Withaferin A inhibits NF-kappaB activation by targeting cysteine 179 in IKKβ

The transcription factor NF-κB is one of the main players involved in inflammatory responses during which NF-κB becomes rapidly activated. However to maintain homeostasis, this NF-κB activation profile is only transient. Nevertheless deregulation of NF-κB activity is often observed and can lead to chronic inflammatory diseases as well as cancer. Therefore various research projects focus on the development of therapeutics that target the NF-κB activation pathway. One such compound is Withaferin A from the Ayurvedic plant Withania somnifera. Several reports already described the NF-κB inhibiting, anti-inflammatory capacity of WA, either in vitro as well as in vivo. However the underlying molecular mechanism remains largely unknown. In this paper we demonstrate a direct interaction of WA with the IKK-complex, more specifically with IKKβ, a kinase which is indispensable for the nuclear translocation of NF-κB. Hereby WA directly inhibits IKK catalytic activity. By mutation of Cys179 in IKKβ we could demonstrate loss of interaction between IKKβ and WA indicating that WA exerts its anti-inflammatory effects by targeting the crucial Cys179 residue located in the catalytic site of IKKβ. Upon docking of WA to a IKKβ homology structure model, WA was found to fit nicely into the groove of IKKβ where it can form hydrogen bond to stabilize its interaction with Cys179.