Inhibition of the NEMO/IKKβ association complex formation, a novel mechanism associated with the NF-κB activation suppression by Withania somnifera’s key metabolite withaferin A

Abstract

BackgroundNuclear Factor kappa B (NF-κB) 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-κB 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-κB 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-κB activation, the mechanism behind this is still eluding the researchers. The study conducted here is an attempt to explore NF-κB 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.ResultsFormation of active IKK (IκB kinase) complex comprising NEMO (NF-κB Essential Modulator) and IKKβ subunits is one of the essential steps for NF-κB 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 IKKβ subunits, which in turn pave way to naive complex formation capability of NEMO with IKKβ. Docking of WA into active NEMO/IKKβ 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/IKKβ complex conducted in this study provides significant evidence in support of the proposed mechanism of NF-κB activation suppression by inhibition or disruption of active NEMO/IKKβ complex formation being accounted by non-assembly of the catalytically active NEMO/IKKβ 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.ConclusionsNF-κB 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-κB signalling pathway along with its proposed mode of inhibition of...

Covalent Modification of the NF-κB Essential Modulator (NEMO) by a Chemical Compound Can Regulate Its Ubiquitin Binding Properties in Vitro

“…Recently, it was hypothesized that WA could disrupt the formation of the IKK complex in silico; however, this appears to not be the case based on previous literature (26,27). Instead, we decided to test the hypothesis that WA could disrupt the NEMO-ubiquitin interaction.…”

Anti-cancer activity of withaferin A in B-cell lymphoma

“…19 In most cancer cell lines WA inhibits the proliferation of tumor cells through a G 2 /M phase cell cycle arrest, 20 and inhibits the activation of NF-kB via interaction with the IKKg subunit preventing IkB phosphorylation. 21,22 In pancreatic cancer cells, Yu et. al.…”

Withaferin A inhibits pro-inflammatory cytokine-induced damage to islets in culture and following transplantation

“…Intraportal transplantation of syngeneic islets in diabetic C57BL/6 mice, a model representing inflammatory response to transplanted islets, showed that the engraftment of islets is significantly improved by WA. While several reports have shown that WA inhibits the NF-κB pathway through its binding to NEMO/ IKKβ complex [14,15], no study has been undertaken on the use of WA to protect islets from inflammatory damage in islet transplants into diabetic mice. Despite significant work on islet reaction to inflammation, there has been little research into the contribution of islets to the immediate immune reaction.…”

“…There are several extracts from the leaves of W. somnifera, but a study by Kaileh et al identified the steroidal lactone, WA, as the most potent inhibitor of NF-κB based on the hyperphosphorylation of inhibitor of κB (IκB) kinase beta (IKKβ) preventing IκB degradation [14]. Grover et al suggested that WA inhibits NF-κB by preventing the formation of NF-κB essential modulator (NEMO)/IKKβ complex [15]. WA has been studied as an anti-inflammatory compound in cystic fibrosis [16] with further potential to treat chronic inflammatory diseases such as rheumatoid arthritis, asthma and inflammatory bowel disease [17,18].…”