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

Protease-activated receptor 2 (PAR₂) 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 PAR₂ 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 PAR₂ antagonism and anti-inflammatory effects resulted in a promising candidate, compound 43, which demonstrated an IC₅₀ value of 8.22 µM against PAR₂. In next step, a PAR₂ homology model was constructed using the crystal structure of the PAR₁ 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 PAR₂ 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 PAR₂ ligands to exert antagonistic activity. MD simulation results supported the predicted docking poses that PAR₂ antagonist blocked a conformational rearrangement of Na⁺ allosteric site in contrast to PAR₂ agonist that showed Na⁺ relocation upon GPCR activation. In conclusion, we identified new a PAR₂ antagonist together with its binding mode, which provides useful insights for the design and development of PAR₂ ligands.