Development of a High-Throughput Assay for Identifying Inhibitors of TBK 1 and IKK e

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 small molecule TBK1/IKKe inhibitors possessing therapeutic potential for both inflammatory diseases and cancer. Citation: Hutti JE, Porter MA, Cheely AW, Cantley LC, Wang X, et al. (2012) Development of a High-Throughput Assay for Identifying Inhibitors of TBK1 and IKKe. PLoS ONE 7(7): e41494. doi:10.1371/journal.pone.0041494 Editor: Edward Harhaj, Johns Hopkins School of Medicine, United States of America Received April 18, 2012; Accepted June 22, 2012; Published July 30, 2012 Copyright: 2012 Hutti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) grants CA75080 (Dr. Baldwin), AI35098 (Dr. Baldwin), CA73756 (Dr. Baldwin), and R01GM56203 (Dr. Cantley). Additional support was provided by Department of Defense CDMRP BCRP grant W81XWH-10-1-0342 (Dr. Hutti), the Samuel Waxman Cancer Research Foundation (Dr. Baldwin), and NC TraCS grant 3-13400 (Dr. Wang). Dr. Hutti is a Lilly Research Labs fellow of the Life Sciences Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bjanzen@email.unc.edu