Transcript Document
Life Sciences High throughput screening and assay development – fit for purpose? Richard M. Eglen ELRIG, Manchester, UK September 7-8, 2011 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’? Life Sciences ELRIG 2 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’? Life Sciences ELRIG 3 Novel trends in drug discovery targets • 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 Rask-Andersen et al., 2011 Nature revs drug Disc. 10, 579. Life Sciences ELRIG 4 Novel drug targets – emerging trends • 435 drug targets in human genome… • …modulated by 989 unique drugs… • …via 2,242 drug-target interactions. Target Class Number Percentage Receptors 193 44% Enzymes 124 29% Transporters 67 15% Other 51 12% Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579. Life Sciences ELRIG 5 New drugs affecting human genome targets – structural classes Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579. Life Sciences ELRIG 6 Targeting drug networks …not targets e.g. PDEs Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579. Life Sciences ELRIG 8 Targeting drug networks e.g. EDGR/PDGR Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579. Life Sciences ELRIG 9 Smaller networks suggest both novel targets and mechanisms for intervention Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579. Life Sciences ELRIG 10 Novel drug targets – emerging trends (1982 – 2010) • 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 One drug/disease, one target or… One drug/disease, target network? Life Sciences ELRIG 11 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’? Life Sciences ELRIG 12 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. Life Sciences ELRIG 13 Discovering first-in-class NPIs Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507. Life Sciences ELRIG 14 Pharmacology of novel NPIs – approx. 50% target are enzymes Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507. Life Sciences ELRIG 15 NPIs – first in class vs. followers Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507. Life Sciences ELRIG 16 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. Life Sciences ELRIG 17 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. Life Sciences ELRIG 18 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’? Life Sciences ELRIG 19 Evolution of HTS e.g. Pfizer ADMET HTS Target based HTS HTS concept DMSO + synthetic cpmds 96 well plates 1984 1986 HTS Centralized Full file screening 1989 1987 Natural products screening Automation 10,000 assays/wk HTS ADMET P450, CACO2 binding 96 well Applied Biotech/Screening 7,200 cmpds/wk 20 concurrent HTS Cell based + biochemical RT-PCR 2880 cmpds/wk All data recorded 96 pipettors + harvesters after Pereira and Williams 2007, Br J Pharmacol 207, 152 Life Sciences 1995 1996 Recent advances Miniaturization Nanotechnology Academic entry NIH roadmap 1997 2000 2002- Pre Candidate tech 90 cmpds/wk HT LC-MS Cytotox MTT 180 cmpds/wk HT LC-MS Cytotox MTT 360 cmpds/wk HT LC-MS Cytotox MTT ELRIG 20 Evolution of HTS assay strategies Coupled protein readouts Isolated membrane studies Isolated protein activity Cell based phenotypic approaches Inglese et al 2007 Nature Chem Biol 3 466. Life Sciences ELRIG 21 Moving from bench top to HTS assays Parameter Bench top HTS Protocol Complex; heterogeneous Simple, homogeneous, automatable Assay volume Large (0.1 – 1 ml) Small (<1ul – 100ul) Reagents Quantity limited, variable quality Large reproducible quantity & quality Assay container Vial, cuvette, large well plates Microtiter plate Time of measurement msecs to months mins 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. Life Sciences ELRIG 22 Assay technologies: HTS & Dx compared Technology High Throughput Screening Diagnostics Absorbance Yes Yes Alpha, LOCI Yes Yes DELFIA Yes Yes ECL Yes Yes EFC, CEDIA Yes Yes FP Yes Yes FRET, TR-FRET Yes Yes Fl Yes Yes SPA, FlashPlate Yes Yes Life Sciences ELRIG 23 Emerging trends in HTS… In vivo (animal) screening In vitro (biochemical) screening In vivo (immortalized cells) screening Phenomenological Low throughput Disease relevance? Target based High throughput Disease relevance - low Target based High throughput Disease relevance? screening In vivo (ES & iPS cells) screening In vivo (primary cells) screening Phenotypically based Throughput? Disease relevance - high Phenotypically based High throughput Disease relevance - high Phenotypically based Low throughput Disease relevance - high In vivo (3D tissue assemblies) Life Sciences ELRIG 24 Converging trends in HTS … Imaging & phenotypic Microfluidic Label-free Disease relevant cells Life Sciences ELRIG 25 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? Life Sciences ELRIG 26 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’? Life Sciences ELRIG 27 Assay technologies: HTS & Dx compared Technology High Throughput Screening Diagnostics No Yes Yes? Yes Microarrays; DNA/RNA/Protein No Yes Microfluidics ??? Yes Biochemical Label-free Yes Yes Cellular Label-free Yes ??? Cellular Imaging Yes Yes Animal Imaging No Yes Next Gen Sequencing Mass Spectrometry Life Sciences ELRIG 28 Example 1 - stepping away from the microtiter plate • Microfluidics – Networks of channels, 10100nm 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. Life Sciences ELRIG 29 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. Life Sciences ELRIG 30 Example 3 - Cell trapping & sequential array cytometry • 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. Gossett et al. 2010, Ann Biomed Eng 39, 1328. Life Sciences ELRIG 31 Hand held Dx devices … implications for HTS? “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”. Dell et al., 2011NSDR’11, June 28, 2011, Bethesda, USA. Chin et al., 2011 Nat Med DOI 10.1038/nm.2408 Life Sciences ELRIG 33 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’? Life Sciences ELRIG 34 HTS contributions in Pharma drug development Macarron et al 2011 Nature Revs Drug Disc. 10, 188. Life Sciences ELRIG 35 Recently approved drugs with HTS origins Macarron et al 2011 Nature Revs Drug Disc. 10, 188. Life Sciences ELRIG 36 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 Common & incorrect HTS myths Why are so few drugs from HTS? • • • • • • • • Data is poor quality Expensive & time consuming Anti intellectual and irrational Fails to find leads for many targets 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. Life Sciences ELRIG 37 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? Life Sciences ELRIG 38 Questions?