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To prepare for rapidly mutating viruses and for the forthcoming pandemic, effective therapies targeting the critical stages of the viral life cycle need to be developed. Viruses are dependent on the interaction between the receptor-binding domain (RBD) of the viral Spike (S) protein (S-RBD) and the angiotensin-converting enzyme 2 (ACE2) receptor to efficiently establish infection and the following replicate. Targeting this interaction provides a promising strategy to inhibit the entry process of the virus, which in turn has both preventive and therapeutic effects. In this study, we developed a robust and straightforward assay based on the Yeast-Two Hybrid system (Y2H) for identifying inhibitors targeting the S-RBD-ACE2 interaction of SARS-CoV-2. Through high-throughput screening, two compounds were identified as potential entry inhibitors. Among them, IMB-1C was superior in terms of pseudovirus entry inhibition and toxicity. It could bind to both ACE2 and S-RBD and induce conformational change in the S-RBD+ACE2 complex. This is the first study to verify the feasibility of utilizing the Y2H system to discover potent SARS-CoV-2 inhibitors targeting the receptor recognition stage. This approach may also be applied in the discovery of other virus receptor recognition inhibitors.<\/jats:p>","DOI":"10.3390\/ijms26020678","type":"journal-article","created":{"date-parts":[[2025,1,15]],"date-time":"2025-01-15T05:59:42Z","timestamp":1736920782000},"page":"678","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Establishment of a Yeast Two-Hybrid-Based High-Throughput Screening Model for Selection of SARS-CoV-2 Spike-ACE2 Interaction Inhibitors"],"prefix":"10.3390","volume":"26","author":[{"given":"Dongsheng","family":"Li","sequence":"first","affiliation":[{"name":"State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China"},{"name":"Research Team of Molecular Medicine, The First Clinical Medical School of Shanxi Medical University, Taiyuan 030001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-5977-2537","authenticated-orcid":false,"given":"Baoqing","family":"You","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keyu","family":"Guo","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenwen","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenyin","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaofang","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6377-3890","authenticated-orcid":false,"given":"Zhen","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuyi","family":"Si","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1967","DOI":"10.1056\/NEJMoa030747","article-title":"Identification of a novel coronavirus in patients with severe acute respiratory syndrome","volume":"348","author":"Drosten","year":"2003","journal-title":"N. 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