Identification of endogenous surrogate ligands for human P2Y₁₂ receptors by in silico and in vitro methods

Endogenous ligands acting on a human P2Y₁₂ receptor, one of the G-protein coupled receptors, were searched by in silico screening against our own database, which contains more than 500 animal metabolites. The in silico screening using the docking software AutoDock resulted in selection of cysteinylleukotrienes (CysLTs) and 5-phosphoribosyl 1-pyrophosphate (PRPP), with high free energy changes, in addition to the known P2Y₁₂ ligands such as 2MeSADP and ADP. These candidates were subjected to an in vitro Ca ²⁺ assay using the CHO cells stably expressing P2Y ₁₂-G₁₆α fusion proteins. We found that CysLTE4 and PRPP acted on the P2Y₁₂ receptor as agonists with the EC₅₀ values of 1.3 and 7.8 nM, respectively. Furthermore, we analyzed the phylogenetic relationship of the P2Y, P2Y-like, and CysLT receptors based on sequence alignment followed by evolutionary analyses. The analyses showed that the P2Y₁₂, P2Y₁₃, P2Y₁₄, GPR87, CysLT-1, and CysLT-2 receptors formed a P2Y-related receptor subfamily with common sequence motifs in the transmembrane regions.