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

用于筛选 Keap1-Nrf2 相互作用抑制剂的同质 HTS 测定

基本信息

批准号：
7902191
负责人：
LONGQIN HU
金额：
$ 28.76万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-25 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7902191
关键词：
Address Adverse effects Affinity Alkenes Amides Amines Antioxidants Binding Binding Sites Biological Assay Biological Factors Biosensor Cancer Biology Carcinogens Cell Culture Techniques Cell Nucleus Cells Chemicals Chemistry Chemopreventive Agent Complex Coupling Curcumin Cyclic Peptides Cysteine Defense Mechanisms Development Disease Dissociation Disulfides Dose Employee Strikes Enzymes Epigallocatechin Gallate Europium Evaluation Exhibits Fluorescence Fluorescence Polarization Fluorescence Resonance Energy Transfer Fluorescent Dyes Genes Human Inflammation Inflammatory Isothiocyanates Knockout Mice Label Lanthanoid Series Elements Lead Link Maleimides Malignant Neoplasms Modification Mus NF-E2-related factor 2 Oxidation-Reduction Oxidative Stress Pathway interactions Peptides Pharmaceutical Chemistry Pharmaceutical Preparations Play Population Preclinical Drug Evaluation Proteins Public Health Reactive Oxygen Species Recombinants Research Research Project Grants Resistance Response Elements Role Safety Screening procedure Signal Pathway Solutions Source Stress Sulfhydryl Compounds Sulforaphane Surface Plasmon Resonance System Tea Terbium Tertiary Protein Structure Time Tracer Transcriptional Activation Triazoles Up-Regulation Work X-Ray Crystallography assay development base biological adaptation to stress cancer chemoprevention chromophore computational chemistry fluorophore fruits and vegetables high risk high throughput screening inhibitor/antagonist mouse model novel programs protein protein interaction research study response sensor small molecule small molecule libraries

项目摘要

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.

描述（由申请人提供）：Keap 1-Nrf 2-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。研究表明，Nrf 2基因敲除小鼠对致癌物、药物和炎症应激的毒理学效应更敏感，而Keap 1基因敲除小鼠则表现出高水平的Nrf 2、细胞保护酶的高组成性表达和对环境应激的显著抵抗力。Keap 1-Nrf 2相互作用在癌症化学预防中起着关键作用，许多化学物质如萝卜硫素，姜黄素和表没食子儿茶素没食子酸酯来自天然来源，如水果，蔬菜和茶产品。这些天然亲电体对氧化还原“传感器”蛋白Keap 1中发现的敏感半胱氨酸残基的修饰被认为是导致Nrf 2易位到细胞核和随后上调抗氧化应激细胞保护酶的原因。解决纯化的天然硫醇反应性化合物作为化学预防剂的一般用途的问题的一种方法是使用化学文库的高通量筛选测定来发现和开发新的小分子作为蛋白质-蛋白质界面处Keap 1-Nrf 2相互作用的直接抑制剂。这些抑制剂将模拟亲电体如异硫氰酸酯和迈克尔受体在诱导细胞保护酶中的作用，但可能对Keap-Nrf 2轴更具选择性和特异性。两个解决方案的荧光为基础的高通量筛选试验将开发和验证在这个新的R 01应用程序使用荧光偏振（FP）和时间分辨荧光共振能量转移（TR-FRET）。这些分析将应用于筛选化学库，以发现涉及药物化学，计算化学，X射线晶体学和癌症生物学的跨学科计划中的新线索。这些研究将提供有关Keap 1和Nrf 2之间蛋白质-蛋白质相互作用的重要信息，并促进开发更有效和选择性的Keap 1-Nrf 2抑制剂作为氧化应激反应酶的诱导剂。公共卫生宣传：本项目中开发的HTS检测方法将有助于发现新的Keap 1-Nrf 2抑制剂的开发。特异性和有效的Keap 1-Nrf 2抑制剂将作为重要的药理学探针，用于阐明细胞保护途径，并作为潜在的癌症化学预防剂在高危人群中。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Optimization of the C2 substituents on the 1,4-bis(arylsulfonamido)naphthalene-N,N'-diacetic acid scaffold for better inhibition of Keap1-Nrf2 protein-protein interaction.

DOI：
10.1016/j.ejmech.2023.115302
发表时间：
2023-03
期刊：
European journal of medicinal chemistry
影响因子：
6.7
作者：
Dhulfiqar Ali Abed;Ahmed R. Ali;Sumi Lee;Mai-Uyen Nguyen;M. Verzi;Longqin Hu
通讯作者：
Dhulfiqar Ali Abed;Ahmed R. Ali;Sumi Lee;Mai-Uyen Nguyen;M. Verzi;Longqin Hu

Discovery of a small-molecule inhibitor and cellular probe of Keap1-Nrf2 protein-protein interaction.

DOI：
10.1016/j.bmcl.2013.03.013
发表时间：
2013-05-15
期刊：
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
影响因子：
2.7
作者：
Hu, Longqin;Magesh, Sadagopan;Chen, Lin;Wang, Lili;Lewis, Timothy A.;Chen, Yu;Khodier, Carol;Inoyama, Daigo;Beamer, Lesa J.;Emge, Thomas J.;Shen, Jian;Kerrigan, John E.;Ah-Ng Tony Kong;Dandapani, Sivaraman;Palmer, Michelle;Schreiber, Stuart L.;Munoz, Benito
通讯作者：
Munoz, Benito

Development of a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for the Inhibition of Keap1-Nrf2 Protein-Protein Interaction.

开发均匀的时间分辨荧光共振能量转移（TR-FRET）测定，以抑制Keap1-NRF2蛋白 - 蛋白质相互作用。