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

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. 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 compound 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 therapeutic 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 mechanism by which target compound attenuate OA, expressions of the major transcription factors involved in osteoclast differentiation were detected by western blot, while markers of nerve damage in dorsal root ganglion (DRG) were evaluated by RT-qPCR and immunofluorescence techniques. 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 IC 50 of 8.287 μM. 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-κB ligand (RANKL)-mediated osteoclastogenesis. More importantly, paederosidic acid suppressed osteoclast formation by downregulating expressions of NFAT2 and ATP6V0D2, as well as relieved neuropathic pain by decreasing expressions of CGRP, CSF1 and galanin in DRG. 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. [Display omitted] [ABSTRACT FROM AUTHOR]