Structure-based virtual screening for discovery of paederosidic acid from Paederia scandens as novel P2Y₁₄R antagonist

Background: The activation of P2Y(14) receptor (P2Y(14)R) promotes osteoclast formation and causes neuropathic pain, exhibiting possible link to osteoarthritis (OA). Given lack of P2Y(14)R antagonist, the present study aims to search a novel P2Y(14)R antagonist with low toxicity and high activity from natural products as a possible drug candidate in treatment of OA. Methods: The role of P2Y(14)R on OA was verified using P2Y(14)R knockout (KO) rats. Molecular docking virtual screening strategy and activity test in P2Y(14)R stably-expressed HEK293 cells were used to screen target com-pound from natural product library. The MM/GBSA free energy calculation/decomposition technique was used to determine the principal interaction mechanism. Next, the binding of target compound to P2Y(14)R was examined using cellular thermal shift assay and drug affinity responsive target stability test. Finally, the ther-apeutic effect of target compound was performed in monosodium iodoacetate (MIA)-induced OA mouse model. To verify whether the effect of target compound was attributed to P2Y(14)R, we establish the osteoarthritis model in P2Y(14)R KO mice to perform pharmacodynamic evaluation. Importantly, to investigate the potential mecha-nism by which target compound attenuate OA, expressions of the major transcription factors involved in oste-oclast differentiation were detected by western blot, while markers of nerve damage in dorsal root ganglion (DRG) were evaluated by RT-qPCR and immunofluorescence techniques. Results: Deficiency of P2Y(14)R alleviated pain behavior and cartilage destruction in MIA-induced OA rats. 14 natural compounds were screened by Glide docking-based virtual screening, among which paederosidic acid exhibited the highest antagonistic activity to P2Y(14)R with IC50 of 8.287 mu M. As a bioactive component extracted from Paederia scandens, paederosidic acid directly interacted with P2Y(14)R to enhance the thermostability and decrease the protease sensitivity of target protein, which significantly inhibited receptor activator for nuclear factor-kappa B ligand (RANKL)-mediated osteoclastogenesis. More importantly, paederosidic acid suppressed osteo-clast formation by downregulating expressions of NFAT2 and ATP6V0D2, as well as relieved neuropathic pain by decreasing expressions of CGRP, CSF1 and galanin in DRG. Conclusions: Paederosidic acid targeted P2Y(14)R to improve OA through alleviating osteoclast formation and neuropathic pain, which provided an available strategy for developing novel drug leads for treatment of OA.