Assay Development for Screening of Specific IKKalpha Kinase Inhibitors

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DESCRIPTION (provided by applicant): Although IKK1 and IKK2 are highly homologous, their functions are very unique. Specifically, IKK2 locates in the cytoplasm and functions through NF-kB pathways whereas IKK1 can translocate to and function in the nucleus that is independent of classical and alterative NF-kB pathways. Recent studies have demonstrated that IKK1 kinase plays a pivotal role in breast and prostate cancer development and cancer therapy resistance, suggesting that IKK1 kinase is an excellent target for cancer therapy. However, currently there are no IKK1 kinase-specific inhibitors or the methods for the discovery of IKK1 kinase-specific inhibitors available. In the proposed project we plan to develop a functional cell-based high-throughput screening (HTS) assay to identify specific inhibitors for IKK1 kinase. This assay utilizes the newly discovered function of nuclear IKK1 kinase that suppresses maspin transcription. We transfected human breast cancer cell line MCF7 with plasmid containing maspin promoter-driven luciferase reporter and established two stable reporter cell lines. These cell lines will be further characterized for HTS development. The density, incubation time, dimethyl sulfoxide (DMSO) concentration used to screen the IKK1 kinase specific inhibitors will also be optimized. In addition, we will establish a complementary assay and a series of counter-screen assays for the validation and prioritization of IKK1 kinase inhibitor candidates. Our studies will establish a high-selective and high-efficient functional cell- based HTS assay for the discovery of IKK1 kinase specific inhibitors. We will submit this assay to MLPCN when the project is completed. In our follow-up studies, promising candidates from screens will be examined further for their ability to inhibit IKK1 kinase activity, maspin expression, cell proliferation and/or motility in human breast and prostate cancer cell lines, and their ability to suppress breast and prostate cancer development, progression, metastasis, and therapy-resistance in animal models. PUBLIC HEALTH RELEVANCE: IKK1 kinase plays a pivotal role in breast and prostate cancer development and therapy-resistance, suggesting that IKK1 kinase is an excellent target for breast and prostate cancer treatment. However, currently there are no IKK1 kinase-specific inhibitors or the methods for the discovery of IKK1 kinase-specific inhibitors available. Our studies will establish a high-selective and high-efficient functional cell-based high-throughput screen system for the discovery of IKK1 kinase-specific inhibitors.