HTS fluorescence polarization assay for inhibitors of Keap1-Nrf2 interaction

描述（由申请人提供）：Keap1-Nrf2相互作用抑制剂的HTS荧光偏振测定Keap1-Nrf2抗氧化反应元件（ARE）系统调节细胞防御机制，保护细胞免受氧化应激。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剂进行测试。然而，由于其化学反应性和假定的不可逆作用机制，人们普遍担心将其用作化学预防剂。高通量筛选化学文库是发现小分子先导物作为蛋白质相互作用的直接可逆抑制剂（如Keap1-Nrf2）的有效方法；这种Keap1-Nrf2相互作用的直接抑制剂将解决一般使用纯化的天然硫醇反应性抑制剂作为化学预防剂的问题。本应用的目的是将我们的荧光偏振（FP）分析转化为MLPCN筛选中心，用于MLPCN文库的筛选。FP分析检测荧光标记的nrf2肽与Keap1 kelch结构域结合的竞争性抑制剂。开发荧光探针和FP测定的部分资金由R01 CA133791， Hu L （PI）提供。我们的实验室已经建立了二级和三级检测，包括SPR结合检测和基于细胞的are -荧光素酶报告基因检测。有限的化学修饰也将在第二年进行，以获得初始SAR，以便选择命中点作为未来优化成Keap1-Nrf2相互作用的有效直接抑制剂的先导。这些抑制剂将成为阐明细胞保护途径的重要药理学探针，并在高危人群中作为癌症和其他疾病的潜在化学预防剂。

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.