richard m. eglen elrig, manchester, uk september 7-8, 2011
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High throughput screening and assay development – fit for purpose?. Richard M. Eglen ELRIG, Manchester, UK September 7-8, 2011. Agenda. Novel drug target trends Single target vs. cellular networks Target based vs. phenotypic screening HTS assay development - PowerPoint PPT PresentationTRANSCRIPT
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LifeSciences
Life Sciences
Richard M. EglenELRIG, Manchester, UKSeptember 7-8, 2011
High throughput screening and assay development – fit for purpose?
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2ELRIGLife 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’?
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3ELRIGLife 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’?
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• 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.
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• 435 drug targets in human genome…
• …modulated by 989 unique drugs…
• …via 2,242 drug-target interactions.
Novel drug targets – emerging trends
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.
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New drugs affecting human genome targets– structural classes
Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579.
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Targeting drug networks …not targetse.g. PDEs
Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579.
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Targeting drug networks e.g. EDGR/PDGR
Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579.
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Smaller networks suggest both novel targets and mechanisms for intervention
Rask-Andersen et al., 2011 Nature Revs Drug Disc. 10, 579.
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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)
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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’?
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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.
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Discovering first-in-class NPIs
Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507.
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Pharmacology of novel NPIs– approx. 50% target are enzymes
Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507.
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NPIs – first in class vs. followers
Swinney and Anthony 2011 Nature Revs Drug Disc. 10, 507.
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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.
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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.
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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’?
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Evolution of HTS e.g. Pfizer
360 cmpds/wkHT LC-MSCytotox MTTafter Pereira and Williams 2007, Br J Pharmacol 207, 152
1984 1986 1987 1989 1995 1996 1997 2002-2000
Natural products screeningAutomation10,000 assays/wk
HTS conceptDMSO + synthetic cpmds96 well plates
2880 cmpds/wkAll data recorded96 pipettors + harvesters
Applied Biotech/Screening7,200 cmpds/wk20 concurrent HTSCell based + biochemicalRT-PCR
Pre Candidate tech90 cmpds/wkHT LC-MSCytotox MTT
180 cmpds/wkHT LC-MSCytotox MTT
HTSCentralizedFull file screening
HTS ADMETP450, CACO2 binding96 well
Recent advancesMiniaturizationNanotechnologyAcademic entryNIH roadmap
Target based HTS ADMET HTS
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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
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Moving from bench top to HTS assays
Parameter Bench top HTS
Protocol Complex; heterogeneousSimple, homogeneous, automatable
Assay volume Large (0.1 – 1 ml) Small (<1ul – 100ul)
Reagents Quantity limited, variable qualityLarge reproducible quantity & quality
Assay container Vial, cuvette, large well plates Microtiter plate
Time of measurement msecs to months mins to hours
Output formatRadioactive, 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.
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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
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Emerging trends in HTS…
In vivo (animal)
screening
PhenomenologicalLow throughput
Disease relevance?
In vitro (biochemical)
screening
Target basedHigh throughput
Disease relevance - low
In vivo (immortalized cells)
screening
Target basedHigh throughput
Disease relevance?
In vivo (primary cells)
screening
Phenotypically basedLow throughput
Disease relevance - high
In vivo (ES & iPS cells)
screening
Phenotypically basedHigh throughput
Disease relevance - high
In vivo (3D tissue assemblies)
screening
Phenotypically basedThroughput?
Disease relevance - high
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Converging trends in HTS …
Microfluidic
Imaging & phenotypic
Label-free
Diseaserelevant
cells
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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?
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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’?
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Assay technologies: HTS & Dx compared
Technology High Throughput Screening Diagnostics
Next Gen Sequencing No Yes
Mass Spectrometry 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
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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.
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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.
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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.
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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 immunoassaytest data on a smart phone. The speed and accuracy demonstrated by the applicationsuggest 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
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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’?
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HTS contributions in Pharma drug development
Macarron et al 2011 Nature Revs Drug Disc. 10, 188.
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Recently approved drugs with HTS origins
Macarron et al 2011 Nature Revs Drug Disc. 10, 188.
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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
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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?
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Questions?
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