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Despite the rapid development of antiviral vaccines and some approved treatments such as remdesivir and paxlovid, effective antiviral pharmacological treatments for COVID\u201019 patients remain limited. This study explores Nsp15, a 3\u2032\u2010uridylate\u2010specific RNA endonuclease, which has a critical role in immune system evasion and hence in escaping the innate immune sensors. We conducted a comprehensive drug repurposing screen and identified 44 compounds that showed more than 55% inhibition of Nsp15 activity in a real\u2010time fluorescence assay. A validation pipeline was employed to exclude unspecific interactions, and dose\u2013response assays confirmed 29 compounds with an IC<jats:sub>50<\/jats:sub> below 10\u2009\u03bcM. Structural studies, including molecular docking and x\u2010ray crystallography, revealed key interactions of identified inhibitors, such as TAS\u2010103 and YM\u2010155, with the Nsp15 active site and other critical regions. Our findings show that the identified compounds, particularly those retaining potency under different assay conditions, could serve as promising hits for developing Nsp15 inhibitors. Additionally, the study emphasizes the potential of combination therapies targeting multiple viral processes to enhance treatment efficacy and reduce the risk of drug resistance. This research contributes to the ongoing efforts to develop effective antiviral therapies for SARS\u2010CoV\u20102 and possibly other coronaviruses.<\/jats:p>","DOI":"10.1002\/pro.70156","type":"journal-article","created":{"date-parts":[[2025,5,15]],"date-time":"2025-05-15T10:59:03Z","timestamp":1747306743000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Identification, validation, and characterization of approved and investigational drugs interfering with the <scp>SARS<\/scp>\u2010<scp>CoV<\/scp>\u20102 endoribonuclease Nsp15"],"prefix":"10.1002","volume":"34","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7569-5474","authenticated-orcid":false,"given":"Spyros D.","family":"Chatziefthymiou","sequence":"first","affiliation":[{"name":"Photon Science, DESY  Hamburg Germany"},{"name":"Department for Structural Infection Biology Centre for Structural Systems Biology (CSSB) &amp; Helmholtz\u2010Centre for 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