Use of the Pharmacological Inhibitor BX795 to Study the Regulation and Physiological Roles of TBK1 and IκB Kinase ϵ

TANK-binding kinase 1 (TBK1) and IκB kinase ϵ (IKKϵ) regulate the production of Type 1 interferons during bacterial and viral infection, but the lack of useful pharmacological inhibitors has hampered progress in identifying additional physiological roles of these protein kinases and how they are regulated. Here we demonstrate that BX795, a potent and relatively specific inhibitor of TBK1 and IKKϵ, blocked the phosphorylation, nuclear translocation, and transcriptional activity of interferon regulatory factor 3 and, hence, the production of interferon-β in macrophages stimulated with poly(I:C) or lipopolysaccharide (LPS). In contrast, BX795 had no effect on the canonical NFκB signaling pathway. Although BX795 blocked the autophosphorylation of overexpressed TBK1 and IKKϵ at Ser-172 and, hence, the autoactivation of these protein kinases, it did not inhibit the phosphorylation of endogenous TBK1 and IKKϵ at Ser-172 in response to LPS, poly(I:C), interleukin-1α (IL-1α), or tumor necrosis factor α and actually enhanced the LPS, poly(I:C), and IL-1α-stimulated phosphorylation of this residue. These results demonstrate that the phosphorylation of Ser-172 and the activation of TBK1 and IKKϵ are catalyzed by a distinct protein kinase(s) in vivo and that TBK1 and IKKϵ control a feedback loop that limits their activation by LPS, poly(I:C) and IL-1α (but not tumor necrosis factor α) to prevent the hyperactivation of these enzymes.