In Vitro Evaluation and Mitigation of Niclosamide’s Liabilities as a COVID-19 Treatment
Jesse W Wotring, Sean M Mccarty, Khadija Shafiq, Charles J Zhang, Theophilus Nguyen, Sophia R Meyer, Reid Fursmidt, Carmen Mirabelli, Martin C Clasby, Christiane E Wobus, Matthew J O’meara, Jonathan Z Sexton
Vaccines, doi:10.3390/vaccines10081284
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 the B.1.1.7 (alpha) variant. 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.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/vaccines10081284/s1 , Supplementary Figure S1 : Control data for VeroE6 and H1437 SARS-CoV-2 long-term exposure bioassays; Supplementary Figure S2: Niclosamide analog structures; Supplementary Figure S3 : Some niclosamide analogs cause dose-dependent exacerbation of infection in H1437. Author Contributions: Conceptualization, J.W.W., S.M.M., J.Z.S. and M.J.O.; methodology, J.W.W., S.M.M. and C.M.; formal analysis, J.W.W., S.M.M., M.C.C. and J.Z.S., writing-original draft preparation, J.W.W., S.M.M., K.S. and J.Z.S.; writing-review and editing, J.W.W., S.M.M., K.S., C.J.Z., T.N., S.R.M., R.F., C.M., M.C.C., C.E.W., M.J.O. and J.Z.S.; visualization, J.W.W., S.M.M. and J.Z.S.; supervision, J.Z.S., M.C.C., C.E.W. and M.J.O.; project administration, J.Z.S.; funding acquisition, J.Z.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Center for Advancing Translational Science grant number UL1TR002240. The APC was funded by the same grant. Institutional Review Board Statement: This work was performed under the approval of the University of Michigan Institutional Biosafety Committee under protocol IBCA00001528.
Informed Consent Statement: Not applicable. Data Availability Statement: All relevant data are within the paper and its Supplementary Materials files.
Conflicts of..
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doi:10.1038/s41392-022-00941-zDOI record:
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"abstract": "<jats:p>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 the B.1.1.7 (alpha) variant. 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.</jats:p>",
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