{"title":"用于鉴定靶向热点残基的Nrf2-Kep1蛋白-蛋白相互作用抑制剂的筛选方法。","authors":"Wataru Asano , Rie Hantani , Toru Uhara , François Debaene , Akihiro Nomura , Keishi Yamaguchi , Tsuyoshi Adachi , Kazuki Otake , Kazuhito Harada , Yoshiji Hantani","doi":"10.1016/j.slasd.2023.11.001","DOIUrl":null,"url":null,"abstract":"<div><p>Protein-protein interactions (PPIs) play a crucial role in most biological processes and are important targets in the development of therapeutic agents. However, small molecule drug discovery that targets PPIs remains very challenging. Targeting hot spot residues is considered the best option for inhibiting such interactions, but there are few examples of how knowledge of hot spots can be used in high throughput screening to find hit compounds. A substrate adaptor protein for a ubiquitin ligase complex, Kelch-like ECH-associated protein 1 (Keap1), negatively modulates the expression of genes involved in cellular protection against oxidative stress. Here, we focused on three arginine hot spot residues in the Keap1 substrate binding pocket (Arg380, Arg415, and Arg483), and screened the carboxylic acid library owned by Japan Tobacco Inc. for compounds that interact with the arginine residues in differential scanning fluorescence assays. Furthermore, we identified several small molecule compounds that specifically bind to the Keap1 Kelch domain hot spots by comparing binding to alanine mutant proteins (R380A, R415A, and R483A) with binding to the wild-type protein using surface plasmon resonance (SPR) screening. These compounds inhibited the protein-protein interaction between the Keap1 Kelch domain and the nuclear factor erythroid 2-related factor 2 (Nrf2) peptide, and the ubiquitination of Nrf2 catalyzed by the Cul3/RINGBox 1 E3 ligase. In addition, the binding mode of one compound (Compound 4) was determined by X-ray crystallography after validation of binding by isothermal titration calorimetry, native mass spectrometry, and nuclear magnetic resonance. Compound 4 had favorable thermodynamic properties, and noncovalently bound to Keap1 with a stoichiometry of 1:1. Our results suggest that Compound 4 could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions such as oxidative stress response, inflammation, and carcinogenesis. We believe that the use of a set of complementary biophysical techniques including the SPR assay with single alanine mutant of hot spots provides opportunities to identify hit compounds for developing inhibitors of PPIs.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 2","pages":"Article 100125"},"PeriodicalIF":2.7000,"publicationDate":"2024-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555223000795/pdfft?md5=15c038b4f2ad2614e505129c72ef0593&pid=1-s2.0-S2472555223000795-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Screening approaches for the identification of Nrf2-Keap1 protein-protein interaction inhibitors targeting hot spot residues\",\"authors\":\"Wataru Asano , Rie Hantani , Toru Uhara , François Debaene , Akihiro Nomura , Keishi Yamaguchi , Tsuyoshi Adachi , Kazuki Otake , Kazuhito Harada , Yoshiji Hantani\",\"doi\":\"10.1016/j.slasd.2023.11.001\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Protein-protein interactions (PPIs) play a crucial role in most biological processes and are important targets in the development of therapeutic agents. However, small molecule drug discovery that targets PPIs remains very challenging. Targeting hot spot residues is considered the best option for inhibiting such interactions, but there are few examples of how knowledge of hot spots can be used in high throughput screening to find hit compounds. A substrate adaptor protein for a ubiquitin ligase complex, Kelch-like ECH-associated protein 1 (Keap1), negatively modulates the expression of genes involved in cellular protection against oxidative stress. Here, we focused on three arginine hot spot residues in the Keap1 substrate binding pocket (Arg380, Arg415, and Arg483), and screened the carboxylic acid library owned by Japan Tobacco Inc. for compounds that interact with the arginine residues in differential scanning fluorescence assays. Furthermore, we identified several small molecule compounds that specifically bind to the Keap1 Kelch domain hot spots by comparing binding to alanine mutant proteins (R380A, R415A, and R483A) with binding to the wild-type protein using surface plasmon resonance (SPR) screening. These compounds inhibited the protein-protein interaction between the Keap1 Kelch domain and the nuclear factor erythroid 2-related factor 2 (Nrf2) peptide, and the ubiquitination of Nrf2 catalyzed by the Cul3/RINGBox 1 E3 ligase. In addition, the binding mode of one compound (Compound 4) was determined by X-ray crystallography after validation of binding by isothermal titration calorimetry, native mass spectrometry, and nuclear magnetic resonance. Compound 4 had favorable thermodynamic properties, and noncovalently bound to Keap1 with a stoichiometry of 1:1. Our results suggest that Compound 4 could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions such as oxidative stress response, inflammation, and carcinogenesis. We believe that the use of a set of complementary biophysical techniques including the SPR assay with single alanine mutant of hot spots provides opportunities to identify hit compounds for developing inhibitors of PPIs.</p></div>\",\"PeriodicalId\":21764,\"journal\":{\"name\":\"SLAS Discovery\",\"volume\":\"29 2\",\"pages\":\"Article 100125\"},\"PeriodicalIF\":2.7000,\"publicationDate\":\"2024-03-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S2472555223000795/pdfft?md5=15c038b4f2ad2614e505129c72ef0593&pid=1-s2.0-S2472555223000795-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"SLAS Discovery\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2472555223000795\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"SLAS Discovery","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2472555223000795","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Screening approaches for the identification of Nrf2-Keap1 protein-protein interaction inhibitors targeting hot spot residues
Protein-protein interactions (PPIs) play a crucial role in most biological processes and are important targets in the development of therapeutic agents. However, small molecule drug discovery that targets PPIs remains very challenging. Targeting hot spot residues is considered the best option for inhibiting such interactions, but there are few examples of how knowledge of hot spots can be used in high throughput screening to find hit compounds. A substrate adaptor protein for a ubiquitin ligase complex, Kelch-like ECH-associated protein 1 (Keap1), negatively modulates the expression of genes involved in cellular protection against oxidative stress. Here, we focused on three arginine hot spot residues in the Keap1 substrate binding pocket (Arg380, Arg415, and Arg483), and screened the carboxylic acid library owned by Japan Tobacco Inc. for compounds that interact with the arginine residues in differential scanning fluorescence assays. Furthermore, we identified several small molecule compounds that specifically bind to the Keap1 Kelch domain hot spots by comparing binding to alanine mutant proteins (R380A, R415A, and R483A) with binding to the wild-type protein using surface plasmon resonance (SPR) screening. These compounds inhibited the protein-protein interaction between the Keap1 Kelch domain and the nuclear factor erythroid 2-related factor 2 (Nrf2) peptide, and the ubiquitination of Nrf2 catalyzed by the Cul3/RINGBox 1 E3 ligase. In addition, the binding mode of one compound (Compound 4) was determined by X-ray crystallography after validation of binding by isothermal titration calorimetry, native mass spectrometry, and nuclear magnetic resonance. Compound 4 had favorable thermodynamic properties, and noncovalently bound to Keap1 with a stoichiometry of 1:1. Our results suggest that Compound 4 could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions such as oxidative stress response, inflammation, and carcinogenesis. We believe that the use of a set of complementary biophysical techniques including the SPR assay with single alanine mutant of hot spots provides opportunities to identify hit compounds for developing inhibitors of PPIs.
期刊介绍:
Advancing Life Sciences R&D: SLAS Discovery reports how scientists develop and utilize novel technologies and/or approaches to provide and characterize chemical and biological tools to understand and treat human disease.
SLAS Discovery is a peer-reviewed journal that publishes scientific reports that enable and improve target validation, evaluate current drug discovery technologies, provide novel research tools, and incorporate research approaches that enhance depth of knowledge and drug discovery success.
SLAS Discovery emphasizes scientific and technical advances in target identification/validation (including chemical probes, RNA silencing, gene editing technologies); biomarker discovery; assay development; virtual, medium- or high-throughput screening (biochemical and biological, biophysical, phenotypic, toxicological, ADME); lead generation/optimization; chemical biology; and informatics (data analysis, image analysis, statistics, bio- and chemo-informatics). Review articles on target biology, new paradigms in drug discovery and advances in drug discovery technologies.
SLAS Discovery is of particular interest to those involved in analytical chemistry, applied microbiology, automation, biochemistry, bioengineering, biomedical optics, biotechnology, bioinformatics, cell biology, DNA science and technology, genetics, information technology, medicinal chemistry, molecular biology, natural products chemistry, organic chemistry, pharmacology, spectroscopy, and toxicology.
SLAS Discovery is a member of the Committee on Publication Ethics (COPE) and was published previously (1996-2016) as the Journal of Biomolecular Screening (JBS).