title: Discovery of novel P2Y14 agonist and antagonist using conventional and nonconventional methods.
abstract: P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 µM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.
authors.affiliation: Department of Biochemistry and Molecular Biology, Merck Frosst Canada, Inc., Kirkland, Quebec, Canada.
authors.forename: Martine
authors.initials: M
authors.lastname: Hamel
authors.forename: Martin
authors.initials: M
authors.lastname: Henault
authors.forename: Huda
authors.initials: H
authors.lastname: Hyjazie
authors.forename: Nicolas
authors.initials: N
authors.lastname: Morin
authors.forename: Christopher
authors.initials: C
authors.lastname: Bayly
authors.forename: Kathryn
authors.initials: K
authors.lastname: Skorey
authors.forename: Alex G
authors.initials: AG
authors.lastname: Therien
authors.forename: Joseph
authors.initials: J
authors.lastname: Mancini
authors.forename: Christine
authors.initials: C
authors.lastname: Brideau
authors.forename: Stacia
authors.initials: S
authors.lastname: Kargman
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D051379:Mice; D002679:Pan troglodytes; D011485:Protein Binding; D058910:Purinergic P2 Receptor Agonists / Q000494:pharmacology*; D058919:Purinergic P2 Receptor Antagonists / Q000494:pharmacology*; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D008024:Ligands; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2