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J Biol Chem, Vol. 274, Issue 46, 32655-32661, November 12, 1999

IB Kinases  and  Show a Random Sequential Kinetic Mechanism and Are Inhibited by Staurosporine and Quercetin

Boehringer Ingelheim Pharmaceuticals, Research and Development Center, Ridgefield, Connecticut 06877-0368

Activation of transcription factor NF-B is regulated by phosphorylation and subsequent degradation of its inhibitory subunit IB. The signal-induced phosphorylation of IB involves two IB kinases, IKK and IKK. In the present study, we investigated the kinetic mechanisms of IKK and IKK by substrate and product inhibition. For both IKK and IKK, the product ADP was a competitive inhibitor versus ATP and a non-competitive inhibitor versus IB. An alternative peptide substrate, IB-(21-41), was a competitive inhibitor versus IB 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-B 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 IKK and IKK. Daidzein, a quercetin analogue that does not inhibit NF-B activation, showed no significant inhibition of either enzyme. This suggests that the inhibitory properties of quercetin and staurosporine in the NF-B pathway are mediated in part by their inhibition of IKK and IKK. 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 IKK 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 IKK, which is an attractive drug target for inflammatory diseases.

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