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. 2023 Aug 29:14:1206951.
doi: 10.3389/fmicb.2023.1206951. eCollection 2023.

The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein

Sara Piacentini  1 , Anna Riccio  1 , Silvia Santopolo  1 , Silvia Pauciullo  1 , Simone La Frazia  1 , Antonio Rossi  2 , Jean-Francois Rossignol  3 , M Gabriella Santoro  1   2
Affiliations

The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein

Sara Piacentini et al. Front Microbiol. .

Abstract

Coronaviridae is recognized as one of the most rapidly evolving virus family as a consequence of the high genomic nucleotide substitution rates and recombination. The family comprises a large number of enveloped, positive-sense single-stranded RNA viruses, causing an array of diseases of varying severity in animals and humans. To date, seven human coronaviruses (HCoV) have been identified, namely HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, which are globally circulating in the human population (seasonal HCoV, sHCoV), and the highly pathogenic SARS-CoV, MERS-CoV and SARS-CoV-2. Seasonal HCoV are estimated to contribute to 15-30% of common cold cases in humans; although diseases are generally self-limiting, sHCoV can sometimes cause severe lower respiratory infections and life-threatening diseases in a subset of patients. No specific treatment is presently available for sHCoV infections. Herein we show that the anti-infective drug nitazoxanide has a potent antiviral activity against three human endemic coronaviruses, the Alpha-coronaviruses HCoV-229E and HCoV-NL63, and the Beta-coronavirus HCoV-OC43 in cell culture with IC50 ranging between 0.05 and 0.15 μg/mL and high selectivity indexes. We found that nitazoxanide does not affect HCoV adsorption, entry or uncoating, but acts at postentry level and interferes with the spike glycoprotein maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Altogether the results indicate that nitazoxanide, due to its broad-spectrum anti-coronavirus activity, may represent a readily available useful tool in the treatment of seasonal coronavirus infections.

Keywords: HCoV-229E; HCoV-NL63; HCoV-OC43; antiviral; nitazoxanide; spike glycoprotein.

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Conflict of interest statement

Financial support for this study was in part provided by Romark Laboratories LC, the company that owns the intellectual property rights related to nitazoxanide. J-FR is an employee and stockholder of Romark Laboratories, LC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Antiviral activity of nitazoxanide against human seasonal coronaviruses. (A) Schematic representation of genome structure, classification and receptors of the human coronaviruses HCoV-229E, HCoV-OC43 and HCoV-NL63. ORF1a and ORF1b are represented as yellow boxes; genes encoding structural proteins spike (S), nucleocapsid (N), envelope (E), membrane (M), and hemagglutinin-esterase (HE) are shown as blue boxes, and genes encoding accessory proteins are shown as red boxes. hAPN, human aminopeptidase N; 9-O-Ac-Sia, N-acetyl-9-O-acetylneuraminic acid; ACE2, angiotensin-converting enzyme 2. (B–D) MRC-5 (B,C) and LLC-MK2 (D) cells mock-infected or infected with HCoV-229E (B), HCoV-OC43 (C) or HCoV-NL63 (D) at an MOI of 0.1 TCID50/cell were treated with different concentrations of NTZ or vehicle immediately after the adsorption period. In the case of HCoV-NL63, NTZ was removed at 48 h after infection. Virus yield in cell supernatants was determined at 48 (B), 96 (C) or 120 (D) hours p.i. by infectivity assay (B,C) or RNA quantification by qRT-PCR (D). Data, expressed as TCID50/ml (B,C) or percent of untreated control (D), represent the mean ± S.D. of duplicate samples. (E) MRC-5 cells infected with HCoV-229E or HCoV-OC43 and LLC-MK2 cells infected with HCoV-NL63 (0.1 TCID50/cell) were treated with 3 μM nitazoxanide, the NTZ active metabolite tizoxanide, remdesivir, chloroquine and ribavirin after virus adsorption. In the case of HCoV-NL63, NTZ and tizoxanide were removed at 48 h after infection. Virus yields were determined at 48 (HCoV-229E), 72 (HCoV-OC43) or 120 (HCoV-NL63) hours p.i. by infectivity assay. Data, expressed as TCID50/ml, represent the mean ± S.D. of duplicate samples. *p < 0.05; ANOVA test.
Figure 2
Figure 2
Effect of short treatment with nitazoxanide in OC43 and 229E HCoV-infected human lung cells. (A,B) MRC-5 cell monolayers mock-infected or infected with HCoV-229E and HCoV-OC43 (0.5 TCID50/cell) were treated with different concentrations of NTZ or vehicle immediately after the adsorption period. Virus yield (Ο, red line) was determined at 24 h p.i. by infectivity assay. In parallel, cell viability (△, black line) was determined by MTT assay in mock-infected cells (A). Absorbance (O.D.) of converted dye was measured at λ = 570 nm. IC50 and LD50 in μg/ml (B) were calculated using Prism 5.0 software. Data represent the mean ± S.D. of duplicate samples. Selectivity indexes (SI) are indicated. *p < 0.05; ANOVA test.
Figure 3
Figure 3
Nitazoxanide acts at postentry level. (A,B) MRC-5 cells mock-infected or infected with HCoV-OC43 (A) or HCoV-229E (B) (0.5 TCID50/cell) were treated with NTZ 1 μg/mL [(A,B) top; filled bars], NTZ 2.5 μg/mL [(A,B) bottom; filled bars] or vehicle (empty bars) 2 h before infection (Pre), after the adsorption period (Post), or 2 h before infection and treatment was continued during and after the adsorption period (Pre + Post). Virus yield was determined at 24 h p.i. by TCID50 infectivity assay. (C) MRC-5 cells mock-infected or infected with HCoV-OC43 or HCoV-229E were treated with NTZ (1 μg/mL) or vehicle only during the adsorption period (Ads). Virus yield was determined at 24 h p.i. by infectivity assay. (A–C) Data represent the mean ± S.D. of duplicate samples. *p < 0.01; Student’s t-test. (D) Schematic representation of HCoV genomic RNA transfection assay. (E) MRC-5 cells were transfected with HCoV-OC43 or HCoV-229E genomic RNA for 4 h and treated with NTZ (2.5 μg/mL) or vehicle for 24 h. Virus yield was determined at 24 h after treatment in the supernatant of transfected cells by TCID50 infectivity assay. Nucleocapsid (N) protein levels in HCoV-OC43 or HCoV-229E RNA-transfected cells are shown (insets). (F) MRC-5 cells infected with HCoV-OC43 were treated with NTZ (1 μg/mL) or vehicle at the indicated times after infection. Virus yield was determined at 24 h p.i. by infectivity assay. (E,F) Data represent the mean ± S.D. of duplicate samples. *p < 0.01; Student’s t-test.
Figure 4
Figure 4
Effect of Nitazoxanide on HCoV-OC43 spike expression. (A,B) MRC-5 cells were mock-infected or infected with HCoV-OC43 at an MOI of 0.5 TCID50/cell and treated with different concentrations of NTZ or vehicle immediately after the virus adsorption period. After 24 h, whole cell extracts (WCE) were analyzed for levels of viral S and N proteins by IB using anti-spike and anti-N HCoV-OC43 antibodies [(A) top], and quantitated by scanning densitometry. Relative amounts of S and N proteins were determined after normalizing to β-actin [(A) bottom]. The faster-migrating form of the S protein in NTZ-treated cells is indicated by red arrowheads. In the same experiment virus yield was determined at 24 h p.i. in the supernatant of infected cells by TCID50 infectivity assay (B). Data represent the mean ± S.D. of duplicate samples. *p < 0.01; ANOVA test. (C) Confocal images of HCoV-OC43 spike glycoprotein (red) in MRC-5 cells mock-infected or infected with HCoV-OC43 at an MOI of 0.5 TCID50/cell and treated with NTZ (1 μg/mL) or vehicle for 24 h. Nuclei are stained with Hoechst (blue). Merge images are shown. Scale bar, 20 μm.
Figure 5
Figure 5
Nitazoxanide impairs HCoV-OC43 and HCoV-229E spike maturation at an Endo-H sensitive stage. (A–D) MRC-5 cells were mock-infected or infected with HCoV-OC43 (A,C) or HCoV-229E (B,D) at an MOI of 0.5 TCID50/cell and treated with NTZ (2.5 μg/mL) or vehicle immediately after the virus adsorption period. After 24 h, WCE were analyzed for levels of S protein by IB using anti-S HCoV-OC43 (A,C) or anti-S HCoV-229E (B,D) antibodies. Red arrowheads indicate the faster-migrating forms of the HCoV-OC43 (A) and HCoV-229E (B) spike proteins in NTZ-treated cells. Spike proteins densitometric and molecular weight analysis (C,D) are shown. (E) Diagram of substrate specificity of endoglycosidase-H (Endo-H). Mannose (green circles), N-acetylglucosamine (GlcNAc, blue squares) and asparagine (Asn, red triangle) residues are shown. Scissors represent the cleavage site of Endo-H. (F,G) MRC-5 cells were mock-infected or infected with HCoV-OC43 (F) or HCoV-229E (G) at an MOI of 0.5 TCID50/cell and treated with NTZ (1 μg/mL) or vehicle immediately after the virus adsorption period. After 24 h proteins were digested with Endo-H (+) or left untreated (−) and processed for IB analysis using anti-S HCoV-OC43 (F) or anti-S HCoV-229E (G) antibodies, and quantitated by scanning densitometry. The Endo-H-cleaved faster-migrating S forms are indicated by white arrowheads. The percentage of Endo-H-resistant (Endo-H R) S protein in the different samples is indicated.
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