Cathleen E. Hanau

A Selective IKK-2 Inhibitor Blocks NF-κB-dependent Gene Expression in Interleukin-1β-stimulated Synovial Fibroblasts

NF-κB-induced gene expression contributes significantly to the pathogenesis of inflammatory diseases such as arthritis. IκB kinase (IKK) is the converging point for the activation of NF-κB 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-κB-dependent genes in IL-1β-induced rheumatoid arthritis-derived synovial fibroblasts in a dose-dependent manner. When the mechanism of NF-κB 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 IκBα 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 IκBα and p65 were comparable substrates for IKK-2, with similar Km and Kcat 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 IκBα phosphorylation/degradation, affecting NF-κB nuclear import/export as well as the phosphorylation and transactivation of p65.