Hit and Lead Discovery in an Academic Setting

1. Hit and Lead Discovery in an Academic Setting Julie Frearson Iain Collie University of Dundee Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

2. Introduction • The facility • Current approaches & technologies • Progress Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

3. A unique, fully integrated drug discovery team within a world class research environment DDU Drug Discovery Unit College of Life Sciences University of Dundee Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

4. Sir James Black Centre Screening laboratory with modular workstation layout The recently constructed James Black Centre housing, on level 1, the Drug Discovery Unit www.drugdiscovery.dundee.ac.uk Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

5. Vision, Mission, Key Objectives Vision To be an internationally recognised drug discovery group and partner of repute for the biopharmaceutical sector in developing new therapeutics for unmet need Mission To increase the efficiency and success of translating life sciences research into therapeutic application Key Objectives To translate basic biomedical research into candidate medicines for neglected diseases and To vastly improve intellectual property positions on novel therapeutic targets through small molecule discovery Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

6. Primary Activities Novel Targets & Pathways Neglected Diseases DDU Hits & Leads Leads & preclinical candidates Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

7. Drug Discovery @ Dundee • Total Funding over 5 years: ~£12m • ~67% Wellcome Trust • Remainder from • Scottish Funding Council • Scottish Enterprise • ITI Life Sciences Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

8. Key Successes ● ● ● Target Prioritisation 384 MTS/HTS Robotics Standard readouts Validation Druggability Assay Feasibility Toxicity Resistance potential Structural Information Compound Sets in vitro models in vivo models DMPK Data Management Medicinal & Computational Chemistry Structural Biology • Created a small Biotech style drug discovery group • Operational within 4 months of opening Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

9. Key Successes • Created a small Biotech style drug discovery group • Operational within 4 months of opening • Creation of purpose designed compound libraries • >97,000 compounds: diversity, bioactives, gene family focused • Prosecution of 18 target or phenotypic screens through hit discovery • 7 projects through hit to lead Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

10. DDU Portfolio Nov 2008 Nonsense mutation read through agents Stem cell modulation agents Numerous campaigns Target validation Assay Development Hit Discovery Hit Validation Hits to Leads Lead Optimization Sugar dehydrogenase De-N-acetylase PS-Q series Focussed kinase set phenotypic screen hits Phenotypic screening hit N-Myristoyl- transferase Kinase Kinase Chemical target validation PK4 KPS Series 01 Kinase RNA Ligase Chemical target validation GSK GSK 07 Kinase Viral protease PK50 Chemical target validation Replication licensing inhibitors Trypanothione synthetase Chemical target validation Phosphatase inhibitors NFκB repressor RNAi screen Fungal targets 2,3,4 SUMO repressor RNAi screen miRNA regulator RNAi screen Studies on hold and project returned to originating lab to address no go issues PTR1/DHFR UDP-Glc-4’- epimerase CRK3 UDP-GlcNAc diphosphorylase Trypanothione reductase Fungal Target 1 Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

11. Key Successes • Created a small Biotech style drug discovery group • Operational within 4 months of opening • Creation of purpose designed compound libraries • >97,000 compounds: diversity, bioactives, gene family focused • Prosecution of 18 targets/phenotypics screens through hit discovery • 7 projects through hit to lead • Advanced leads developed • Series curing trypanosomiasis in mice • Series under investigation in cancer xenograft models • First group to be routinely screening in high throughput using hES cells Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

12. Balanced Approach to Hit Discovery P. falciparum T. brucei Molecular Target hES cells keratinocytes • Diverse and flexible approach to lead discovery • Not limited to any one technology or target bias • Polypharmacology required for good & sustained efficacy? Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

13. Drug Discovery for Tropical Diseases initiative (DDTDi)- Goals To deliver at least 1 drug candidate for human African trypanosomiasis (HAT) for entry into formal pre-clinical development by March 2011 To deliver high quality lead series for neglected diseases suitable for progression into lead optimisation in-house or by partner organisations Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

14. Tropical Infectious Disease Burden • 35,000 deaths per day in developing world • Many of the deaths preventable • Many current drugs need to be replaced due to toxicity, cost, emerging resistance, suitability to use in the developing world • Health care budget in Sub-Saharan Africa • $13 per capita per year • No commercial market to offset ~$800m cost and risk to develop new drugs • The solution does not lie solely with the commercial sector Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

15. DDTDi Strategy Work in collaboration with academic, industrial and PDP partners Engaging in small focussed consortia to enable Drug Discovery for kinetoplastids from target to clinic Co-ordination with DNDi and SGC for lead generation Aligning ourselves with major downstream partners DNDi MMV Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

16. Gene Family Approach Kinases Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

17. Kinases as Drug Targets for T. brucei • Known druggable targets • Increasing genetic evidence of essentiality of kinases in trypanosomes and other parasites • Evidence of inhibitor selectivity between T. brucei and human homologues • Non-selectivity within tryp kinases an advantage • Acute dosing regimen for HAT could limit toxicity concerns Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

18. DDTDi Strategy • Developed focused kinase library • ~ 5000 compounds • >150 scaffolds with even representation • Tackle known genetically validated kinases in parallel • Collaborate with expert biology groups • Multiple kinase enzyme assays in parallel • Phenotypic screening against T. brucei, malaria and TB in vitro Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

19. ● ● ● Kinase Targets Under Investigation Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

20. TbGSK3 PI > 50% 87 cpds TbPK4 PI > 50% 65 cpds 1 cpds 12 cpds 10 cpds TbPK50 PI >50% 77 cpds Kinase Target Selectivity PK4: 70% confirmation rate Final hit rate 5.4% PK50: 91% confirmation rate Final hit rate 12.8% TbGSK3: 91% confirmation rate Final hit rate 12.6% GSK3 project advanced Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

21. Target vs. parasite proliferation - correlation? Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

22. Lack of trypanosome activity T. brucei potency pEC50 T. brucei potency pEC50 CRK3 potency pIC50 CRK3 potency pIC50 No evidence of Leishmania in vitro activity either (Mottram et al.) Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

23. Kinase Set Phenotypic Screen • Screen • Kinase Set • 3,885 compounds • 121 compounds progressed to potency • Multiple series • <1µM EC50 • >10-fold selectivity • Best • T. brucei EC50 20nM • MRC-5 EC50 >50M • Following up in chemistry and DMPK • Investigating Serenex, Cellzome technology to identify potential targets Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

24. N-Myristoyl Transferase Project Leaders: Stephen Brand, Stuart McElroy Debbie Smith (York) – Parasitology, Mode of action studies Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

25. NMT Introduction • Catalyses the transfer of myristate from MyCoA to the N-terminal glycine NH2 of potentially >60 target proteins • Myristoylation important for localisation at membranes and/or activation • Strong biological + chemical evidence for NMT being a viable target • Screened 63,362 compounds within DDU • Identified one key compound series (5 examples <20μM) • Hit validation: DDD00064558 resynthesised and retested Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

26. Summary so far • Identified sub nanomolar inhibitors of T. brucei NMT and T. brucei proliferation in culture • Good correlation - NMT IC50 and T. brucei activity • Selectivity over human enzyme (>100-fold) reflected in selectivity vs. MRC5 growth assay (>1000-fold) • Enzyme kinetics in line with proposed NMT Bi Bi mechanism • Cidal mode of action • Reduction of activity vs. T. brucei on 20-fold over- expression of TbNMT • Inhibition of protein myristoylation without inhibition of protein synthesis • Binding mode confirmed by X-ray structure Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

27. Good affinity achieved and early selectivity driven by varying off-rates HsNMT1 TbNMT Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

28. Pharmacokinetics of DDD00085646 Oral pharmacokinetics of DDD00085646; good exposure at 50mg/kg Cmax 6712 ng/ml Tmax 2 hours T1/2 2.0 hours AUC0-T 22 µg.h/ml EC90 (free) EC50 2nM (~1ng/ml) Fraction unbound 0.11 (89% ppb) Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

29. Efficacy in mouse model of acute African sleeping sickness Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

30. Conclusions • Balanced approach of molecular target and phenotypic screening vital in a drug discovery-immature area • Need robust assays & systems to support both approaches • Series of secondary assays to support target deconvolution • Particular success experienced with a molecular target leading to pleiotropic effects • Toxicity issues manageable – short term treatment? • Phenotypic hits may well be affecting multiple targets and therefore less susceptible to rapid resistance development • Efficient way forward for Neglected Disease Drug Discovery • Build up chemistries for druggable gene families and cross organism molecular targets to allow therapeutic switching • Leishmaniasis, Chagas, malaria etc. Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

31. Interactions • CRK3 - Jeremy Mottram (Univ. of Glasgow) • NMT - Debbie Smith (Univ. of York) • PK4, PK50,PLK - Tansy Hammarton (Univ. of Glasgow) • GSK3 - University of Washington; GSK • Quinols - Malcolm Stevens (Nottingham); Pharminox • Kinase Phenotypic Screening - Swiss Tropical Institute • Compound sets – Imperial College London, MRCT, WEHI, Max Planck (Dresden) • Companies – GlaxoSmithKline, Pfizer, Pharminox, Genzyme, Schering-Plough Scotland, BioFocus DPI, Vernalis, iTi Life Sciences • PDPs – DNDi, MMV • Drug Discovery Consortium – MRCT, CRT, ICR Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

32. Acknowledgements Core Group: Daan van Aalten, Ruth Brenk, Alan Fairlamb, Mike Ferguson, Julie Frearson, Ian Gilbert, Andrew Hopkins, Bill Hunter, Kevin Read, Paul Wyatt Lifting the Lid on Academic Laboratory Automation Hinxton Hall, Cambridge

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