Virtual screening leads to the discovery of novel non-nucleotide P2Y1 receptor antagonists.

The P2Y(1) receptor (P2Y(1)R) is a G protein-coupled receptor naturally activated by extracellular ADP. Its stimulation is an essential requirement of ADP-induced platelet aggregation, thus making antagonists highly sought compounds for the development of antithrombotic agents. Here, through a virtual screening campaign based on a pharmacophoric representation of the common characteristics of known P2Y(1)R ligands and the putative shape and size of the receptor binding pocket, we have identified novel antagonist hits of µM affinity derived from a N,N'-bis-arylurea chemotype. Unlike the vast majority of known P2Y(1)R antagonists, these drug-like compounds do not have a nucleotidic scaffold or highly negatively charged phosphate groups. Hence, our compounds may provide a direction for the development of receptor probes with altered physicochemical properties.

Synthesis and P2Y2 receptor agonist activities of uridine 5'-phosphonate analogues.

We explored the influence of modifications of uridine 5'-methylenephosphonate on biological activity at the human P2Y(2) receptor. Key steps in the synthesis of a series of 5-substituted uridine 5'-methylenephosphonates were the reaction of a suitably protected uridine 5'-aldehyde with [(diethoxyphosphinyl)methylidene]triphenylphosphorane, C-5 bromination and a Suzuki-Miyaura coupling. These analogues behaved as selective agonists at the P2Y(2) receptor, with three analogues exhibiting potencies in the submicromolar range. Although maximal activities observed with the phosphonate analogues were much less than observed with UTP, high concentrations of the phosphonates had no effect on the stimulatory effect of UTP. These results suggest that these phosphonates bind to an allosteric site of the P2Y(2) receptor.

Synthesis and P2Y receptor activity of nucleoside 5'-phosphonate derivatives.

Ribose-based nucleoside 5'-diphosphates and triphosphates and related nucleotides were compared in their potency at the P2Y receptors with the corresponding nucleoside 5'-phosphonate derivatives. Phosphonate derivatives of UTP and ATP activated the P2Y(2) receptor but were inactive or weakly active at P2Y(4) receptor. Uridine 5'-(diphospho)phosphonate was approximately as potent at the P2Y(2) receptor as at the UDP-activated P2Y(6) receptor. These results suggest that removal of the 5'-oxygen atom from nucleotide agonist derivatives reduces but does not prevent interaction with the P2Y(2) receptor. Uridine 5'-(phospho)phosphonate as well as the 5'-methylenephosphonate equivalent of UMP were inactive at the P2Y(4) receptor and exhibited maximal effects at the P2Y(2) receptor that were 50% of that of UTP suggesting novel action of these analogues.

Molecular dynamics simulation of the P2Y14 receptor. Ligand docking and identification of a putative binding site of the distal hexose moiety.

A rhodopsin-based homology model of the P2Y14 receptor was inserted into a phospholipid bilayer and refined by molecular dynamics (MD) simulation. The binding modes of several known agonists, namely UDP-glucose and its analogues, were proposed using automatic molecular docking combined with Monte Carlo Multiple Minimum calculations. Compared to other P2Y receptors, the P2Y14 receptor has an atypical binding mode of the nucleobase, ribose, and phosphate moieties. The diphosphate moiety interacts with only one cationic residue, namely Lys171 of EL2, while in other P2Y receptor subtypes three Arg or Lys residues interact with the phosphate chain. Two other conserved cationic residues, namely Arg253 (6.55) and Lys277 (7.35) of the P2Y14 receptor together with two anionic residues (Glu166 and Glu174, located in EL2), are likely involved in interactions with the distal hexose moiety.