small molecule screens clifford stephan, ph.d. research assistant professor john s. dunn gcc...

SMALL MOLECULE SCREENS

Clifford Stephan, Ph.D.Research Assistant Professor

John S. Dunn GCC Chemical Genomics Research Consortium

A Workshop on High Throughput / High Content Screening Applications to Target-based Drug Discovery Research

Scott R. Gilbertson, Ph.D.Professor

M.D. Anderson Chair in the College of Natural Sciences and MathematicsDepartment of Chemistry, University of Houston

Stages of Commercial Drug Development

Drug Development is a game of attrition. The Challenge … Select 1-2 compounds from the millions of possibilities that will be safe and efficacious in humans

Basic ResearchBasic Research Target ValidationTarget Validation HTS and Lead IdHTS and Lead Id Lead OptLead Opt

Preclinical ToxPreclinical Tox File IND File IND Clinical Phase 0/1Clinical Phase 0/1 Clinical Phase 2Clinical Phase 2

Clinical Phase 3Clinical Phase 3 File NDAFile NDA ApprovalApproval Sales Marketing Phase 4Sales Marketing Phase 4

Stages of Academic Drug Development

Still a game of attrition. The Challenge … Identify agents that increase the fundamental scientific knowledgefor a particular target with the possibility of providing further validationof the target as a ‘druggable’ target.

Retain the possibility of identifying a lead series of compoundsthat could take our research in new, unexpected directions.

The possibility of establishing intellectual property and the basis for afuture pharmaceutical.

Basic ResearchBasic Research HTS and Lead IdHTS and Lead Id Lead OptLead Opt

Why perform High Throughput Screening?

HTS enables the testing of large numbers of chemicalsubstances for activity in diverse areas of biology ina relatively short time.

The entire chemical space of small organic molecules isestimated to be > 1060. Of those, ~ 2.7 x 107 compoundshave been registered and made. (Nature Insight, 2004)

Responses studied can range from biochemicalsystems of purified proteins or enzymes to signaltransduction pathways to complex cellular networks(Systems Biology).

UltraHTS test >1,000,000 data points/day

10,000 Compounds/day


96-Well plate(80 compounds/plate) 125 Plates/day 1250 Plates/day



High Throughput Screening: A relative term

HTS in Pharma and Biotech is a process loosely defined astesting 10,000 to 100,000 data points/day using ‘industrialized’ methods


96-Well plate(80 compounds/plate) 125 Plates/day



HTS in the Dunn Screening Core the potential of screening100’s to >10,000 data points/day following Pharma industry standards

The Screening Continuum

From a poster by RR Tice et al of the National Toxicology Program HTS Initiative, 2007

Homogeneous‘Mix and Read” style assaysSimple with addition steps only, higher throughputExamples:

Cell viabilityLive cell imagingProximity (e.g., radioisotope, FRET, ALPHA)Enzyme Kinetics

HeterogeneousTraditional style assaysMultiple steps, more manipulations, slower throughputExamples:

Traditional binding assaysTraditional sandwich ELISA

Classes of HTS Assays

Parameter Traditional Assay HTS Assay

Protocol Complex, numerous steps•Multiple Additions•Multiple Aspirations•Multiple Washes

Simple, few steps (3-5)•Additions•Aspirations - discouraged•Washes - discouraged




Assay Container VariedTubes/slides/dishes/cuvettes/animals

Microtiter plates96/384/1536-Wells






Assay Volume 0.1 to 1 mL < 1 μl to 100 μl







Reagents Varied•Limited quantity•Various batches•Limited stability

Consistent•QS for complete screen•Single batch - preferred•Prolonged stability









Variables Many•Time/temperature•Ligand/substrate concentration•Cell type

Compound










Compound

Readout Time Milliseconds to months Minutes to hours










Compound

Readout Time Milliseconds to months Minutes to hours

Output Formats VariedReaders/scoring/image interpretation

Plate readerAbsorbance/fluorescence/luminescence

Compare Traditional Assays with HTS Assays


HTSScreen

Time

CostsQuality

time/wellwells/dayscreens/yearproject time

reagentsconsumablesinstrumentationpersonnel

few false positivesfew false negativesS:N,SW,z’-FactorValidated ‘Hits’

Key factors for successful HTS

HTS: An Iterative Process

HTS GroupSecondary ScreenPurpose: Validate initial ‘Hits’Method: Selection of compoundsor medicinal chemistry

Chemistry groupsAnalysis and interpretation of Data for

Structure Activity RelationshipsRefine and improve identified ‘Hits’

Modeling and medicinal chemistrySelection of compounds for screening via

virtual screening, focused libraries

HTS GroupPerform Primary ScreenPurpose: Identify a starting placeMethod: Interrogate libraries of

compounds/genes

Research groupsTarget Id and ValidationDevelop Primary and Secondary assaysDefine criteria for active compoundsDirect ‘Hit’ improvement process

Critical Issues to be Addressed Prior to Testing the First Compound

Key factors that must be addressed prior to screening:

• Assay protocol (miniaturization/simplification)• DMSO resistance (test 0.1 - 5%), standard compound vehicle• Reagent quantity and batch consistency• Reagent stability for storage and use under assay conditions• Appropriate positive and negative controls• Assay reproducibility and signal stability• Available secondary or counter screen to test target

specificity and selectivity

Consider: Reagent QuantityCompare traditional assays with HTS assays

Traditional Assay 96-Well HTS 384-Well HTS

Plates/Day 1 40 40

Total Plates 5 375 94

Cells/Day 1 x 106 40 x 106 40 x 106

Total Cells 5 x 106 375 x 106 94 x 106

Total Assay

Volume (μL)100 100 25

μL/Well User purified

reagent10 10 2.5

mL Purified rgt/plate 1 1 1

mL Total purified rgt 5 375 94

30,000 Compounds Traditional Assay 96-Well HTS

Plates/Day 1 40

Total Plates 5 375

Cells/Day 1 x 106 40 x 106

Total Cells 5 x 106 375 x 106

Total Assay

Volume (μL)100 100


reagent10 10

mL Purified rgt/plate 1 1

mL Total purified rgt 5 375

Traditional Assay

Plates/Day 1

Total Plates 5

Cells/Day 1 x 106

Total Cells 5 x 106

Total Assay

Volume (μL)100


reagent10

mL Purified rgt/plate 1

mL Total purified rgt 5

Consider: Reagent StabilityCompare traditional assays with HTS assays

Traditional assay:Reagent stability

30-minutes to manually setup plate2-hr incubation for an end-point measurement5-minutes to read plate

Reagents need to be stable for up to ~2.5hr

HTS assay:Reagent stability

5-minutes to setup each plate2-hr incubation for an end-point measurement5-minutes to read plate, 40 plates/run total

Reagents need to be stable from first to last plateUp to ~3 hrs for plate setup, ~3hr read timeUp to 8 hrs from start to finishCan remaining reagents be reused

Consider: Availability of appropriatepositive and negative controls

Traditional assessments of assay quality

S/B = Mean PosCtl / Mean NegCtl

S/N = (Mean PosCtl - Mean NegCtl) / StdDev NegCtl

0

10

20

30

40

50

60

70

0 8 16 24 32 40 48 56 64 72 80 88 96

ve+

ve-

Assay 1Ve+ mean 50, Ve- mean 10S/B = 5, S/N = 13

Assay 2Ve+ mean 112, Ve- mean 10S/B = 11, S/N = 39

0

50

100

150

200

250

0 8 16 24 32 40 48 56 64 72 80 88 96

ve+

ve-

Availability of appropriatepositive and negative controls

Common HTS assessment of assay quality

(3 * StdDev PosCtl) + ( 3 * StdDev NegCtl)z’ = 1 - ----------------------------------------------------------- Mean PosCtl - Mean NegCtl


0

10

20

30

40

50

60

70

0 8 16 24 32 40 48 56 64 72 80 88 96

ve+

ve-

Ve+ mean 50, Ve- mean 10S/B = 5, S/N = 13, z’ = 0.5


Separation Band


Ve+ mean 112, Ve- mean 10S/B = 11, S/N = 39, z’ = 0.0

0

50

100

150

200

250

0 8 16 24 32 40 48 56 64 72 80 88 96

ve+

ve-



z’ = 0.5 z’ = 0.1

z’ = 1 An ideal assay

1 > z’ ≥ 0.5 A good assay

0.5 > z’ > 0 Assay will require replicates

z’ = 0 “Yes/No” type assay


Assay Issues to be Addressed Prior toTesting the First Compound

Plate Uniformity and Signal Variability Testing

Critical testing of an assay system prior to screening for all assaysrun in the core

These assays test the performance of the following controls:

Maximum signal reference (highest assay end point)

Minimum signal reference (background/lowest assay end point)

Midrange signal reference (signal variability assessment)

For all assays run in the core, similar results must be obtainedover three separate days (independent experiments in triplicate)using all equipment and compound vehicle that will be usedduring the screen.

Acceptance criteria:

Intraplate variability:No apparent edge effects or drift%CVmax and %CVmin < 20%z’ ≥ 0.4

Interplate and Inter-Day variability:Midrange control < 2-fold within a single dayMidrange control < 2-fold across any two days

Assay Issues to be Addressed Prior toTesting the First Compound

What Are These Small Molecules We Test?

They are not DNA, RNA, or protein macromolecules

Practical Definition:An organic molecule of less than 1000 DaltonsTypically in the range of 300-700 Daltons

Small organic molecules made by living organisms(e.g., natural products)

Small organic molecules made by chemists(e.g., organic compounds, RNAi)

In all cases one is looking for a small ‘drug-like’ organicmolecule that displays a biological activity(e.g., agonist, antagonist) with the target of interest.

How Does One Select a Library to Screen?

Random SelectionRandom high throughput screeningLittle is known about the targetFew or no active compounds as guides

Computational Chemistry/Virtual ScreeningCreation of ‘Focused Libraries’Requires prior knowledge about target

Active compounds, 3D-StructureSequence homology

Prior ExperienceLibrary successfully used for similar

or related targets

Core Lab HTS Hit Guidelines

z’ = 0.5 z’ = 0.1

On each screening day, z’-factor (controls) is evaluatedfor every plate

Controls must meet original acceptance criteriapreviously defined for the assay

Only outliers dropped are those created becauseof assay error or those > 3 SD from mean for all ofthat particular control

No more than 10-25% of a particular control will bedropped for any particular plate

Active compounds are those outside 3 SD from the meanfor all test agents on a valid plate.

If replicates are performed, a test agent must be active on≥ 66% of all replicates to be classified as active.

‘Hits’ are those active compounds that demonstrateconcentration response upon reorder and retest.

Core Lab HTS Hit Guidelines

Helpful References:

Inglese J, Johnson RL, Simeonov A, Xia M, Zheng W, Austin CP, Auld DS.High-throughput screening assays for the identification of chemical probes.Nat Chem Biol. 2007 Aug;3(8):466-79.

Zhang JH, Chung TD, Oldenburg KR.A Simple Statistical Parameter for Use in Evaluation and Validation of

High Throughput Screening Assays.J Biomol Screen. 1999;4(2):67-73.

Inglese J, Shamu CE, Guy RK.Reporting data from high-throughput screening of small-molecule libraries.Nat Chem Biol. 2007 Aug;3(8):438-41.

Iversen PW, Eastwood BJ, Sittampalam GS, Cox KL.A comparison of assay performance measures in screening assays:

signal window, Z' factor, and assay variability ratio.J Biomol Screen. 2006 Apr;11(3):247-52.

National Institutes of Health (NIH) Chemical Genomics Center (NCGC), a member of the Molecular Libraries Probe Production Center Network.http://ncgc.nih.gov/

Cliff Stephan, Ph.D.

B.A. Chemistry andMolecular Biology

Ph.D. Pharmacology

Postdoctoral training

Cardiovascular Division

Research Instructor, Cardiovascular Department, Hypertension Division

Director of High ThroughputScreening

Head of the John S Dunn Central ScreeningCore Laboratory

small molecule screens clifford stephan, ph.d. research assistant professor john s. dunn gcc...

Documents

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