Development and validation of a high-throughput calcium mobilization assay for the orphan receptor GPR88

Abstract

BackgroundGPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders. However, the receptor functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for central nervous system investigation. Development of a high-throughput screening assay for GPR88 should facilitate the discovery of novel ligands to probe GPR88 functions.MethodsIn this paper, we describe the development of a CHO-Gαqi5-GPR88 cell-based calcium mobilization assay. The assay takes advantage of functional coupling of GPR88 with the promiscuous Gαqi5 protein and consequent mobilization of intracellular calcium, which can be measured in a 384-well format with a Fluorescent Imaging Plate Reader.ResultsThe CHO-Gαqi5-GPR88 cell-based calcium mobilization assay was validated by the structure-activity relationship study of known GPR88 agonist (1R,2R)-2-PCCA analogues. The assay was automated and miniaturized to a 384-well format, and was deemed robust and reproducible with a Z’-factor of 0.72 and tolerated dimethyl sulfoxide to a final concentration of 2%. Screening a pilot neurotransmitter library consisting of 228 compounds yielded 10 hits, but none of the hits were confirmed as GPR88 agonists in follow-up assays.ConclusionsWe have developed a high-throughput calcium mobilization assay for the orphan receptor GPR88. This calcium mobilization assay can be used to identify several different types of GPR88 ligands including agonists, competitive and noncompetitive antagonists, inverse agonists, and allosteric modulators. These ligands will serve as valuable tools to probe signaling mechanisms and in vivo functions of GPR88, and could expedite development of novel therapies for diseases potentially mediated by GPR88.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-017-0330-3) contains supplementary material, which is available to authorized users.

Discovery of a Potent, Selective, and Brain-Penetrant Small Molecule that Activates the Orphan Receptor GPR88 and Reduces Alcohol Intake

“…24 Later, we observed that, over time, the HEK293 cells were physically unhealthy after transfection with GPR88 and subsequently led to deteriorated responses to 2-PCCA. 26 To facilitate the SAR study, we recently created a stable CHO-PPLS-HA-GPR88 cell line and measured cAMP levels using the PerkinElmer Lance assay kit. 27 The new assay system is reliable and sensitive and therefore was used in subsequent SAR studies.…”

Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists

“…Because the NPFF1 assay was originally developed in 96-well format, we miniaturized the assay to 384-well format and determined the cell density that provided a confluent cell monolayer and NPFF potency/efficacy consistent with the 96-well assay. We then tested the calcium mobilization assay using our automated liquid handling equipment, which includes a Biotek cell dispenser, Beckman Coulter Biomek NX, and FLIPR Tetra to determine whether automation resulted in adverse effects; no changes in assay performance were observed . Using the automated and miniaturized assay, we generated a Z ′-factor by testing positive (10 μM final NPFF) and negative controls (1% DMSO/assay buffer) on three individual days .…”

“…We then tested the calcium mobilization assay using our automated liquid handling equipment, which includes a Biotek cell dispenser, Beckman Coulter Biomek NX, and FLIPR Tetra to determine whether automation resulted in adverse effects; no changes in assay performance were observed. 43 Using the automated and miniaturized assay, we generated a Z′-factor by testing positive (10 μM final NPFF) and negative controls (1% DMSO/assay buffer) on three individual days. 44 The Z′-factor (N = 3) was determined to be 0.75 with a standard deviation of 0.06; a Z′-factor greater than 0.5 is considered appropriate for HTS.…”

Validation of a High-Throughput Calcium Mobilization Assay for the Human Trace Amine-Associated Receptor 1

“…33 Since then, our group has developed numerous calcium mobilization assays for multiple targets and found that pretreating cells for 15 min prior to compound addition and simultaneous data acquisition dramatically reduces the initial cellular response to DMSO. [35][36][37][38] Thus, when this pretreatment protocol was applied to the hTAAR1 assay during the initial development stage, a concentration of 1% DMSO did not elevate the baseline (baseline RFU = 243 for 1% DMSO, compared with 250 RFU for 0% DMSO).…”