Quantitative High‐Throughput Screening Using a Coincidence Reporter Biocircuit

Brittany W. Schuck1, Ryan MacArthur1, James Inglese2

1 National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, 2 National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.32
DOI:  10.1002/cpns.27
Online Posting Date:  April, 2017
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Abstract

Reporter‐biased artifacts—i.e., compounds that interact directly with the reporter enzyme used in a high‐throughput screening (HTS) assay and not the biological process or pharmacology being interrogated—are now widely recognized to reduce the efficiency and quality of HTS used for chemical probe and therapeutic development. Furthermore, narrow or single‐concentration HTS perpetuates false negatives during primary screening campaigns. Titration‐based HTS, or quantitative HTS (qHTS), and coincidence reporter technology can be employed to reduce false negatives and false positives, respectively, thereby increasing the quality and efficiency of primary screening efforts, where the number of compounds investigated can range from tens of thousands to millions. The three protocols described here allow for generation of a coincidence reporter (CR) biocircuit to interrogate a biological or pharmacological question of interest, generation of a stable cell line expressing the CR biocircuit, and qHTS using the CR biocircuit to efficiently identify high‐quality biologically active small molecules. © 2017 by John Wiley & Sons, Inc.

Keywords: assay development; coincidence reporter; quantitative high‐throughput screening

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Construction and Validation of the Coincidence Reporter Biocircuit
  • Basic Protocol 2: Development of Cell Lines for qHTS
  • Basic Protocol 3: qHTS Using A Coincidence Reporter Biocircuit
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Construction and Validation of the Coincidence Reporter Biocircuit

  Materials
  • Vector plasmid: pNLCoI1 (Promega, cat. no. N1461) or pCI 9.0 (Addgene, James Inglese Lab Plasmids, cat. no. 74229)
  • Geneblock/Gene synthesis, PCR product, or plasmid vector containing RE or promoter region of interest
  • DNA restriction enzymes (see Fig.  for multiple cloning sites)
  • Calf intestinal alkaline phosphatase (CIAP; New England Biolabs, cat. no. M0290)
  • QIAquick PCR Purification kit (Qiagen, cat. no. 28104) or equivalent
  • Quick Ligation kit (New England Biolabs, cat. no. M2200) or equivalent
  • Competent E. coli, e.g., DH5α (New England Biolabs) or Top10 (Life Technologies)
  • Transfection reagent (e.g., Lipofectamine 2000)
  • SOC medium
  • LB agar plates containing 100 µg/ml ampicillin
  • 50% (v/v) glycerol
  • QIAprep Spin Miniprep kit (Qiagen, cat. no. 27104) or equivalent
  • HiSpeed Plasmid Maxi kit (Qiagen, cat. no. 12662) or equivalent
  • Dimethyl sulfoxide (DMSO)
  • 10 mM PTC124 (see recipe)
  • 20 mM cilnidipine (see recipe)
  • Biological control compound(s) specific for RE/promoter (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Tissue culture hood
  • 37°C, 5% CO 2, 95% humidity incubator
  • 2‐ml cryovials
  • 6‐well tissue culture–treated plates (Corning, cat. no. 3506)
  • 96‐well solid white tissue culture–treated assay plates (Corning, cat. no. 3917)
  • Nano‐Glo Dual‐Luciferase Reporter (NanoDLR) Assay System (Promega, cat. no. N1610 or N1620)
  • Plate reader with luminescence capabilities and amenability for HTS (see Table 5.32.1)
  • Graphing software (e.g., GraphPad Prism)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ) and Sanger sequencing (Chapter 7 in Ausubel et al., 2017)

Basic Protocol 2: Development of Cell Lines for qHTS

  Materials
  • Cells of interest (see Strategic Planning)
  • Growth medium appropriate for cells
  • Coincidence reporter (see protocol 1)
  • Green fluorescent protein (GFP) vector lacking hygromycin B or puromycin resistance gene
  • Transfection reagent (e.g., Lipofectamine 2000 or Nucleofector kit)
  • Hygromycin B or puromycin
  • Dimethyl sulfoxide (DMSO)
  • 10 mM PTC124 (see recipe)
  • 20 mM cilnidipine (see recipe)
  • Biological control compound(s) specific for RE/promoter (see recipe)
  • Mouse anti‐FLAG antibody (Sigma, cat. no. F1804, RRID: AB_262044; for pCI 9.0 only)
  • Tissue culture hood
  • 37°C, 5% CO 2, 95% humidity incubator
  • 6‐well tissue culture–treated plates (Corning, cat. no. 3506)
  • 96‐well clear‐bottom tissue culture–treated plates (Corning, cat. no. 3596)
  • T25, T75, and T175 cell culture flasks with vented caps
  • 96‐well white solid‐bottom tissue culture–treated plates (Corning, 3917)
  • Nano‐Glo Dual‐Luciferase Reporter (NanoDLR) Assay System (Promega, cat. no. N1610 or N1620)
  • Reagent Dispenser for NanoDLR (see Table 5.32.1)
  • 384‐ or 1536‐well white solid‐bottom tissue culture–treated plates (Greiner Bio‐One)
  • Multidrop Combi Reagent Dispenser with small cassette (Thermo Scientific) or equivalent
  • Stainless‐steel lids (Kalypsys) containing pinholes for gas exchange (1536‐well plates only)
  • Liquid handling instrument for transferring compounds to assay plate (e.g., Multimek, PinTool, Mosquito; see Table 5.32.1)
  • Plate reader with luminescence capabilities and amenability for HTS (see Table 5.32.1)
  • Graphing software (e.g., GraphPad Prism)
  • Additional reagents and equipment for western blotting (Ni et al., )

Basic Protocol 3: qHTS Using A Coincidence Reporter Biocircuit

  Materials
  • Dimethyl sulfoxide (DMSO)
  • 10 mM PTC124 (see recipe)
  • 20 mM cilnidipine (see recipe)
  • Biological control compound(s) specific for RE/promoter (see recipe)
  • Coincidence reporter cell line (see protocol 2)
  • Growth medium appropriate for cell line
  • Library of Pharmacologically Active Compounds (LOPAC) titrated in 100% DMSO
  • Compound libraries titrated in 100% DMSO (7‐ to 11‐point inter‐ or intra‐plate titrations)
  • 384‐ or 1536‐well white solid‐bottom tissue culture–treated plates (Greiner Bio‐One)
  • Tissue culture hood
  • 37°C, 5% CO 2, 95% humidity incubator
  • T175 or T225 cell culture flasks with vented filter caps
  • Multidrop Combi Reagent Dispenser with small cassette (Thermo Scientific) or equivalent
  • Stainless‐steel lids containing pinholes for gas exchange (1536‐well plates only)
  • Liquid handling instrument for transferring compounds to assay plate (e.g., Multimek, PinTool, Mosquito; see Table 5.32.1)
  • Reagent Dispenser for NanoDLR (see Table 5.32.1)
  • Nano‐Glo Dual‐Luciferase Reporter (NanoDLR) Assay System (Promega, cat. no. N1610 or N1620)
  • Plate reader with luminescence capabilities and amenability for HTS (see Table 5.32.1)
  • Analysis software for qHTS:
    • For guided workflow programs with more user‐friendly interfaces: Collaborative Drug Discovery Vault, Dotmatics Studies, Genedata Screener, IDBS ActivityBase, or Screenable
    • For programs that require user ability to pivot incoming plate data: NCGC Curve Fit, scripting in GraphPad software and Tibco Spotfire
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.ncbi.nlm.nih.gov/books/NBK92014/
  Assay Guidance Manual. The entire manual should be read prior to beginning experiments.
  https://www.promega.com/resources/protocols/technical‐manuals/101/nanoglo‐dual‐luciferase‐reporter‐assay‐protocol/
  Nano‐Glo Dual‐Luciferase Reporter Assay Technical Manual.
  https://www.lifetechnologies.com/us/en/home/references/gibco‐cell‐culture‐basics.html
  Provides information on cell culture basics.
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