HTSF

High-throughput Screening
and the HTS Facility
Chen Zhang
HTSF Director
What is HTS
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Wanted: Small molecule antagonist/ binder/
inhibitor/etc. for your macromolecule of interest
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Problem: No known small molecule, Not
enough information for rational design
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Solution: Use robotic system to screen large
collections of diverse compound libraries to
identify and then validate hits
HTS Applications
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Enzymes
Micro RNAs
DNA/RNA-protein interaction
Protein-protein interaction
Receptor-ligand binding
Bacteria/ mammalian cell growth, gene expression
Cell differentiation and signaling pathway
…
Compound Libraries
Compound collections
(~180,000 total):
Chembridge Library
~150,000 compounds
National Cancer Institute ~10,000 compounds
Marvel Library
HTSF House Library
~10,000 compounds
~6,000 compounds
> 500
384-well plates
Assay Design and optimization
 Appropriate controls
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Sufficient assay quality (Z’ ≥ 0.5)
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Compatible with the robotic system instrumentation
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Stability and reproducibility
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Reagent costs
Assay Design and optimization
False negatives
False Positives
Determination of assay robustness:
3 x (δ p + δ n)
Z-factor (Z’) = 1- |µ – µ |
p
n
µp - Mean of postive controls
µn - Mean of negative controls
δ p - Standard deviation of postive controls
δ n - Standard deviation of negative controls
Liquid Handling Robotics
PlateMatePlus
Small one in
Biosafety Cabinet
96/384 Tips
PinTransferTool
Add reagents or cell culture to plates
Transfer compounds from stock plates to assay plates
Readouts
 Absorbance
 Fluorescence
Intensity/ Fluorescence
Resonance Energy Transfer (FRET)
 Fluorescence Polarization
 Luminescence
Examples of Detection Methods
Absorbance
FRET
Donor Acceptor
Emission
Wavelength
Change
FP
Reporter GeneLuminescence
Luciferase
Image Sources:
http://www.zmb.uzh.ch/resources/protocols/FRET/FRET.jpg
http://www.glycoforum.gr.jp/science/word/glycotechnology/GT-C06E.html
http://www.sabiosciences.com/pathway10_QuantitativeSignal_1.php
http://www.biocompare.com/images/bc/006/ArticleImages/Antiviral_Assay_1.jpg
Plate Readers
Luminascence
Fluorescence
Absorbance
Potential
Hits
Positive
Controls
Negative
Controls
Data Analysis & Hit Validation
Screening complete. Now what?
Analyze data for putative hits
Retest putative hits to identify false positives
- hits typically retested under screening conditions
- real hit compounds reordered for further testing
Follow-up Assay
 Efficacy
 Potency
 Specificity
 In
vivo test
 Drug
potential
Synthesize
derivatives
of good hit.
Test
Clinical
trial
ChemSpeed Synthesizer SLTII
Solid
Liquid
Dispensing Dispensing
Sonicator
Robotic
Arm
ChemSpeed Synthesizer SLTII
Solid Phase
Extractor
Reactor
Pressure
Block
Block
Holder Double
Solution
Temperature
Rack
Reactor
Bottle Holder
Shaker
HTSF Summary
HTSF can help with all aspects of:
- Assay development/optimization
- High-throughput screening
- Data analysis/interpretation
- Secondary experiments
- Follow-up assays
-High throughput synthesis
- Mammalian cell culture
What can you do for the HTSF
Compound Submission:
Submit your compounds to the HTSF House library
Compounds will be added to screening plates
Compounds screened in numerous assays
Compounds could provide valuable research tool or new drug
Contact Info
 www.scs.illinois.edu/htsf
 361
Noyes Lab
 244-4198
 [email protected]
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