title: Comparison on functional assays for Gq-coupled GPCRs by measuring inositol monophospate-1 and intracellular calcium in 1536-well plate format.
abstract: Cell-based functional assays used for compound screening and lead optimization play an important role in drug discovery for G-protein coupled receptors (GPCRs). Cell-based assays can define the role of a compound as an agonist, antagonist or inverse agonist and can provide detailed information about the potency and efficacy of a compound. In addition, cell-based screens can be used to identify allosteric modulators that interact with sites other than the binding site of the endogenous ligand. Intracellular calcium assays which use a fluorescent calcium binding dye (such as Fluo-3, Fluo-4 or Fura-2) have been used in compound screening campaigns to measure the activity of Gq-coupled GPCRs. However, such screening methodologies require a special instrumentation to record the rapid change in intracellular free calcium concentration over time. The radioactive inositol 1,4,5- triphosphate (IP(3)) assay measures (3)H-inositol incorporation and is another traditional assay for the assessment of Gq-coupled GPCR activity, but it is not suitable for screening of large size compound collections because it requires a cell wash step and generates radioactive waste. To avoid these limitations, we have optimized and miniaturized a TR-FRET based IP-One assay that measures inositol monophosphate in a 1536-well plate format. This assay is homogenous, non-radioactive and does not require a kinetic readout. It has been tested with the cell lines expressing M(1) acetylcholine, FFAR1, vasopressin V1b, or Neuropeptide S receptors. The activities of antagonists determined in the IP-One assay correlated well with these measured in the intracellular calcium assay while the correlation of agonist activities might vary from cell line to cell line. This IP-One assay offers an alternative method for high throughput screening of Gq-coupled GPCRs without using costly kinetic plate readers.
authors.affiliation: NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, USA.
authors.forename: Ke
authors.initials: K
authors.lastname: Liu
authors.forename: Steve
authors.initials: S
authors.lastname: Titus
authors.forename: Noel
authors.initials: N
authors.lastname: Southall
authors.forename: Pingjun
authors.initials: P
authors.lastname: Zhu
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng