High-throughput Screening and the HTS Facility Chen Zhang HTSF Director What is HTS  Wanted: Small molecule antagonist/ binder/ inhibitor/etc. for your macromolecule of interest  Problem: No known small molecule, Not enough information for rational design  Solution: Use robotic system to screen large collections of diverse compound libraries to identify and then validate hits HTS Applications         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  Sufficient assay quality (Z’ ≥ 0.5)  Compatible with the robotic system instrumentation  Stability and reproducibility  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]