title: Identification of determinants required for agonistic and inverse agonistic ligand properties at the ADP receptor P2Y12.
abstract: The ADP receptor P2Y(12) belongs to the superfamily of G protein-coupled receptors (GPCRs), and its activation triggers platelet aggregation. Therefore, potent antagonists, such as clopidogrel, are of high clinical relevance in prophylaxis and treatment of thromboembolic events. P2Y(12) displays an elevated basal activity in vitro, and as such, inverse agonists may be therapeutically beneficial compared with antagonists. Only a few inverse agonists of P2Y(12) have been described. To expand this limited chemical space and improve understanding of structural determinants of inverse agonist-receptor interaction, this study screened a purine compound library for lead structures using wild-type (WT) human P2Y(12) and 28 constitutively active mutants. Results showed that ATP and ATP derivatives are agonists at P2Y(12). The potency at P2Y(12) was 2-(methylthio)-ADP > 2-(methylthio)-ATP > ADP > ATP. Determinants required for agonistic ligand activity were identified. Molecular docking studies revealed a binding pocket for the ATP derivatives that is bordered by transmembrane helices 3, 5, 6, and 7 in human P2Y(12,) with Y(105), E(188), R(256), Y(259), and K(280) playing a particularly important role in ligand interaction. N-Methyl-anthraniloyl modification at the 3'-OH of the 2'-deoxyribose leads to ligands (mant-deoxy-ATP [dATP], mant-deoxy-ADP) with inverse agonist activity. Inverse agonist activity of mant-dATP was found at the WT human P2Y(12) and half of the constitutive active P2Y(12) mutants. This study showed that, in addition to ADP and ATP, other ATP derivatives are not only ligands of P2Y(12) but also agonists. Modification of the ribose within ATP can result in inverse activity of ATP-derived ligands.
authors.affiliation: Institute of Biochemistry, Molecular Biochemistry, Johannisallee 30, 04103 Leipzig, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Schmidt
authors.forename: Lars
authors.initials: L
authors.lastname: Ritscher
authors.forename: Elizabeth N
authors.initials: EN
authors.lastname: Dong
authors.forename: Thomas
authors.initials: T
authors.lastname: Hermsdorf
authors.forename: Maxi
authors.initials: M
authors.lastname: Cöster
authors.forename: Doreen
authors.initials: D
authors.lastname: Wittkopf
authors.forename: Jens
authors.initials: J
authors.lastname: Meiler
authors.forename: Torsten
authors.initials: T
authors.lastname: Schöneberg
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D016208:Databases, Factual; D054314:Drug Inverse Agonism; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D062105:Molecular Docking Simulation; D009154:Mutation; D058912:Purinergic P2Y Receptor Agonists / Q000737:chemistry* / Q000494:pharmacology; D011687:Purines / Q000737:chemistry* / Q000494:pharmacology; D058925:Receptors, Purinergic P2Y12 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D008024:Ligands; C548576:P2RY12 protein, human; D058912:Purinergic P2Y Receptor Agonists; D011687:Purines; D058925:Receptors, Purinergic P2Y12; D000242:Cyclic AMP