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Development of a high-throughput assay for identifying inhibitors of TBK1 and IKKε
Jessica E Hutti1, Melissa A Porter, Adam W Cheely
1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina, United States of America.
Abstract:
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.
Insights
Researchers identified the optimal substrate motif for TBK1 (TANK-binding kinase 1), finding it identical to IKKε (IκB kinase ε). This enabled a new high-throughput screen to discover potential inhibitors for inflammatory diseases and cancer.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- IKKε (IκB kinase ε) and TBK1 (TANK-binding kinase 1) are key regulators of inflammatory signaling and oncogenesis.
- Limited identification of inhibitors for these kinases hinders therapeutic development.
- The substrate specificity of TBK1 was previously unknown, impeding high-throughput screening.
Purpose of the Study:
- To determine the substrate phosphorylation motif for TBK1.
- To develop a high-throughput screening assay for TBK1 and IKKε inhibitors.
- To identify novel small molecule inhibitors for TBK1 and IKKε.
Main Methods:
- Determined TBK1 substrate phosphorylation motif using biochemical assays.
- Designed an optimal substrate peptide for TBK1/IKKε.
- Screened a library of 6,006 compounds, including 4,727 kinase-focused compounds, for inhibitory activity.
Main Results:
- The optimal substrate phosphorylation motif for TBK1 was identified and found to be identical to that of IKKε.
- A high-throughput screening assay for TBK1/IKKε was successfully developed.
- 227 compounds inhibited TBK1, and 57 compounds inhibited IKKε at 10 µM concentration.
Conclusions:
- The established substrate motif and screening assay facilitate the discovery of TBK1/IKKε inhibitors.
- Identified inhibitors show therapeutic potential for inflammatory diseases and cancer.
- This work provides a foundation for developing novel small molecule therapeutics targeting TBK1 and IKKε.
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