How to Develop an HTS Compatible Assay

Lucile WhiteSouthern Research Institute

High Throughput Screening Center

What 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.

Key System Components

• Compound management

• Precision robotics for liquid and plate handling

• Informatics – Associating data

with a particular compound

– Analyzing data from a screen

• Cheminformatics• People

Since 2006, screened over 3 million compounds each year; 3.5 million in 2009In 30 – 40 assays/year

Percent of Compounds Screened by Assay Type

Percent Compounds Screened by Biocontainment Level

Neils Bohr’s Definition of an Expert

• An expert is someone who has made every mistake possible within a very narrow field of inquiry

• The HTS Center’s role is to help you not make the same mistakes we have already made

Why is this a bottleneck?

Assay Development

When you go from this:

To this:

THE RULES CHANGE

• What are you aiming for in an HTS assay– To have a reasonable chance to believe the results of a

single determination, i.e. one well

• For that you need– Reproducibility from well to well– Reproducibility from assay plate to assay plate– Reproducibility from day to day

Rules Changes

• Mode of Detection• Mode of Manipulation• Reproducibility in prep• No Physical Intervention• No Protocol Changes – No if/then scenarios• Counter Screens and Secondary Assays• Cost for Failure

Mode of Detection• Detection methods simplified (i.e. homogenous mix and read)• Plate readers (not high content)

– Fluorescence– Luminescence– Absorbance– Fluorescence Polarization– FRET; TR-FRET– AlphaScreen (Perkin Elmer)– RT-PCR– siRNA

• Not available at SRI– Radioactivity– Formats that need to be read within seconds after addition of

reagent• Flash luminescence• FLIPR – Calcium channel sensing

Why Not HCS

• Expensive and time consuming especially when 90-99% of the compounds will have no effect

• To run as HTS, cells must be fixed• Fixing and adding dyes and/or antibodies require

wash steps• Clearly some types of questions can only be

answered by HCS; however at this time I do not recommend this approach for HTS– Currently useful for 2nd or 3rd level assays – low

to medium throughput – known actives

New Technologies at SRI2010

• RT-PCR– For HTS it needs to be compatible with 1536-well plates– The 1536 instrument can process a plate in less than an hour allowing

high throughput qPCR for screening operations– Not the best option because of high cost– Available by end of 2010

• siRNA (collaboration with Dr. Bjornsti)– Ambion siRNA human genome library– 21,585 human targets with 3 non-overlapping siRNAs for each target;

Silencer Select v4– Available by end of 2010

• Infectious Diseases– Bacterial Motility – soft agar swarm assay– Bacterial Biofilm– Antivirals- developing new methodology where CPE is not required

Mode of Manipulation

• Robotic• Shakers• Non manual dissociation (can not do it with your

hand, fingers, scrapers, etc)• In general no centrifugation• In general no separation steps• In general no wash steps• If it can't be done by pipetting or shaking

probably not HTS

Reproducibility in Prep

• Every steps adds variability• Minimize 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 signal

• Provide excess time for step when available (eg. lyse for 20 minutes when 5 minutes is usually sufficient)

• Use stable cell lines rather than transient transfections

No Physical Intervention or Protocol Changes

• Robots can’t make judgment call (i.e. it looks like the cells are lysed, it looks like the solution is mixed)

• Can’t let cells grow another day• Can’t incubate longer for an enzyme assay• Ensure solutions pipette without clogging

Other Issues• All HTS assays are subject to some type of

“compound” interference– Interference increases with lower wavelengths– Red shifted dyes preferred, eg GFP not good

endpoint reagent– Time-resolved delays help– Ratiometric methods have not been useful in

our hands• Incubation temperature – room temp• Cost associated with thousands of hits• Average hit rates 0.5-3% so sooner you identify

what is real and what is not the better off you are.

The Rules of HTS

• Begin with the end in mind (Stephen Covey)– Design an HTS assay from the beginning– Don’t try to automate a manual assay after the fact

• KISS- Keep It Simple Stupid (Kelly Johmson)– Simple robust assays are best– Fewer variables increase the chance of success

Two General Types of Assays

• Drug 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

• Biochemical Assays:

• pH• Temperature (room temperature)• Ion Concentration• Reagent Stability• Reagent Aggregation• Reagent Solubility• Order of Reagent Addition• Solvent Effects (DMSO)• Reagent Concentration

Some Causes of Assay Variation

Cell and Organism Based Assays As for Biochemical assays, plus:

• Cell culture plastics• Culture media• Culture conditions• Serum• Cell cycle• Passage number• Solvent effects (DMSO)• Infection• Transient transfections• Heterogenous population of cells

Some Causes of Assay Variation

Statistical Analysis: Z Factor

3SD of sample + 3SD of control mean of sample – mean of control

Z = 1 –

Zhang, Chung and Oldenburgh, J. Biomol. Screening, 4:67-73, 1999

SeparationBand

Data variabilityBand

Data variabilityBand

ms mc

Fre

quen

cy

Assay Signal

3ss 3sc

Statistical Analysis

• Z-plate analysis defines edge and row effects as minimal

• Liquid handling methods perform as expected

• Z = 0.86• S/B = 83• S/N = 24

Excellent Data!

What You Assay is What You Get

• Capture the right biology– Balance 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?

Costs 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 schedule

Useful References for HTS Assay Guidance

• Assay guidance manual– www.ncgc.nih.gov/manual/toc.html– Joint effort of Lilly and NIH scientists– Sections on assay development issues for specific assay

formats• Reporting data from HTS/information I am looking

for to review an ADDA proposal– Inglese et al, Nature Chemical Biology, 3:438, 2007.

• Troubleshooting cell based assays– Maddox et al, J. Assoc. Lab. Automation 13:168-73,

2008.

Compound Libraries

• Which library we will use does not need to be specified for the ADDA proposal– We will determine that in conjunction with the SRI

chemists once we are closer to performing the screen• Southern Research has purchased several libraries from

commercial suppliers for use in its HTS program– One example, a 100,000 compound library from ChemBridge Corporation was

pre-filtered using a number of drug-likeness parameters including molecular weight <500, <5 hydrogen bond donors, and <10 rotatable bonds. Over 28 reactive groups such as carbodiimides, diazonium salts, peroxides, and diazines were also removed, resulting in 100,000 compounds with drug-like properties that meet a number of criteria including the Lipinski rules.

• Specialty libraries– Kinase library– NIH Small Molecule Repository

The Three Cardinal Rulesin

Developing an HTS Compatible

Assay

Do Everything You Can to Minimize Variation

Well to WellPlate to PlateDay to Day

DO EVERYTHING YOU CAN TO MINIMIZE THE CHANCES OF A CATASTROPHIC FAILURE

HTS has the Flexibility of a Mac-Truck

• So unless you have an extra 250k to pay for us to make the changes, it’s best to conform to our process

Properly Designed

Poorly Designed