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
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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’?
Life Sciences
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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.
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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.
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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 targets
e.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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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?
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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’?
Life Sciences
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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’?
Life Sciences
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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
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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.
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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.
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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
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)
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Converging trends in HTS …
Imaging &
phenotypic
Microfluidic
Label-free
Disease
relevant
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’?
Life Sciences
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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
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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.
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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
•
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.
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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
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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’?
Life Sciences
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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
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.
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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?