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Here we report the identification and characterization of RNA-binding compounds that inhibit SARS-CoV-2 replication. The compounds were detected by screening a small library of antiviral compounds previously shown to bind HIV-1 or HCV RNA elements with a live-virus cellular assay detecting inhibition of SARS-CoV-2 replication. These experiments allowed detection of eight compounds with promising anti-SARS-CoV-2 activity in the sub-micromolar to micromolar range and wide selectivity indexes. Examination of the mechanism of action of three selected hit compounds excluded action on the entry or egress stages of the virus replication cycle and confirmed recognition by two of the molecules of conserved RNA elements of the SARS-CoV-2 genome, including the highly conserved S2m hairpin located in the 3\u2019-untranslated region of the virus. While further studies are needed to clarify the mechanism of action responsible for antiviral activity, these results facilitate the discovery of RNA-targeted antivirals and provide new chemical scaffolds for developing therapeutic agents against coronaviruses.<\/jats:p>","DOI":"10.3390\/ph15121448","type":"journal-article","created":{"date-parts":[[2022,11,22]],"date-time":"2022-11-22T08:25:28Z","timestamp":1669105528000},"page":"1448","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["SARS-CoV-2 Inhibitors Identified by Phenotypic Analysis of a Collection of Viral RNA-Binding Molecules"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3970-3873","authenticated-orcid":false,"given":"Alvaro","family":"Simba-Lahuasi","sequence":"first","affiliation":[{"name":"Centro de Investigaci\u00f3n Traslacional San Alberto Magno, Universidad Cat\u00f3lica de Valencia, 46001 Valencia, Spain"},{"name":"Escuela de Doctorado, Universidad Cat\u00f3lica de Valencia, 46001 Valencia, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"\u00c1ngel","family":"Cantero-Camacho","sequence":"additional","affiliation":[{"name":"Centro de Investigaci\u00f3n Traslacional San Alberto Magno, Universidad Cat\u00f3lica de Valencia, 46001 Valencia, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5326-4753","authenticated-orcid":false,"given":"Romel","family":"Rosales","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"},{"name":"Global Health Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0492-9904","authenticated-orcid":false,"given":"Briana Lynn","family":"McGovern","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"},{"name":"Global Health Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M. Luis","family":"Rodr\u00edguez","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"},{"name":"Global Health Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1905-2156","authenticated-orcid":false,"given":"Vicente","family":"March\u00e1n","sequence":"additional","affiliation":[{"name":"Departament de Qu\u00edmica Inorg\u00e0nica i Org\u00e0nica, Secci\u00f3 de Qu\u00edmica Org\u00e0nica, Institut de Biomedicina (IBUB), Universitat de Barcelona (UB), 08028 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0889-0506","authenticated-orcid":false,"given":"Kris M.","family":"White","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"},{"name":"Global Health Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6551-1827","authenticated-orcid":false,"given":"Adolfo","family":"Garc\u00eda-Sastre","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"},{"name":"Global Health Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"},{"name":"Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"},{"name":"Tish Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"},{"name":"Department of Pathology, Molecular and Cell Based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0627-6927","authenticated-orcid":false,"given":"Jos\u00e9","family":"Gallego","sequence":"additional","affiliation":[{"name":"Centro de Investigaci\u00f3n Traslacional San Alberto Magno, Universidad Cat\u00f3lica de Valencia, 46001 Valencia, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,11,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e0010921","DOI":"10.1128\/CMR.00109-21","article-title":"SARS-CoV-2 Antiviral Therapy","volume":"34","author":"Tao","year":"2021","journal-title":"Clin. 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