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
Assay
Development
Why is this a
bottleneck?
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
•
•
•
•
•
•
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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 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 highthroughput screening
Some Causes of Assay Variation
•
Biochemical Assays:
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•
•
•
•
•
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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
Statistical Analysis: Z Factor
Z=1 –
3SD of sample + 3SD of control
mean of sample – mean of control
Zhang, Chung and Oldenburgh, J. Biomol. Screening, 4:67-73, 1999
Statistical Analysis
Frequency
Data variability Separation Data variability
Band
Band
Band
3ss
ms
3sc
Assay Signal
mc
• 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 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
Properly
Designed
Poorly
Designed