Mitigation of the replication of SARS-CoV-2 by nitric oxide in vitro

Abstract

The ongoing SARS-CoV-2 pandemic is a global public health emergency posing a high burden on nations' health care systems and economies. Despite the great effort put in the development of vaccines and specific treatments, no prophylaxis or effective therapeutics are currently available. Nitric oxide (NO) is a broad-spectrum antimicrobial and a potent vasodilator that has proved to be effective in reducing SARS-CoV replication and hypoxia in patients with severe acute respiratory syndrome. Given the potential of NO as treatment for SARS-CoV-2 infection, we have evaluated the in vitro antiviral effect of NO on SARS-CoV-2 replication. The NO-donor S-nitroso-N-acetylpenicillamine (SNAP) had a dose dependent inhibitory effect on SARS-CoV-2 replication, while the non S-nitrosated NAP was not active, as expected. Although the viral replication was not completely abolished (at 200 μM and 400 μM), SNAP delayed or completely prevented the development of viral cytopathic effect in treated cells, and the observed protective effect correlated with the level of inhibition of the viral replication. The capacity of the NO released from SNAP to covalently bind and inhibit SARS-CoV-2 3CL recombinant protease in vitro was also tested. The observed reduction in SARS-CoV-2 protease activity was consistent with S-nitrosation of the enzyme active site cysteine.

Keywords: 3CL protease; COVID-19; FRET; Nitric oxide; SARS-CoV-2.

Graphical abstract

Fig. 1

SARS-CoV-2 replication kinetics in Vero-E6 cells in the presence and absence of NAP (A) and SNAP (B) as expressed by viral RNA copy numbers/μl (mean values from triplicates with standard deviation are indicated at each time point). In the presence of NAP no significant difference in viral replication between treated cells and not-treated controls was observed. A dose dependent inhibitory effect of SNAP on SARS-CoV-2 replication was observed. Data was analyzed by One-Way ANOVA of the calculated mean of the copy numbers for each replicate. Statistical significance is reported as **p < 0.001, ****p < 0.0001 (as compared with the controls).

Fig. 2

SNAP inhibitory effect plotted as percentage of the viral replication reduction over time. At a concentration of 400 μM of SNAP inhibited the viral replication after the treatment was terminated (36 hpi). For the two tested concentrations, mean values from triplicates with standard deviation are indicated at each time point. Average viral replication inhibition values (%) at 12, 24, 36, 48 and 72 hpi were compared by One-Way ANOVA. Statistical significance is reported as *p < 0.05, **p < 0.001, ****p < 0.0001 (as compared with the control).

Fig. 3

Comparison of the CPE development between cells treated with SNAP and untreated controls at 24 h intervals. The shown wells are representative for all replicates, the total magnification used to observe the cells was 100×.

Fig. 4

Effect of SNAP and NAP on the activity of recombinant SARS-CoV-2 protease. The protease activity (A) is shown as the amount of relative fluorescence units (RFU) per minute. SNAP and NAP effect was also quantified (B). SNAP clearly inhibited SARS-CoV-2 protease in a dose dependent manner. In contrast, NAP acts as a reducing agent increasing the protease activity. Observed effects of compounds on SARS-CoV-2 protease activity was evaluated by One-Way ANOVA of the calculated mean area under the curves, the difference in average inhibitory activity (column graph) of the compounds was evaluated by One-Way ANOVA. Statistical significance is reported as ****p < 0.0001, **p < 0.001 (as compared with the controls).