Assay Development for Screening of Specific IKKalpha Kinase Inhibitors

描述(申请人提供)：虽然IKK1和IKK2高度同源，但它们的功能非常独特。具体地说，IKK2位于细胞质中，通过NF-kB途径发挥功能，而IKK1可以转运到细胞核并在细胞核内发挥作用，这与经典的和可变的NF-kB途径无关。最近的研究表明，IKK1激酶在乳腺癌和前列腺癌的发生发展和癌症治疗耐药中起着关键作用，表明IKK1激酶是癌症治疗的良好靶点。然而，目前还没有IKK1激酶特异性抑制剂或发现IKK1激酶特异性抑制剂的方法可用。在拟议的项目中，我们计划开发一种基于细胞的功能性高通量筛选(HTS)试验，以确定IKK1激酶的特定抑制剂。这种分析利用了新发现的核IKK1激酶抑制maspin转录的功能。我们将maspin启动子驱动的荧光素酶报告基因导入人乳腺癌细胞系MCF7，建立了两个稳定的报告细胞系。这些细胞系将为HTS的开发进一步表征。用于筛选IKK1激酶特异性抑制剂的密度、孵育时间、二甲基亚砜(DMSO)浓度也将得到优化。此外，我们将建立一个补充试验和一系列反筛选试验来验证和确定IKK1激酶抑制剂候选的优先顺序。我们的研究将建立一种高选择性和高效率的基于细胞的功能HTS方法，用于发现IKK1激酶特异性抑制剂。当项目完成后，我们将向MLPCN提交这一检测结果。在我们的后续研究中，将进一步检验筛选出的有希望的候选药物在人类乳腺癌和前列腺癌细胞系中抑制IKK1激酶活性、maspin表达、细胞增殖和/或运动的能力，以及在动物模型中抑制乳腺癌和前列腺癌的发展、进展、转移和治疗耐药性的能力。 公共卫生相关性：IKK1激酶在乳腺癌和前列腺癌的发展和治疗耐药中起着关键作用，这表明IKK1激酶是乳腺癌和前列腺癌治疗的极佳靶点。然而，目前还没有IKK1激酶特异性抑制剂或发现IKK1激酶特异性抑制剂的方法可用。我们的研究将建立一个高选择性和高效率的功能细胞为基础的高通量筛选系统，以发现IKK1激酶特异性抑制剂。

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.