Richard M. Eglen ELRIG, Manchester, UK September 7-8, 2011

LifeSciences

Life Sciences

Richard M. EglenELRIG, Manchester, UKSeptember 7-8, 2011

High throughput screening and assay development – fit for purpose?

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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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Agenda





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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


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Targeting drug networks …not targetse.g. PDEs


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Targeting drug networks e.g. EDGR/PDGR


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Smaller networks suggest both novel targets and mechanisms for intervention



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)


Agenda






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.


Discovering first-in-class NPIs



Pharmacology of novel NPIs– approx. 50% target are enzymes



NPIs – first in class vs. followers



NPIs according to mode of discovery

First in class: Novel mechanisms of action (MOAs)

Follower drugs:Established MOAs



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?



Agenda






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


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


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.


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


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


In vivo (3D tissue assemblies)

screening

Phenotypically basedThroughput?



Converging trends in HTS …

Microfluidic

Imaging & phenotypic

Label-free

Diseaserelevant

cells


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?


Agenda






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


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.


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.


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.


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


Agenda






HTS contributions in Pharma drug development

Macarron et al 2011 Nature Revs Drug Disc. 10, 188.


Recently approved drugs with HTS origins



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!


Common & incorrect HTS myths

• Data is poor quality• Expensive & time consuming• Anti intellectual and irrational• Fails to find leads for many targets


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?

Questions?

Documents

Richard M. Eglen ELRIG, Manchester, UK September 7-8, 2011