Discovery of an IKK2 Site that Allosterically Controls Its Activation

I{kappa}B kinase 2/{beta} (IKK2) is a critical regulator of inflammation which is inducibly activated by a host of stimuli. Aberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Efforts to prevent these diseases by small-molecule inhibitors of IKK2 activity have not been successful. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. Here we focused on identifying inhibitors to block IKK2 activity from an allosteric site, not the ATP-binding pocket. Using virtual screening, we first identified several candidate allosteric sites and screened for potential small-molecule binders, and then selected candidates inhibitory to IKK2 activity using cell-based functional assays. Hydrogen deuterium exchange coupled to mass-spectrometry (HDX-MS) and MS-MS assays revealed that a class of benzoyl conjugates of pyrrolidinedione covalently bound to a site located at the interface of the kinase domain (KD) and the helical domain (SDD), and inhibited IKK2 activation allosterically by preventing phosphorylation of its activation loop serines. Additionally, this class of inhibitor partially blocks IKK2s catalytic activity by enhancing dynamics within the ATP binding pocket and likely the general active site. Hydrogen deuterium exchange (HDX) experiments further revealed that while binding of substrate ATP perturbs only the local structure surrounding its binding site, binding to ATP-competitive or allosteric inhibitors induces structural perturbations in an expansive area including the helical domain. We propose that these allosteric sites can act as specific targets for the development of novel potent IKK inhibitors. SIGNIFICANCEAberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. By combination of virtual screening and cell-based functional assays, we identified small-molecule binders of the class of benzoyl conjugates of pyrrolidinedione that block IKK2 activity from an allosteric site through covalent attachment and could be specific only for IKK2. HDX-MS and MS-MS assays identified a binding pocket with a Cys-Cys motif for these inhibitors, and revealed specific differences in IKK2 dynamics upon binding to substrate ATP vs ATP-competitive and allosteric inhibitors. Present work provides a framework for the development of allosteric inhibitors to combat IKK2-induced diseases inhibitors.