HTS fluorescence polarization assay for inhibitors of Keap1-Nrf2 interaction

描述(由申请人提供)：Keap1-Nrf2相互作用抑制物的HTS荧光偏振分析Keap1-Nrf2-抗氧化反应元件(ARE)系统调节保护细胞免受氧化应激的细胞防御机制。Keap1-Nrf2相互作用的间接抑制剂，如萝卜硫素、姜黄素和表没食子儿茶素没食子酸酯，正在作为化学预防药物进行测试。然而，由于它们的化学反应活性和推测的不可逆转的作用机制，人们普遍关注它们作为化学预防药物的使用。高通量筛选化学文库是发现小分子铅作为Keap1-Nrf2等蛋白质-蛋白质相互作用的直接可逆抑制剂的有效方法；这种对Keap1-Nrf2相互作用的直接抑制剂将解决人们对纯化的天然硫醇反应抑制剂作为化学预防药物的普遍使用的担忧。目前这项应用的目标是将我们的荧光偏振(FP)分析转换到MLPCN筛选中心，用于筛选MLPCN文库。FP法检测荧光标记的Nrf2多肽与Keap1 kelch结构域结合的竞争性抑制物。荧光探针和FP分析的开发资金部分由R01CA133791，胡L(PI)提供。我们的实验室已经建立了二级和三级检测方法，包括SPR结合分析和基于细胞的ARE-荧光素酶报告分析。第二年还将进行有限的化学修饰，以获得初始的SAR，以便能够选择HITS作为未来优化的线索，成为有效的Keap1-Nrf2相互作用的直接抑制剂。这些抑制剂将作为重要的药物探针用于阐明细胞保护途径，并在高危人群中作为潜在的癌症和其他疾病的化学预防药物。 公共卫生相关性：使用我们的高通量荧光偏振分析筛选MLPCN文库将能够发现新的小分子作为Keap1-Nrf2相互作用的直接抑制剂。特异和有效的Keap1-Nrf2抑制剂将成为阐明细胞保护途径的重要药理探针，并在高危人群中作为潜在的癌症化学预防药物。

DESCRIPTION (provided by applicant): HTS fluorescence polarization assay for inhibitors of Keap1-Nrf2 interaction Keap1-Nrf2-antioxidant response element (ARE) system regulates cellular defense mechanisms that protect cells from oxidative stress. Indirect inhibitors of Keap1-Nrf2 interaction such as sulforaphane, curcumin, and epigallocatechin gallate are being tested as chemopreventive agents. However, there are general concerns over their use as chemopreventive agents because of their chemical reactivity and their putative irreversible mechanism of action. High throughput screening of chemical libraries is an effective approach to discover small molecule leads as direct reversible inhibitors of protein-protein interactions like Keap1-Nrf2; such direct inhibitors of Keap1-Nrf2 interaction would address concerns over the general use of purified natural thiol- reactive inhibitors as chemopreventive agents. The goal of this current application is to translate our fluorescence polarization (FP) assay to a MLPCN screening center for the screening of MLPCN library. The FP assay detects competitive inhibitors of a fluorescently labeled-Nrf2 peptide from binding to Keap1 kelch domain. Funding for the development of the fluorescent probe and the FP assay has been provided in part by R01 CA133791, Hu L (PI). Secondary and tertiary assays have been established in our labs including an SPR binding assay and a cell-based ARE-luciferase reporter assay. Limited chemical modification will also be performed in the second year to derive initial SAR to enable the selection of hits as leads for future optimization into potent direct inhibitors of Keap1-Nrf2 interaction. Such inhibitors will be useful as important pharmacological probes for the elucidation of cytoprotective pathways and as potential chemopreventive agents of cancer and other diseases in high risk populations. PUBLIC HEALTH RELEVANCE: Screening of the MLPCN library with our high throughput fluorescence polarization assay will enable the discovery of novel small molecules as direct inhibitors of Keap1-Nrf2 interaction. Specific and potent Keap1- Nrf2 inhibitors will be useful as important pharmacological probes for the elucidation of cytoprotective pathways and as potential cancer chemopreventive agents in high risk populations.