development of a high-throughput assay for identifying inhibitors of tbk1 and ikkε发展高通量分析对于识别抑制剂tbk1 ikkε.pdfVIP

Development of a High-Throughput Assay for Identifying Inhibitors of TBK1 and IKKe 1 2 2 3,4 2 Jessica E. Hutti , Melissa A. Porter , Adam W. Cheely , Lewis C. Cantley , Xiaodong Wang , Dmitri Kireev2, Albert S. Baldwin1,5, William P. Janzen1,2* 1 Lineberger Comprehensive Cancer Center, University of North Carolina (UNC) at Chapel Hill, Chapel Hill, North Carolina, United States of America, 2 Division of Medicinal Chemistry and Natural Products, Center for Integrated Chemical Biology and Drug Discovery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 3 Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America, 4 Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America, 5 Department of Biology, UNC at Chapel Hill, Chapel Hill, North Carolina, United States of America Abstract 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