Pharmacophore-based virtual screening, biological evaluation and binding mode analysis of a novel protease-activated receptor 2 antagonist

Abstract

Protease-activated receptor 2 (PAR(2)) is a G protein-coupled receptor, mediating inflammation and pain signaling in neurons, thus it is considered to be a potential therapeutic target for inflammatory diseases. In this study, we performed a ligand-based virtual screening of 1.6 million compounds by employing a common-feature pharmacophore model and two-dimensional similarity search to identify a new PAR(2) antagonist. The common-feature pharmacophore model was established based on the biological screening results of our in-house library. The initial virtual screening yielded a total number of 47 hits, and additional biological activity tests including PAR(2) antagonism and anti-inflammatory effects resulted in a promising candidate, compound 43, which demonstrated an IC50 value of 8.22 A mu M against PAR(2). In next step, a PAR(2) homology model was constructed using the crystal structure of the PAR(1) as a template to explore the binding mode of the identified ligands. A molecular docking method was optimized by comparing the binding modes of a known PAR(2) agonist GB110 and antagonist GB83, and applied to predict the binding mode of our hit compound 43. In-depth docking analyses revealed that the hydrophobic interaction with Phe243(5.39) is crucial for PAR(2) ligands to exert antagonistic activity. MD simulation results supported the predicted docking poses that PAR(2) antagonist blocked a conformational rearrangement of Na+ allosteric site in contrast to PAR(2) agonist that showed Na+ relocation upon GPCR activation. In conclusion, we identified new a PAR(2) antagonist together with its binding mode, which provides useful insights for the design and development of PAR(2) ligands.