AlphaScreen-Based Assays: Ultra-High-Throughput Screening for Small-Molecule Inhibitors of Challenging Enzymes and Protein-Protein Interactions.
AlphaScreen technology has been routinely utilized in high-throughput screening assays to quantify analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range, and sensitivity associated with AlphaScreens as well as the homogenous assay format and reagent stability make the technology particularly well suited for high-throughput screening applications. Here, we describe the development of AlphaScreen assays to identify small-molecule inhibitors of enzymes and protein-protein interactions using the highly miniaturized 1536-well format. The subsequent implementation of counter assays to identify false-positive compounds is also discussed.
National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Building B, Rm. 1034E, Rockville, MD, 20850, USA.
Adam
A
Yasgar
National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Building B, Rm. 1034E, Rockville, MD, 20850, USA.
Ajit
A
Jadhav
National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Building B, Rm. 1034E, Rockville, MD, 20850, USA.
Anton
A
Simeonov
National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Building B, Rm. 1034E, Rockville, MD, 20850, USA. coussensn@mail.nih.gov.
Nathan P
NP
Coussens
D000818:Animals; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D060066:Protein Interaction Maps / Q000187:drug effects*; D054852:Small Molecule Libraries / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries
AlphaLISA; AlphaScreen; Assay; ELISA; Enzyme; HTS; High-throughput screening; Protein-protein interaction