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How to Develop an HTS Compatible AssayLucile WhiteSouthern Research InstituteHigh Throughput Screening Center1What is HTS?It is a system that uses specialized automation equipment and high density microtiter plates to screen a large number of wells in a short period of time. Throughput of 30,000 to 100,000 compounds per day is common.

2Key System ComponentsCompound management Precision robotics for liquid and plate handlingInformatics Associating data with a particular compoundAnalyzing data from a screenCheminformaticsPeople

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Since 2006, screened over 3 million compounds each year; 3.5 million in 2009In 30 40 assays/year4Percent of Compounds Screened by Assay Type

Percent Compounds Screened by Biocontainment Level

5Neils Bohrs Definition of an ExpertAn expert is someone who has made every mistake possible within a very narrow field of inquiry

The HTS Centers role is to help you not make the same mistakes we have already made

Why is this a bottleneck?Assay Development7When you go from this:

8To this:

9THE RULES CHANGE10What are you aiming for in an HTS assayTo have a reasonable chance to believe the results of a single determination, i.e. one well

For that you needReproducibility from well to wellReproducibility from assay plate to assay plateReproducibility from day to day11Rules ChangesMode of DetectionMode of ManipulationReproducibility in prepNo Physical InterventionNo Protocol Changes No if/then scenariosCounter Screens and Secondary AssaysCost for Failure12Mode of DetectionDetection methods simplified (i.e. homogenous mix and read)Plate readers (not high content)FluorescenceLuminescenceAbsorbanceFluorescence PolarizationFRET; TR-FRETAlphaScreen (Perkin Elmer)RT-PCRsiRNANot available at SRIRadioactivityFormats that need to be read within seconds after addition of reagentFlash luminescenceFLIPR Calcium channel sensing

13Why Not HCSExpensive and time consuming especially when 90-99% of the compounds will have no effectTo run as HTS, cells must be fixedFixing and adding dyes and/or antibodies require wash stepsClearly some types of questions can only be answered by HCS; however at this time I do not recommend this approach for HTSCurrently useful for 2nd or 3rd level assays low to medium throughput known actives

14New Technologies at SRI2010RT-PCRFor HTS it needs to be compatible with 1536-well platesThe 1536 instrument can process a plate in less than an hour allowing high throughput qPCR for screening operationsNot the best option because of high costAvailable by end of 2010siRNA (collaboration with Dr. Bjornsti)Ambion siRNA human genome library21,585 human targets with 3 non-overlapping siRNAs for each target; Silencer Select v4Available by end of 2010Infectious DiseasesBacterial Motility soft agar swarm assayBacterial BiofilmAntivirals- developing new methodology where CPE is not required

Mode of ManipulationRoboticShakersNon manual dissociation (can not do it with your hand, fingers, scrapers, etc)In general no centrifugationIn general no separation stepsIn general no wash stepsIf it can't be done by pipetting or shaking probably not HTS

16Reproducibility in PrepEvery steps adds variabilityMinimize variation in steps (eg. dispensing cell suspension minimize clumping)Minimize chance of error (eg. Ease of pipetting solution)Minimizing variability may not mean choosing conditions that give the maximum signal but optimizing around parameters where small changes produce very little change in the signalProvide excess time for step when available (eg. lyse for 20 minutes when 5 minutes is usually sufficient)Use stable cell lines rather than transient transfections17No Physical Intervention or Protocol ChangesRobots cant make judgment call (i.e. it looks like the cells are lysed, it looks like the solution is mixed)Cant let cells grow another dayCant incubate longer for an enzyme assayEnsure solutions pipette without clogging

Other IssuesAll HTS assays are subject to some type of compound interferenceInterference increases with lower wavelengthsRed shifted dyes preferred, eg GFP not good endpoint reagentTime-resolved delays helpRatiometric methods have not been useful in our handsIncubation temperature room tempCost associated with thousands of hitsAverage hit rates 0.5-3% so sooner you identify what is real and what is not the better off you are.19The Rules of HTSBegin with the end in mind (Stephen Covey)Design an HTS assay from the beginningDont try to automate a manual assay after the fact

KISS- Keep It Simple Stupid (Kelly Johmson)Simple robust assays are bestFewer variables increase the chance of successTwo General Types of AssaysDrug discovery assays can be divided into two broad classes: biochemical and cell-based

Both assay formats are amenable to miniaturization and adaptation to high-throughput screening

21Biochemical Assays:

pHTemperature (room temperature)Ion ConcentrationReagent StabilityReagent AggregationReagent SolubilityOrder of Reagent AdditionSolvent Effects (DMSO)Reagent ConcentrationSome Causes of Assay Variation22Cell and Organism Based Assays As for Biochemical assays, plus:

Cell culture plastics Culture media Culture conditions Serum Cell cycle Passage number Solvent effects (DMSO) InfectionTransient transfectionsHeterogenous population of cellsSome Causes of Assay Variation23Statistical Analysis: Z Factor 3SD of sample + 3SD of control mean of sample mean of controlZ = 1 Zhang, Chung and Oldenburgh, J. Biomol. Screening, 4:67-73, 199924SeparationBandData variabilityBandData variabilityBandmsmcFrequencyAssay Signal3ss3scStatistical Analysis25Z-plate analysis defines edge and row effects as minimal

Liquid handling methods perform as expectedZ = 0.86S/B = 83S/N = 24

Excellent Data!

26What You Assay is What You GetCapture the right biologyBalance with making assay so complex it can not be automated

Every assay has artifacts in the form of false positives and false negatives.Design and use counter screens and secondary assays

There is such a thing as a too stringent assay.Very high Z-factors can often indicate that the bar for something to show up as an active is set very high.Is the assay still in the linear range?27Costs for Failure$20-40,000 for every failed day in HTS

HTS schedule usually finalized a week to a month in advance failures cause a negative ripple effect in rearranging the schedule28Useful References for HTS Assay GuidanceAssay guidance manualwww.ncgc.nih.gov/manual/toc.htmlJoint effort of Lilly and NIH scientistsSections on assay development issues for specific assay formatsReporting data from HTS/information I am looking for to review an ADDA proposalInglese et al, Nature Chemical Biology, 3:438, 2007.Troubleshooting cell based assaysMaddox et al, J. Assoc. Lab. Automation 13:168-73, 2008.

29Compound LibrariesWhich library we will use does not need to be specified for the ADDA proposalWe will determine that in conjunction with the SRI chemists once we are closer to performing the screenSouthern Research has purchased several libraries from commercial suppliers for use in its HTS programOne example, a 100,000 compound library from ChemBridge Corporation was pre-filtered using a number of drug-likeness parameters including molecular weight