Homogenous HTS Assays to Screen for Inhibitors of Keap1-Nrf2 Interaction

描述(申请人提供)：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。已有研究表明，Nrf2基因敲除小鼠对致癌物、药物和炎症应激的毒理作用更加敏感，而Keap1基因敲除小鼠则表现出高水平的Nrf2、高结构性的细胞保护酶表达和对环境应激的显著抗性。Keap1-Nrf2相互作用在许多化学物质如萝卜硫素、姜黄素和从水果、蔬菜和茶叶等天然来源中提取的表儿茶素没食子酸酯等化学预防癌症中发挥着关键作用。这些天然亲电体对氧化还原“传感器”蛋白Keap1中发现的敏感半胱氨酸残基的修饰被认为是导致Nrf2移位到细胞核并随后上调抗氧化应激细胞保护酶的原因。为了解决人们对纯化的天然硫醇活性化合物作为化学预防药物的普遍使用的担忧，一种方法是使用化学文库的高通量筛选分析来发现和开发新的小分子，作为蛋白质-蛋白质界面上Keap1-Nrf2相互作用的直接抑制剂。这些抑制剂将模仿异硫氰酸酯和Michael受体等亲电体在诱导细胞保护酶方面的作用，但可能对Keap-Nrf2轴具有更高的选择性和特异性。将开发两种基于溶液荧光的高通量筛选分析，并在这一新的R01应用中使用荧光偏振(FP)和时间分辨荧光共振能量转移(TR-FRET)进行验证。这些分析将被应用于筛选化学库，以在涉及药物化学、计算化学、X射线结晶学和癌症生物学的跨学科计划中发现新的线索。这些研究将提供关于Keap1和Nrf2之间蛋白质-蛋白质相互作用的重要信息，并有助于开发更有效和更具选择性的Keap1-Nrf2抑制剂作为氧化应激反应酶的诱导剂。公共卫生评论：在该项目中开发的HTS分析将有助于发现开发新的Keap1-Nrf2抑制剂的新线索。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): Keap1-Nrf2-antioxidant response element (ARE) system regulates cellular defense mechanisms that protect cells from oxidative stress. It has been demonstrated that Nrf2 knockout mice are more sensitive to toxicological effects of carcinogens, drugs, and inflammatory stresses while Keap1 knockout mice exhibited high levels of Nrf2, high constitutive expression of cytoprotective enzymes and striking resistance to the environmental stresses. Keap1-Nrf2 interaction plays a key role in cancer chemoprevention by many chemicals like sulforaphane, curcumin, and epigallocatechin gallate derived from natural sources such as fruits, vegetables, and tea products. Modification, by these natural electrophiles, of sensitive cysteine residues found in the redox "sensor" protein Keap1 is believed to be responsible for causing the translocation of Nrf2 to the nucleus and subsequent upregulation of anti-oxidative stress cytoprotective enzymes. One approach to address concerns over the general use of purified natural thiol-reactive compounds as chemopreventive agents is to use high throughput screening assays of chemical libraries to discover and develop novel small molecules as direct inhibitors of Keap1-Nrf2 interaction at the protein-protein interface. These inhibitors will mimic the actions of electrophiles like isothiocyanates and Michael acceptors in the induction of cytoprotective enzymes but will potentially be more selective and specific against the Keap-Nrf2 axis. Two solution fluorescence-based high throughput screening assays will be developed and validated in this new R01 application using fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET). These assays will be applied to the screening of chemical libraries for the discovery of new leads in an interdisciplinary program involving medicinal chemistry, computational chemistry, X-ray crystallography, and cancer biology. These studies will provide important information about the protein-protein interaction between Keap1 and Nrf2 and facilitate the development of more potent and selective Keap1-Nrf2 inhibitors as inducers of oxidative stress response enzymes. PUBLIC HEALTH REVELANCE:HTS assays developed in this project will facilitate the discovery of novel leads for the development of new Keap1-Nrf2 inhibitors. 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.