In vitro activity of cysteamine against SARS-CoV-2 variants

Abstract

Global COVID-19 pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Continuous emergence of new variants and their rapid spread are jeopardizing vaccine countermeasures to a significant extent. While currently available vaccines are effective at preventing illness associated with SARS-CoV-2 infection, these have been shown to be less effective at preventing breakthrough infection and transmission from a vaccinated individual to others. Here we demonstrate broad antiviral activity of cysteamine HCl in vitro against major emergent infectious variants of SARS-CoV-2 in a highly permissible Vero cell line. Cysteamine HCl inhibited infection of wild type, alpha, beta, gamma, delta, lambda, and omicron variants effectively. Cysteamine is a very well-tolerated US FDA-approved drug used chronically as a topical ophthalmic solution to treat ocular cystinosis in patients who receive it hourly or QID lifelong at concentrations 6 times higher than that required to inhibit SARS CoV-2 in tissue culture. Application of cysteamine as a topical nasal treatment can potentially1) mitigate existing infection 2) prevent infection in exposed individuals, and 3) limit the contagion in vulnerable populations.

Keywords: Cysteamine; Disulfides; Nasal spray; Orphan drug; SARS-CoV-2; Viral inhibition.

Fig. 1

Cytotoxicity of cysteamine HCl in Vero-TMPRSS2 cells. Cysteamine HCl was preincubated with Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% FBS, Lglutamine, 1% penicillin/streptomycin and puromycin (10 μg/ml) for 2 h at 37 °C in 5% CO2 in 100 μl medium and then transferred to the wells containing Vero-TMPRSS2 cells. After 1 h of incubation medium from each well was removed and 100 μl of complete DMEM medium containing either no cysteamine-HCl (A) or 20% of the original concentration of cysteamine-HCl was added to mimic the condition of the infection assay method (B). Cells were cultured for 72 h at 37 °C in 5% CO2 and cytotoxicity measured as described in the Method section.

Fig. 2

Cysteamine Stability in Tissue Culture: The free thiol content of four concentrations of cysteamine HCl were measured at the initiation of pre-incubation with virus (0 h), after pre-incubation with virus (2 h), during incubation of cysteamine HCl/virus mixture with Vero-TMPRSS2 cells 3, 24 and 72 h post infection as described in the Method Section.

Fig. 3

Dose dependent inhibition of infection of wild type and SARS-CoV-2 variants by cysteamine HCl: (A) Wild type (25 pfu/well), alpha (125 pfu/well); (B) beta (18.75 pfu/well), gamma (25 pfu/well); lambda (60 pfu/well); (C) delta subvariant 1 (37.5 pfu/well), subvariant 2 (48pfu/ml), subvariant 3 (42 pfu/ml); (D) omicron B1.1, subvariant 1 (16.8pfu/well) and subvariant 2 (24pfu/well). Virus was preincubated with varying concentrations of cysteamine HCl at 37 °C for 2 h in a total volume of 600 μl of complete DMEM medium as shown in the figure. Cysteamine HCl/virus mixture was then transferred to each well of Vero-TMPRSS2 cells in a total volume of 250 μl and incubated for 1 h at 37 °C in 5% CO2. Each well was then overlaid with 1 ml of culture medium containing 0.5% methylcellulose and incubated for 3 days at 37 °C in 5% CO2 and plaques were developed and scored as described in the Method section. Mean percent inhibition +/− standard error of infection compared to the untreated control is plotted.

Fig. 4

Kinetics of Inhibition of delta variant infection by cysteamine HCl. Delta variant (Subvariant 1) (37.5 pfu/ml) was preincubated with cysteamine HCl (5 or 10 mM) for 0, 15, 30, 60 and 90 min in a total volume of 600 μl of complete DMEM medium. The cysteamine HCl/virus mixture was then transferred to wells of Vero-TMPRSS2 cells in a total volume of 250 μl and incubated for 1 h at 37 °C in 5% CO2. Wells were then overlaid with 1 ml of culture medium containing 0.5% methylcellulose and plaques were developed as described in the Method section. In both assays plaques were developed and scored as described in the Method section. Mean percent inhibition of infection +/− standard error compared to the untreated control along with the total time of association of virus and cysteamine before addition of 0.5% methyl cellulose overlay was plotted.

Fig. 5

Binding of S1 protein to Vero-TMPRSS2 cells in the presence of cysteamine. (A) Binding of varying concentrations of S1 protein to the Vero-TMPRSS2 cells. The cells were incubated for 60 min at 37 °C with concentrations of S1 protein (Sino Biologicals) from 100 to 1000 ng/ml, followed by anti-S1-Alexa Fluor-488 antibody staining as described in Methods. Percent binding is the % of anti-S1+ cells after gating on singlets and live cells. The concentrations of 250 and 100 ng/ml were selected to perform the subsequent experiments based on the non-saturating level of S1 binding (54.5–85.7%). All titration experiments were replicated twice with similar outcome. (B) Scatter plots and (C) corresponding column graphs showing the effect of cysteamine on the binding of S1 protein to the Vero-TMPRSS2 cells. S1 protein (100 or 250 ng/ml) was preincubated with varying concentrations (0.31 to 10 mM) of cysteamine for 60 min at 37 °C. For control S 1 protein was incubated with FACS binding buffer without cysteamine. Cysteamine-treated or medium treated S1 protein solution was then transferred to a tube containing Vero-TMPRSS2 cells and the binding assay was performed as described in the Methods using anti-S1-Alexa Fluor-488 antibody staining. Percent binding represents the % of anti-S1+ cells after gating on singlets and live cells. All experiments were repeated twice with comparable outcome. (D) Scatter plots evaluated the effect of cysteamine on binding of S1 (250 ng/ml) using above condition. The gating strategy selected for the analysis was done using specific unstained cells treated with medium (control) or with 5 and 10 mM cysteamine-HCL for 60 min for the respective group.

Fig. 6

Binding of virus to the Vero-TMPRSS2 in the presence of cysteamine-HCl. (A) Scatter plots obtained for the binding of S1 protein following infection with varying MOI of infection both in the absence or in the presence of 5 mM cysteamine HCl. Varying infectious units of SARS-CoV2 (Wild type SARS-CoV-2 (P4) isolate USA-WA1/2020) were pre-incubated with medium alone or with 5 mM cysteamine-HCl for 90 min at 37 °C in 5% CO2 and the mixture was incubated with Vero-TMPRSS2 cells for an additional 60 min at 37 °C to initiate viral infection. The level of bound S1 protein was then assayed by flow cytometry after staining with Alexa Four-488 conjugated anti-S1 antibody. Percent binding represents the % of anti-S1+ cells after gating on singlets and live cells. (B) Dose dependent inhibition of the virus binding to Vero-TMPRSS2 cells in the presence of cysteamine. Values were normalized with the background fluorescence obtained from stained uninfected cells under appropriate conditions.