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Enhancing therapeutic proteins with additional covalent binding capabilities, such as minibinders and nanobodies, reportedly potentiates their antiviral efficacy by irreversibly capturing the viral cell\u2010entry protein. However, viral mutations that interfere with covalent bonding or reduce viral affinity with therapeutic proteins might compromise the efficacy of this strategy. Therefore, in this study, we aimed to develop a broadly neutralizing covalent angiotensin\u2010converting enzyme 2\u2010Fc (ACE2\u2010Fc) decoy using a rational design strategy that integrates functional genomics with structural information. Using this approach, we targeted a highly conserved and functionally constrained residue on the viral receptor\u2010binding domain (RBD) and identified tyrosine 473 (Y473) as an optimal target. We engineered ACE2\u2010Fc constructs by replacing glutamate 23 (E23) and threonine 27 (T27) with the non\u2010canonical amino acid\u2014fluorosulfate\u2010L\u2010tyrosine (FSY), generating E23FSY and T27FSY constructs. These constructs formed a specific and efficient covalent bond with Y473 of the RBD. Notably, this covalent capture was retained against the highly mutated Omicron BA.5 RBD. In pseudovirus neutralization assays, both E23FSY and T27FSY exhibited markedly enhanced potency against both wild\u2010type\u2010like (D614G) and Omicron variants compared to their non\u2010covalent counterparts. These results demonstrate that using an inherently escape\u2010resistant decoy receptor to covalently target evolutionarily constrained residues on the viral RBD is a highly efficient strategy for creating potent, broad\u2010spectrum covalent inhibitors against rapidly evolving viruses such as SARS\u2010CoV\u20102.<\/jats:p>","DOI":"10.1002\/pro.70306","type":"journal-article","created":{"date-parts":[[2025,9,13]],"date-time":"2025-09-13T08:23:46Z","timestamp":1757751826000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Rational design of a covalent <scp>ACE2<\/scp> decoy receptor that broadly neutralizes <scp>SARS<\/scp>\u2010<scp>CoV<\/scp>\u20102 variants"],"prefix":"10.1002","volume":"34","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7072-1183","authenticated-orcid":false,"given":"Nobumasa","family":"Hino","sequence":"first","affiliation":[{"name":"Laboratory of Biopharmaceutics, Graduate School of 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