title: IkappaB kinases alpha and beta show a random sequential kinetic mechanism and are inhibited by staurosporine and quercetin.
abstract: Activation of transcription factor NF-kappaB is regulated by phosphorylation and subsequent degradation of its inhibitory subunit IkappaB. The signal-induced phosphorylation of IkappaB involves two IkappaB kinases, IKKalpha and IKKbeta. In the present study, we investigated the kinetic mechanisms of IKKalpha and IKKbeta by substrate and product inhibition. For both IKKalpha and IKKbeta, the product ADP was a competitive inhibitor versus ATP and a non-competitive inhibitor versus IkappaBalpha. An alternative peptide substrate, IkappaBalpha-(21-41), was a competitive inhibitor versus IkappaBalpha and a non-competitive inhibitor versus ATP for both kinases. These results rigorously eliminate the possibility of an ordered sequential mechanism and demonstrate that both kinases have a random sequential bi bi mechanism. Two natural compounds, quercetin and staurosporine, had previously been shown to inhibit the NF-kappaB pathway, but the molecular target(s) of these compounds in the event had not been established. Here we demonstrate that quercetin and staurosporine potently inhibit both IKKalpha and IKKbeta. Daidzein, a quercetin analogue that does not inhibit NF-kappaB activation, showed no significant inhibition of either enzyme. This suggests that the inhibitory properties of quercetin and staurosporine in the NF-kappaB pathway are mediated in part by their inhibition of IKKalpha and IKKbeta. Mechanism studies reveal that staurosporine is a competitive inhibitor versus ATP, whereas quercetin serves as a mixed type inhibitor versus ATP. The strong inhibition of IKKbeta by staurosporine (K(i) = 172 nM) and ADP (K(i) = 136 nM) provides a rationale and structural framework for designing potent ATP-site inhibitors of IKKbeta, which is an attractive drug target for inflammatory diseases.
authors.affiliation: Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut 06877-0368, USA.
authors.forename: G W
authors.initials: GW
authors.lastname: Peet
authors.forename: J
authors.initials: J
authors.lastname: Li
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051550:I-kappa B Kinase; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics; D015394:Molecular Structure; D016328:NF-kappa B / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011794:Quercetin / Q000494:pharmacology*; D019311:Staurosporine / Q000494:pharmacology*; D015533:Transcriptional Activation
chemical_list: D004791:Enzyme Inhibitors; D007529:Isoflavones; D016328:NF-kappa B; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; C004742:daidzein; D000255:Adenosine Triphosphate; D011794:Quercetin; D017346:Protein Serine-Threonine Kinases; D051550:I-kappa B Kinase; D019311:Staurosporine