title: Development and validation of a high-throughput calcium mobilization assay for the orphan receptor GPR88.
abstract: BACKGROUND
  GPR88 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.
  
  
  METHODS
  In 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 Gαqi5protein and consequent mobilization of intracellular calcium, which can be measured in a 384-well format with a Fluorescent Imaging Plate Reader.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Center for Drug Discovery, Research Triangle Institute, Post Office Box 12194, Research Triangle Park, NC, 27709, USA.
authors.forename: Ann M
authors.initials: AM
authors.lastname: Decker
authors.affiliation: Center for Drug Discovery, Research Triangle Institute, Post Office Box 12194, Research Triangle Park, NC, 27709, USA.
authors.forename: Elaine A
authors.initials: EA
authors.lastname: Gay
authors.affiliation: Center for Drug Discovery, Research Triangle Institute, Post Office Box 12194, Research Triangle Park, NC, 27709, USA.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Mathews
authors.affiliation: Center for Drug Discovery, Research Triangle Institute, Post Office Box 12194, Research Triangle Park, NC, 27709, USA.
authors.forename: Taylor C
authors.initials: TC
authors.lastname: Rosa
authors.affiliation: Center for Drug Discovery, Research Triangle Institute, Post Office Box 12194, Research Triangle Park, NC, 27709, USA.
authors.forename: Tiffany L
authors.initials: TL
authors.lastname: Langston
authors.affiliation: Center for Drug Discovery, Research Triangle Institute, Post Office Box 12194, Research Triangle Park, NC, 27709, USA.
authors.forename: Rangan
authors.initials: R
authors.lastname: Maitra
authors.affiliation: Center for Drug Discovery, Research Triangle Institute, Post Office Box 12194, Research Triangle Park, NC, 27709, USA. cjin@rti.org.
authors.forename: Chunyang
authors.identifier: http://orcid.org/0000-0001-6733-3094
authors.initials: C
authors.lastname: Jin
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002839:Chromans / Q000494:pharmacology*; D003412:Cricetulus; D057166:High-Throughput Screening Assays / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists*; D013329:Structure-Activity Relationship; D010133:p-Chloroamphetamine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D002839:Chromans; C418740:GPR88 protein, human; D043562:Receptors, G-Protein-Coupled; C029201:2-(4-chlorophenyl)cyclopropylamine; D010133:p-Chloroamphetamine; D002118:Calcium
keywords: 2-PCCA; Calcium mobilization; GPR88; Gαqi5; High-throughput screen