept:
Altered Half‐lifeMaintain Steady State
+ compound
‐ Can the same principles be applied to cell‐based assays?
‐ compound
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InCELL Hunter: How It Works
Cell‐based Bromodomain Assays Measure compound permeability Binding to intracellular protein Determine cellular potency
• Compound activity in a native cellular environment
• Simple chemiluminescent protocol
• Bridges gap between biochemical and cellular phenotypic assays
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Ideal for studying bromodomain protein:protein interactions
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Compound Binding to BRD4 in Live CellsJQ1 and IBET
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JQ1IBET
JQ1 > IBET
CMPD Lit* InCELL
JQ1 120 230
C 100n
M
1000
nM
Western Blot Confirmation
Concentration of JQ1
*Filippakopoulos, et al. Nature 468: 1067‐1073, 2010.
Ligand [M]
RLU
-12 -10 -8 -6 -40
100000
200000
300000
400000
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Target Specificity and Selectivity
• Differentiate target‐selective stereoisomers and determine target specificity
• Multiple target classes available, ideal for counter‐screening
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Enantiomer Testing Counter Screening
BRD4A
Ligand [M]
RLU
-12 -10 -8 -6 -40
100000
200000
300000
400000
JQ-1 (+)
JQ-1 (-)
BRD4(1)
BRD4A
Ligand [M]
RLU
-12 -10 -8 -6 -40
100000
200000
300000 IBET (+)
IBET (-)
BRD4(1)
G9A
UNC0638 [M]
RLU
10- 1 1 10- 1 0 10- 9 10- 8 10- 7 10- 6 10- 5 10- 40
500000
1000000
1.5100 6
UNC0638JQ1(+)JQ(-)
G9A
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Expansion to Tandem Domains
Domain Compound Kd (nM)BRD3(1) I‐BET 34BRD3(1) JQ1 25BRD3(1) PFI‐1 87
BRD3(2) I‐BET 27BRD3(2) JQ1 26BRD3(2) PFI‐1 72
Organization of Tandem Bromodomains
Anna C. Belkina & Gerald V. DenisNature Reviews Cancer 12, 465-477 (July 2012)
BROMOscan
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BRD3 Tandem
[M]
RLU
10- 1 1 10- 1 0 10- 9 10- 8 10- 7 10- 6 10- 5 10- 40
200000
400000
600000
800000(+)JQ-1I-BETPFI-1
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BRD9 Assay Development
Kd (nM)E12348 40JQ1 >100000
Wild‐Type Sequence Binding Deficient Mutant
Point mutant studies confirm the specificity of the compound binding reaction
RLU
BottomTopLogEC50HillSlopeEC50
E12348 188537600771-5.7981.1291.594e-006
JQ1 165184191889-7.35714.874.392e-008
Compound [M]10-10 10-9 10-8 10-7 10-6 10-5 10-40
100000
200000
300000
400000
500000E12348JQ1(+)
Best-fit values
TopLogEC50HillSlopeEC50
E12348
156000220747-5.2503.1625.624e-006
JQ1(+)
173137209674-5.9342.4511.165e-006
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TAF1L(2) Assay Development
Kd (nM)E12348 15BDOIA383 14,000
Wild‐Type Sequence Binding Deficient Mutant
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RLU
BottomTopLogEC50HillSlopeEC50
E12348 [M]163561744973-5.6521.2552.231e-006
PF1 BD01A383156116200123-5.46918.853.399e-006
Mutant Studies in progress
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InCELL Hunter ‐ Summary
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• InCELL Hunter is novel, ligand‐induced stabilization approach to cellular assay development
• Proven EFC technology• Robust & quantitative
• InCELL Hunter assays can be used to guide selection of lead compounds based on:
• Cell permeability• Intracellular protein binding• Cellular potency
• InCELL Hunter technology can be applied to druggable target classes including:
• Bromodomains• Methyltransferases• Kinases
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Integrated Strategy for Therapeutic DevelopmentPrimary Human Cell Systems
Quantitative Binding
• Is my compound potent and selective?
• Does my compound bind the target in a cellular environment?
Enzyme Complementation
CellularActivity
31
Primary Human Cell Systems
• What is the phenotypic impact of my compound in the context of disease?
• What efficacy and safety biomarkers are detected?
Primary Cell Model Systems
HTSSAR
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BioMAP® Technology PlatformOverview
BioMAPAssay Systems
ReferenceProfile Database
Predictive Informatics Tools
Human primary cells Disease‐models30+ systems
Biomarker responses to drugs are stored in the database
>3000 drugs
Specialized informatics tools are used to predict clinical outcomes
Human Biology Integrated into a Robust, Scalable Platform
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Each drug induces a signature profile
3C
SAg
BE3C
HDF3CGF
Fibroblasts (HDF)
PBMC
HT-29 Cell Line (Colon adenocarcinoma)
TCR stimulation
Tissue Primary Cell System BioMAP Profile
BioMAP® Systems Model Complex BiologyPrimary Human Cell Systems
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BioMAP Technology PlatformReference Database
BioMAP database contains profiles of >3000 agents• Drugs – Clinical stage, approved, and failed
• Experimental Chemicals ‐ Research tool compounds, environmental chemicals
• Biologics ‐ Cytokines, factors, peptides, antibodies, soluble receptors
Compound TargetI‐BET BRDJQ1 BRD
I‐BET‐151 BRDPFI‐1 BRD
Panobinostat HDACVorinostat, HDACEntinostat HDACScriptaid, HDACTrichostatin HDAC
M344 HDACUNC0624 G9a/GLP methyltransferaseGSK‐J4 Lysine Demethylase (KDM)GSK2801 BAZ2B/A BRDSGC‐CBP30 CREBBP (CBP) and EP300
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BET Inhibitors Have Unique BioMAP Signature Common Activities of I‐BET Inhibitors at Lowest Active Dose
• BET inhibitors have highly similar BioMAP profiles (r ≥ 0.91)• Overlay signature shows broad anti‐proliferative effects on EC, T cells, CASMC
and Fibs indicating impact on vascular, stromal and immune biology• Additional anti‐inflammatory and matrix modulating activities support potential
for development in oncology• May also have anti‐fibrotic potential but likely not suitable for autoimmune
diseases
TF, HLA-DR, Proliferation
VCAM VCAM IL-8, Proliferation
sIgG, IL-6 uPAR, uPA VCAM, MIG, Proliferation
EGFR, PAI-1, MMP-1, TIMP-1, Proliferation
PAI-1, SRB MMP-1, TIMP-1 VCAM, sIL-10
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Clinical HDACi Class Signature in BioMAPVorinostat, Entinostat and Panobinostat
• HDACi signature in BioMAP at lower more clinically relevant doses reveals activities related to efficacy and safety including• Strong immune suppressive effects in SAg and BT with anti‐proliferation activity
detected on HDF• Decrease in TNFα (LPS) shows strong anti‐inflammatory effects at these low
doses plus matrix modulation effects also indicate Potential for hematological oncology application
• Safety : Increased SAA and TF detected at higher doses CV risk
SAA
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Similarity Clustering Confirms Target PhenotypeCompounds Compared To Database Profiles for Signature Match
p38MAPK
BET
JAK
HDAC
• Statistical measures of profile similarity are used to cluster compounds with shared phenotypic impact irrespective of their molecular target(s). • Dot color and size depicts
compound name and dose respectively.
• Compound profiles for compounds that are statistically significantly similar (Pearson 0.9) are represented by dots connected by a line to form a cluster over multiple doses.
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BioMAP Summary of Epigenetic Inhibitors
• BioMAP profiles can be used to classify compounds based on phenotypic impact on primary human cell systems• Determine target or mechanism related BioMAP signatures
• BioMAP activities can be used to predict efficacy to guide indication selection and identify potential safety and efficacy related biomarkers • Dose response effects can also guide therapeutic window for on‐
versus off‐target effects
• BioMAP can detect activities that are associated with adverse effects or safety concerns early in development program
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DiscoveRx Epigenetics Solution Portfolio
39
• Is my compound potent and selective?
• What is the phenotypic impact of my compound in the context of disease?
• What efficacy and safety biomarkers are detected?
• Does my compound bind the target in a cellular environment?
CellularActivity
Primary Cell Model Systems
HTSSAR
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InCELL Hunter Product Menu
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Available Now Coming SoonBRD2(1) BRD9BRD3(1) TAF1L(2)BRD3(1,2) TAF1(2)BRD4(1)BRD2(1)BRDT(1)BRDT(1,2)
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Assay Development Services
41
Options A B C
Timeline 8‐10 weeks 10‐14 weeks 18‐24 weeks
Deliverable Expression validated cell pool
Functionally validated cell pool
Functionally validated clonal cell line
What You Get• Select target• Select cell background• EFC expression validation
• All Option A deliverablesPlus
• Functional validation and optimization of compound/ target interaction
• Option A and B deliverablesPlus
• Isolation of single cell clone
• Clonal stability testing
25 Bromodomain pools now offered through CAD Services
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Complete Bromodomain Menu
• 34 BROMOscan targets‐ 24 MAX panel‐10 Kd mode‐Tandems‐Other isoforms
• 6 ICH clones• 25 ICH pools
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Thank You
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