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 (IP3) assay measures 3H-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 M1 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.
Highlights
Guanine nucleotide triphosphate binding protein (G protein)-coupled receptors (GPCRs) are the largest and most important family of cell surface receptors for drug development
When binding to appropriate ligands, G-protein coupled receptors (GPCRs) transduce these extracellular stimuli into intracellular second messengers through activation of one or several G proteins including the subtypes of Gs, Gi, and Gq
We report here the miniaturization and optimization of this IP-One assay with four GPCRs as well as a comparison of antagonist screens against M1 muscarinic acetylcholine receptor expressed in Chinese hamster ovary (CHO) cells using both the IP-One assay and the intracellular calcium assay
Summary
Guanine nucleotide triphosphate binding protein (G protein)-coupled receptors (GPCRs) are the largest and most important family of cell surface receptors for drug development. We report here the miniaturization and optimization of this IP-One assay with four GPCRs as well as a comparison of antagonist screens against M1 muscarinic acetylcholine receptor expressed in Chinese hamster ovary (CHO) cells using both the IP-One assay and the intracellular calcium assay.
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