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
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
Epigenetic Drug Discovery“….epigenetics is emerging not just as a discipline with a solidtheoretical and mechanistic foundation, but as a highly promising ifstill confusing source of new drug targets”
Epigenetic Drug Discovery“….epigenetics is emerging not just as a discipline with a solidtheoretical and mechanistic foundation, but as a highly promising ifstill 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
A wide range of epigenetic modulator compounds are now known
The changing therapeutic target landscapeDramatic 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
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
How were current epigenetic modulator compounds discovered?
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?
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
Phenotypic approaches played a significant role in first in class drug discovery
Phenotypic approach; epigenetics examplesHDAC inhibitors – discovered and optimized as inhibitors of proliferationbefore mechanism was identifiedBromodomain 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 N molecules => Chemoproteomics N N R2 N -1.4kb Firefly luciferase R1 Human ApoA1 promoter 5’-UTR 3’-UTR Benzodiazepines ApoA1 ApoA1
Bromodomain Target IdentificationHepG2/THP1 cellsBZD 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)
High Content screening to measure cellular histone marks
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
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
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”
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“SGC aims to develop "chemical probes", small molecules that can selectively stimulate or block the activity ofa protein, specifically designed to affect the activity of proteins involved in epigenetic control. They willcomplement genetic knockouts and RNAi approaches to understand the cellular role of these proteins. Theprobes need to be selective for their target protein, and suitable for use in cellular settings. It is hoped thatsome 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 ofNorth Carolina at Chapel Hill, the Departments of Chemistry and Biochemistry at the University ofOxford and the Department of Chemistry at Umeå University (Sweden).
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?
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
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
AcknowledgementsChun-Wa ChungDeepak BandyophyayMartin BrandtMurray BrownElizabeth DavenportLorena KallalAlan GravesEnoch GaoTony Jurewicz …..plus the numerousGlenn HoffmanBob HertzbergMike Hann other authors whoseTom HeightmanRoy KatsoQuinn Lu work was citedCarl MachuttaBill MillerGordon McIntryeBarry MorganMehul PatelSimon SemusSharon SweitzerPeter TuminoSara ThrallAmy QuinnZining WuJess Schneck