ABSTRACT: Nuclear Factor kappa B (NF-?B) is a transcription factor involved in the regulation of cell signaling responses and is a key regulator of cellular processes involved in the immune response, differentiation, cell proliferation, and apoptosis. The constitutive activation of NF-?B contributes to multiple cellular outcomes and pathophysiological conditions such as rheumatoid arthritis, asthma, inflammatory bowel disease, AIDS and cancer. Thus there lies a huge therapeutic potential beneath inhibition of NF-?B signalling pathway for reducing these chronic ailments. Withania somnifera, a reputed herb in ayurvedic medicine, comprises a large number of steroidal lactones known as withanolides which show plethora of pharmacological activities like anti- inflammatory, antitumor, antibacterial, antioxidant, anticonvulsive, and immunosuppressive. Though a few studies have been reported depicting the effect of WA (withaferin A) on suppression of NF-?B activation, the mechanism behind this is still eluding the researchers. The study conducted here is an attempt to explore NF-?B signalling pathway modulating capability of Withania somnifera's major constituent WA and to elucidate its possible mode of action using molecular docking and molecular dynamics simulations studies.Formation of active IKK (I?B kinase) complex comprising NEMO (NF-?B Essential Modulator) and IKK? subunits is one of the essential steps for NF-?B signalling pathway, non-assembly of which can lead to prevention of the above mentioned vulnerable disorders. As observed from our semi-flexible docking analysis, WA forms strong intermolecular interactions with the NEMO chains thus building steric as well as thermodynamic barriers to the incoming IKK? subunits, which in turn pave way to naive complex formation capability of NEMO with IKK?. Docking of WA into active NEMO/IKK? complex using flexible docking in which key residues of the complex were kept flexible also suggest the disruption of the active complex. Thus the molecular docking analysis of WA into NEMO and active NEMO/IKK? complex conducted in this study provides significant evidence in support of the proposed mechanism of NF-?B activation suppression by inhibition or disruption of active NEMO/IKK? complex formation being accounted by non-assembly of the catalytically active NEMO/IKK? complex. Results from the molecular dynamics simulations in water show that the trajectories of the native protein and the protein complexed with WA are stable over a considerably long time period of 2.6 ns.NF-?B is one of the most attractive topics in current biological, biochemical, and pharmacological research, and in the recent years the number of studies focusing on its inhibition/regulation has increased manifolds. Small ligands (both natural and synthetic) are gaining particular attention in this context. Our computational analysis provided a rationalization of the ability of naturally occurring withaferin A to alter the NF-?B signalling pathway along with its proposed mode of inhibition of the pathway. The absence of active IKK multisubunit complex would prevent degradation of I?B proteins, as the I?B proteins would not get phosphorylated by IKK. This would ultimately lead to non-release of NF-?B and its further translocation to the nucleus thus arresting its nefarious acts. Conclusively our results strongly suggest that withaferin A is a potent anticancer agent as ascertained by its potent NF-?B modulating capability. Moreover the present MD simulations made clear the dynamic structural stability of NEMO/IKK? in complex with the drug WA, together with the inhibitory mechanism.
