Presentation on theme: "How to Develop an HTS Compatible Assay Lucile White Southern Research Institute High Throughput Screening Center."— Presentation transcript:
How to Develop an HTS Compatible Assay Lucile White Southern 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 2009 In 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:
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 2 nd or 3 rd level assays – low to medium throughput – known actives
New Technologies at SRI 2010 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
Separation Band Data variability Band Data variability Band msms mcmc Frequency Assay Signal 3s s 3s c 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.htmlwww.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, 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 Rules in Developing an HTS Compatible Assay
Do Everything You Can to Minimize Variation Well to Well Plate to Plate Day 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