Development of a high-throughput assay for identifying inhibitors of TBK1 and IKKepsilon

IKKe and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKe has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKe. This information enabled the design of an optimal TBK1/IKKe substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKe. 227 compounds in this library inhibited TBK1 at a concentration of 10 mM, while 57 compounds inhibited IKKe. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of smal