Repurposing Niclosamide as a Novel Anti-SARS-CoV-2 Drug by Restricting Entry Protein CD147

Figures

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  Figure 1. HuR binds BSG mRNA and regulates its expression. (a) CD147 expression in COVID-19 patients’ blood samples. Here, 6 out of 19 early patients, 6 out of 36 middle patients, and 0 out of 22 late patients are severely ill (the rest are considered as mild/moderate). (b) Western blotting analysis of HuR and CD147 protein in HuR knock-out clones of MDA-MB-231 cells with or without niclosamide treatment. Tubulin was used as the loading control. NIC, niclosamide. (b’) Quantified relative level of CD147 HG and LG in (b). (c) Western blotting analysis of HuR and CD147 protein in doxycycline-inducible HuR TET-off system in Hela cells. Dox, doxycycline. (c’) Quantified relative level of CD147 and HuR in (c). (d–f) RNP-IP analysis of relative enrichment of BSG transcripts in HuR-immunoprecipitation in (d) H460, (e) NL20, and (f) WI-38 cells. (g) Western blotting of HuR protein in the pull-down complex by BSG RNA probes in NL20 and WI-38 cells. *** p < 0.001; ** 0.001 < p < 0.01; * 0.01 < p < 0.05. NS, not significant. Two-tailed T-test was applied for statistics.

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  Figure 2. Immunocytochemistry and immunohistochemistry staining for HuR and the effect of niclosamide on HuR translocation. (a–d’) Immunofluorescence staining for the detection of HuR and the effect of niclosamide on HuR cytoplasmic translocation. WI-38 (a–b’) and NL20 (c–d’) cells were exposed to niclosamide or DMSO for 48 h. HuR is presented in green. Nuclei were counterstained with DAPI (blue). (e,e’) Immunohistochemistry staining for HuR protein in the LL/2 tumor tissue with (e) PBS or (e’) niclosamide treatment for 5 days. Arrows point to the cytoplasmic HuR.

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  Figure 3. Niclosamide inhibits HuR nucleocytoplasmic translocation. (a–f) Western blot of HuR and CD147 in nuclear and cytoplasmic fractions with niclosamide treatment at different concentrations and different times in NL20 (a,b), H460 (c,d), and WI-38 (e,f). Quantified relative levels of cytoplasmic HuR in NL20 (as in blue box), H460 (as in red box), and WI-38 (as in green box) are shown in (a’–f’), respectively. (g) Western blot of HuR and CD147 protein in Hela, 293FT, RKO, and MDA-MB-231 cells treated with niclosamide at different concentrations. NIC, niclosamide.

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  Figure 4. Niclosamide effectively inhibits SARS-CoV-2-induced CD147. (a) Western blotting analysis of CD147 protein in A549-ACE2 cells with or without SARS-CoV-2 infection and niclosamide treatment. Spike protein was used as the virus marker and tubulin was the loading control. (b,c) Quantification of relative band intensity of HG CD147 and LG CD147. (d) The proposed working model of niclosamide restricting SARS-CoV-2 in vitro. Schematic diagram of the mechanism: niclosamide disrupts the nucleocytoplasmic shuttling of HuR, thereby inhibiting the post-transcription of CD147 mRNA and abolishing the glycosylation and maturation of CD147. In addition, niclosamide effectively restrains SARS-CoV-2-induced CD147. Therefore, niclosamide can be a promising anti-COVID-19 drug by blocking HuR nucleocytoplasmic translocation and SARS-CoV-2 entry route CD147. ** 0.001 < p < 0.01; * 0.01 < p < 0.05. NS, not significant. Two-tailed T-test was applied for statistics.