Direct Interaction of Minocycline to p47phox Contributes to its Attenuation of TNF-α-Mediated Neuronal PC12 Cell Death: Experimental and Simulation Validation.

Abstract

Kaempferol illustrates an example of attempting to discover new treatments for neurodegeneration by investigating the potential efficacy of natural products. Despite the identification of several molecular targets for this bio-active compound, the precise underlying pathways are not well elucidated yet. Recently, it has been shown through pulldown assay that kaemferol directly interacts with p47phox, the organizer subunit of NADPH oxidase-2 (NOX2) complex. Hence, in this study, we used homology modelling, computational docking, mutation analysis, molecular dynamics simulations and free energy calculations to determine how kaempferol interacts with p47phox. Firstly, 3D structure of p47phox was generated using x-ray structures of its domains. Then, it was docked with kaempferol, and finally 100-ns molecular dynamics (MD) simulations were performed and the global properties like root-mean square deviation (RMSD) and root-mean square fluctuations (RMSF) were calculated. Literature survey and computational analysis of key interacting amino acid residues of p47phox provided insights into a possible binding site for kaempferol, approximately around Trp193 and Cys196 located within the N-terminal SH3 domain of p47phox. Moreover, free energy calculations indicated that in silico substitution of Trp193 and Cys196 with arginine and alanine, respectively, results in less favorable interaction corroborating their importance in binding with kaempferol. Taken together, these findings suggest that kaempferol directly attaches to N-SH3 domain p47 phox, with a subsequent diminution of p47phox protein-protein interaction and possibly attenuation of NOX2 complex assembly, which reduces reactive oxygen species (ROS) generation. These observations will be beneficial for researchers exploring neuroprotection and for the development of p47phox inhibitors.