1. Life Sciences Life Sciences Richard M. Eglen ELRIG, Manchester, UK September 7-8, 2011 High throughput screening and assay development – fit for purpose?

2. 2ELRIG Life Sciences Agenda Novel drug target trends –Single target vs. cellular networks –Target based vs. phenotypic screening HTS assay development –Parallels & differences with diagnostic assays Emerging trends –Predicting the future Assay development –…still ‘fit for purpose’?

3. 3ELRIG Life Sciences Agenda Novel drug target trends –Single target vs. cellular networks –Target based vs. phenotypic screening HTS assay development –Parallels & differences with diagnostic assays Emerging trends –Predicting the future Assay development –…still ‘fit for purpose’?

4. 4ELRIG Life Sciences 1996 - Drews & Ryser –483 drug targets exploited –5 - 10,000 targets in human genome (prevailing view was 300,000 genes in genome) 2002 - Hopkins & Groom –120 drug targets for marketed small molecule drugs –399 targets druggable 10% of human genes pharmacologically tractable 2002 - Human genome sequenced –30,000 genes; 3,000 linked to disease; 600 – 1500 druggable targets 2006 - Imming et al –218 drug targets 2006 - Overington et al –324 pharmacological targets assigned to 1065 pharmacological agents Novel trends in drug discovery targets Rask-Andersen et al., 2011 Nature revs drug Disc. 10, 579.

5. 5ELRIG Life Sciences 435 drug targets in human genome… …modulated by 989 unique drugs… …via 2,242 drug-target interactions. Novel drug targets – emerging trends Target ClassNumberPercentage Receptors19344% Enzymes12429% Transporters6715% Other5112% Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579.

6. 6ELRIG Life Sciences New drugs affecting human genome targets – structural classes Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579.

7. 7ELRIG Life Sciences New drugs affecting human genome targets – drugs at novel targets Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579.

8. 8ELRIG Life Sciences Targeting drug networks …not targets e.g. PDEs Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579.

9. 9ELRIG Life Sciences Targeting drug networks e.g. EDGR/PDGR Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579.

10. 10ELRIG Life Sciences Smaller networks suggest both novel targets and mechanisms for intervention Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579.

11. 11ELRIG Life Sciences One drug/disease, one target or… One drug/disease, target network? A constant rate of NPI introduction …but not in line with increases in R&D investments Most drugs approved were acting at previously exploited targets Older drugs were most ‘connected’ in terms of drug networks Newer drugs (2005 – 2010) directed at smaller novel networks Novel drug targets – emerging trends (1982 – 2010)

12. 12ELRIG Life Sciences Agenda Novel drug target trends –Single target vs. cellular networks –Target based vs. phenotypic screening HTS assay development –Parallels & differences with diagnostic assays Emerging trends –Predicting the future Assay development –…still ‘fit for purpose’?

13. 13ELRIG Life Sciences Target based vs. network based screening – the rise and rise of phenotypic screening? Target based drug discovery –Accelerated by the human genome sequencing revolution & advances in structure based analysis, in vitro and in silico –Target identification & validation a ‘sine qua non’ …how successful has this approach been? 2011 - Swinney and Anthony –1998 - 2008; 259 NPIs approved –75 had novel mechanisms of action 67% small molecules; 33% Biologics 28 by phenotypic screening; 17 by target based screening Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507.

14. 14ELRIG Life Sciences Discovering first-in-class NPIs Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507.

15. 15ELRIG Life Sciences Pharmacology of novel NPIs – approx. 50% target are enzymes Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507.

16. 16ELRIG Life Sciences NPIs – first in class vs. followers Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507.

17. 17ELRIG Life Sciences NPIs according to mode of discovery First in class: Novel mechanisms of action (MOAs) Follower drugs: Established MOAs Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507.

18. 18ELRIG Life Sciences Target based vs. phenotypic drug screening Phenotypic screening more successful that historically realized –Can give rise to more successful first in class NPIs Robust MOA knowledge is required to optimize back ups –Fast follower NPIs often target based as a result. But… –Robust knowledge of MOAs yields new drug approaches allosterism, kinetics, complex binding phenomena etc. –Generally, critical for Biologic-based drug design 33% of first in class drugs were Biologics e.g. mAbs How well do phenotypic assays translate to human disease? How fit for purpose are current assay development & screening? Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507.

19. 19ELRIG Life Sciences Agenda Novel drug target trends –Single target vs. cellular networks –Target based vs. phenotypic screening HTS assay development –Parallels & differences with diagnostic assays Emerging trends –Predicting the future Assay development –…still ‘fit for purpose’?

20. 20ELRIG Life Sciences Evolution of HTS e.g. Pfizer 360 cmpds/wk HT LC-MS Cytotox MTT after Pereira and Williams 2007, Br J Pharmacol 207, 152 198419861987 198919951996 19972002-2000 Natural products screening Automation 10,000 assays/wk HTS concept DMSO + synthetic cpmds 96 well plates 2880 cmpds/wk All data recorded 96 pipettors + harvesters Applied Biotech/Screening 7,200 cmpds/wk 20 concurrent HTS Cell based + biochemical RT-PCR Pre Candidate tech 90 cmpds/wk HT LC-MS Cytotox MTT 180 cmpds/wk HT LC-MS Cytotox MTT HTS Centralized Full file screening HTS ADMET P450, CACO2 binding 96 well Recent advances Miniaturization Nanotechnology Academic entry NIH roadmap Target based HTS ADMET HTS

21. 21ELRIG Life Sciences Evolution of HTS assay strategies Inglese et al 2007 Nature Chem Biol 3 466. Isolated membrane studies Coupled protein readouts Cell based phenotypic approaches Isolated protein activity

22. 22ELRIG Life Sciences Moving from bench top to HTS assays ParameterBench topHTS ProtocolComplex; heterogeneous Simple, homogeneous, automatable Assay volumeLarge (0.1 – 1 ml)Small (<1ul – 100ul) ReagentsQuantity limited, variable quality Large reproducible quantity & quality Assay containerVial, cuvette, large well platesMicrotiter plate Time of measurementmsecs to monthsmins to hours Output format Radioactive, size separation, imaging Plate reader based, Fl, imaging, label free Reporting format “Representative data”, manually curated datasets Automated data analysis Inglese et al 2007 Nature Chem Biol 3 466.

23. 23ELRIG Life Sciences Assay technologies: HTS & Dx compared TechnologyHigh Throughput ScreeningDiagnostics AbsorbanceYes Alpha, LOCIYes DELFIAYes ECLYes EFC, CEDIAYes FPYes FRET, TR-FRETYes FlYes SPA, FlashPlateYes

24. 24ELRIG Life Sciences Emerging trends in HTS… In vivo (animal) screening Phenomenological Low throughput Disease relevance? In vitro (biochemical) screening Target based High throughput Disease relevance - low In vivo (immortalized cells) screening Target based High throughput Disease relevance? In vivo (primary cells) screening Phenotypically based Low throughput Disease relevance - high In vivo (ES & iPS cells) screening Phenotypically based High throughput Disease relevance - high In vivo (3D tissue assemblies) screening Phenotypically based Throughput? Disease relevance - high

25. 25ELRIG Life Sciences Converging trends in HTS … Microfluidic Imaging & phenotypic Label-free Disease relevant cells

26. 26ELRIG Life Sciences Questions… Adopting diagnostic (Dx) assay platforms, automation and detection systems has provided a strong basis for HTS assay development. Drug network, cell based and phenotypic screening approaches are being more widely adopted. Will the historical parallels of Dx technologies providing HTS assay formats hold going forward? How fit for purpose are classical HTS assays for the next generation of drug targets?

27. 27ELRIG Life Sciences Agenda Novel drug target trends –Single target vs. cellular networks –Target based vs. phenotypic screening HTS assay development –Parallels & differences with diagnostic assays Emerging trends –Predicting the future Assay development –…still ‘fit for purpose’?

28. 28ELRIG Life Sciences Assay technologies: HTS & Dx compared TechnologyHigh Throughput ScreeningDiagnostics Next Gen SequencingNoYes Mass SpectrometryYes?Yes Microarrays; DNA/RNA/ProteinNoYes Microfluidics???Yes Biochemical Label-freeYes Cellular Label-freeYes??? Cellular ImagingYes Animal ImagingNoYes

29. 29ELRIG Life Sciences Example 1 - stepping away from the microtiter plate Microfluidics –Networks of channels, 10- 100nm in diameter –Biochemical & cell based rapidly being developed for diagnostics, notably PCR systems e.g. Droplet based microfluidics –overcomes mixing issues, laminar flow issues Allows physiologically relevant cell-based assays (Chapman, 2004) –e.g. Assays with primary human cells Clausell-Tormos et al. 2008, Chem & Biol 15, 427.

30. 30ELRIG Life Sciences Example 2 - Single cell flow cytometry and immune profiling Cells stained with epitope-specific antibodies conjugated to transition element isotope reporters, each with a different mass. Cells nebulized into single-cell droplets, and an elemental mass spectrum is acquired for each. The integrated elemental reporter signals for each cell can then be analyzed by using flow cytometry. Bendall et al. 2011, Science 332, 687.

31. 31ELRIG Life Sciences Example 3 - Cell trapping & sequential array cytometry Gossett et al. 2010, Ann Biomed Eng 39, 1328. Hydrodynamic cell trapping for exchange of solutions and imaging. (a) Three-dimensional hydrodynamic cell traps were created in massive arrays (b) Cell traps are raised to allow fluid streamlines to pass beneath them, dragging in cells. No external forces other than the fluid driving force are needed. (c) Hydrodynamically trapped cells can have fluid solutions exchanged around them, allowing for sequential staining and imaging of a constant set of cells.

32. 32ELRIG Life Sciences Antibody based diagnostics and cancer personalized medicine Brennan et al. 2010, Nature Revs Cancer August, on line.

33. 33ELRIG Life Sciences Hand held Dx devices … implications for HTS? Dell et al., 2011NSDR’11, June 28, 2011, Bethesda, USA. “In this paper, we present a mobile application that automatically quantifies immunoassay test data on a smart phone. The speed and accuracy demonstrated by the application suggest that cell-phone based analysis could aid disease diagnosis at the point of care”. Chin et al., 2011 Nat Med DOI 10.1038/nm.2408

34. 34ELRIG Life Sciences Agenda Novel drug target trends –Single target vs. cellular networks –Target based vs. phenotypic screening HTS assay development –Parallels & differences with diagnostic assays Emerging trends –Predicting the future Assay development –…still ‘fit for purpose’?

35. 35ELRIG Life Sciences HTS contributions in Pharma drug development Macarron et al 2011 Nature Revs Drug Disc. 10, 188.

36. 36ELRIG Life Sciences Recently approved drugs with HTS origins Macarron et al 2011 Nature Revs Drug Disc. 10, 188.

37. 37ELRIG Life Sciences HTS …views from the front line Fit for purpose: …something that is ‘fit for purpose’ is good enough to do the job it was designed to do… Webster Why are so few drugs from HTS? Poorly validated targets Non physiological screens Limited informatics Unpredictable ADME&T Misplaced and naïve expectations! Macarron et al 2011 Nature Revs Drug Disc. 10, 188. Common & incorrect HTS myths Data is poor quality Expensive & time consuming Anti intellectual and irrational Fails to find leads for many targets

38. 38ELRIG Life Sciences Assay development & HTS – still ‘fit for purpose’? HTS is historically a successful and well integrated activity in drug discovery… …adapting many technologies initially developed for the in vitro diagnostic (IVD) industry. As novel target types - and target networks - are validated, it is likely that new assay technologies (non microtiter plate based?) will need to be adopted. The impact of phenotypic screening may have been underestimated…this fact, plus near universal adoption of cellular assays…suggests HTS assay development will no longer mirror technologies developed for IVDs. Next generation HTS technologies? – what, when & where?

39. Questions?