Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina, United States of America.
PLoS One. 2012;7(7):e41494. doi: 10.1371/journal.pone.0041494. Epub 2012 Jul 30.
IKKε 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 IKKε 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 IKKε. This information enabled the design of an optimal TBK1/IKKε 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 IKKε. 227 compounds in this library inhibited TBK1 at a concentration of 10 µM, while 57 compounds inhibited IKKε. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKε inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.
IKKε 和 TBK1 是非典型的 IKK 家族成员，它们调节炎症信号通路，在肿瘤发生中也发挥着重要作用。然而，目前已经鉴定出的这些激酶抑制剂很少。尽管最近已经描述了 IKKε 的底物特异性，但 TBK1 的底物特异性尚不清楚，这阻碍了高通量筛选技术用于抑制剂鉴定的发展。在这里，我们描述了 TBK1 的最佳底物磷酸化基序，并表明它与先前为 IKKε 描述的磷酸化基序相同。这些信息使得设计一种适合高通量筛选的最佳 TBK1/IKKε 底物肽成为可能，我们对一个包含 4,727 种激酶靶向化合物的 6,006 种化合物文库进行了检测，以发现 TBK1 和 IKKε 的体外抑制剂。该文库中有 227 种化合物在 10µM 浓度下抑制 TBK1，而 57 种化合物抑制 IKKε。总之，这些数据描述了一种新的高通量筛选测定法，这将有助于发现具有治疗炎症性疾病和癌症潜力的小分子 TBK1/IKKε 抑制剂。
