A selective IKK-2 inhibitor blocks NF-κB-dependent gene expression in interleukin-1β-stimulated synovial fibroblasts

NF-kappaB-induced gene expression contributes significantly to the pathogenesis of inflammatory diseases such as arthritis. IkappaB kinase (IKK) is the converging point for the activation of NF-kappaB by a broad spectrum of inflammatory agonists and is thus a novel target for therapeutic intervention. We describe a small molecule, selective inhibitor of IKK-2, SC-514, which does not inhibit other IKK isoforms or other serine-threonine and tyrosine kinases. SC-514 inhibits the native IKK complex or recombinant human IKK-1/IKK-2 heterodimer and IKK-2 homodimer similarly. IKK-2 inhibition by SC-514 is selective, reversible, and competitive with ATP. SC-514 inhibits transcription of NF-kappaB-dependent genes in IL-1beta-induced rheumatoid arthritis-derived synovial fibroblasts in a dose-dependent manner. When the mechanism of NF-kappaB activation was evaluated in the presence of this inhibitor, several interesting observations were found. First, SC-514 did not inhibit the phosphorylation and activation of the IKK complex. Second, there was a delay but not a complete blockade in IkappaBalpha phosphorylation and degradation; likewise there was a slightly slowed, decreased import of p65 into the nucleus and a faster export of p65 from the nucleus. Finally, both IkappaBalpha and p65 were comparable substrates for IKK-2, with similar K-m and K-cat values, and SC-514 inhibited the phosphorylation of either substrate similarly. Thus, the effect of SC-514 on cytokine gene expression may be a combination of inhibiting IkappaBalpha phosphorylation/ degradation, affecting NF-kappaB nuclear import/ export as well as the phosphorylation and transactivation of p65.