Discovery and characterization of P2Y₂ receptor antagonists supported by site-directed mutagenesis studies

Abstract

The purinergic P2Y₂ receptor (P2Y₂R) is the only member of the G protein-coupled P2Y receptor family that is activated by both UTP and ATP at equal concentrations. The receptor is Gq protein-coupled leading to an increase in intracellular calcium concentrations upon activation. It is expressed in various cells and organs and associated with inflammation, obesity, and cancer metastasis. Therefore, it has been proposed as a potential therapeutic target for several diseases including cystic fibrosis, chronic bronchitis, viral infections, myocardial infarction, obesity and Alzheimer's disease. However, only few nucleotide-derived agonists and even fewer moderately potent antagonists with poor P2Y₂R-selectivity have been described so far, most of which lack drug-like properties. Potent and selective ligands are therefore urgently needed as pharmacological tools and lead compounds to develop drugs targeting the P2Y₂R. <br /> To this end, we established and validated intracellular calcium mobilization and β-arrestin recruitment assays for the P2Y₂R to undertake high-throughput screening (HTS) of target-focused commercial and in-house (Pharma-Zentrum Bonn; PZB) compound libraries. Analogues of cherry-picked hits were synthesized, tested and their structure–activity relationship (SAR) analysis performed. Compounds 85, a urea derivative (IC50 1.31 µM) and 247, a diindolymethane derivative (IC50 1.91 µM), were identified and characterized as allosteric modulators of the P2Y₂R receptor, which should be further optimized in the future. <br /> Next, we undertook virtual screening of the ZINC library to identify novel P2Y₂R antagonist scaffolds using a homology model of the P2Y₂R since no crystal structures are currently available. In silico hits were then purchased and validated in vitro by calcium assays at the P2Y₂R and for selectivity versus the closely related P2Y1, P2Y₄ and P2Y6 receptor subtypes. Several hits were identified for the P2Y₂R, but also novel antagonists the P2Y₄ and P2Y6 receptors were discovered. Preliminary SARs were analyzed and further optimization of these compounds is ongoing. <br /> To gain insights into the receptor-binding pockets of the P2Y₂R and its closest relative, the P2Y₄R, we undertook a site-directed mutagenesis study investigating structurally and pharmacologically diverse ligands at the expressed mutant receptors. Overall, 13 new mutants for both the P2Y₂R and P2Y₄R were engineered and expressed in 1321N1 astrocytoma cells. Through homology modeling, docking studies and in vitro calcium assays, we investigated binding interactions with specific amino acids by agonists such as UTP, ATP, Ap4A and MRS4062 as well as antagonists such as AR-C118925 and selected anthraquinone derivatives. Interactions of the residues Phe113 and Phe195 with agonists were found to be important for P2Y₂R activation. The ionic lock formed by Asp185 was discovered to be important for P2Y₂R activation by Ap4A and MRS4062. At the P2Y₄R, interaction of UTP and MRS4062 with Arg190 and Tyr197 were crucial for receptor activation. Mutation of Tyr197 to alanine sensitized the P2Y₄R to activation by ATP. The binding pose and site (allosteric or orthosteric) of the anthraquinone derivatives in the P2Y₂R and P2Y₄ receptor pockets were dependent on their structures. The aromatic residues Val1684.60 and Phe1955.35 of the P2Y₂R were found to contribute to the selectivity of AR-C118925 for the P2Y₂ over the P2Y₄R. <br /> Altogether, the results of both in silico- and in vitro-screening of compounds at the P2Y₂R coupled to insights from site-directed mutagenesis studies contributed to the rational design, optimization and development of selective and potent P2Y₂R antagonists as potential drug candidates.