Development of a high-throughput calcium flux assay for identification of all ligand types including positive, negative, and silent allosteric modulators for G protein-coupled receptors

Assay Drug Dev Technol. 2012 Oct;10(5):457-67. doi: 10.1089/adt.2011.443. Epub 2012 Jun 29.

Abstract

In recent years, the increased use of cell-based functional assays for G protein-coupled receptors in high-throughput screening has enabled the design of robust assays to identify allosteric modulators (AMs) in addition to the more traditional orthosteric agonists and antagonists. In this article, the authors describe a screening format able to identify all ligand types using a triple-add assay that measures changes in cytosolic calcium concentration with three separate additions and reads in the same assay plate. This triple-add assay captures more small molecule ligand types than previously described assay formats without a significant increase in screening cost. Finally, the customizability of the triple-add assay to suit the needs of various AM screening programs is demonstrated.

MeSH terms

  • Allosteric Regulation / physiology
  • Animals
  • CHO Cells
  • Calcium Signaling / physiology*
  • Cricetinae
  • Cricetulus
  • High-Throughput Screening Assays / methods*
  • Humans
  • Ligands
  • Protein Binding / physiology
  • Receptors, G-Protein-Coupled / physiology*

Substances

  • Ligands
  • Receptors, G-Protein-Coupled