TY - JOUR T1 - In Vitro Evaluation and Mitigation of Niclosamide’s Liabilities as a COVID-19 Treatment JF - bioRxiv DO - 10.1101/2022.06.24.497526 SP - 2022.06.24.497526 AU - Jesse W. Wotring AU - Sean M. McCarty AU - Khadija Shafiq AU - Charles J. Zhang AU - Theophilus Nguyen AU - Sophia R. Meyer AU - Reid Fursmidt AU - Carmen Mirabelli AU - Martin C. Clasby AU - Christiane E. Wobus AU - Matthew J. O’Meara AU - Jonathan Z. Sexton Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/06/24/2022.06.24.497526.abstract N2 - Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro, generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including B.1.1.7. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure-activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future.Competing Interest StatementThe authors have declared no competing interest. ER -