Erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin inhibit cytopathic effect, papain-like protease, and MPRO enzymes of SARS-CoV-2

Abstract

Background: Although tremendous success has been achieved in the development and deployment of effective COVID-19 vaccines, developing effective therapeutics for the treatment of those who do come down with the disease has been with limited success. To repurpose existing drugs for COVID-19, we previously showed, qualitatively, that erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin inhibit SARS-COV-2-induced cytopathic effect (CPE) in Vero cells.

Aim: This study aimed to quantitatively explore the inhibition of SARS-CoV-2-induced CPE by erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin and to determine the effect of these drugs on SARS-CoV-2 papain-like protease and 3CL protease (M^PRO) enzymes.

Methods: Neutral red (3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride) cell viability assay was used to quantify CPE after infecting pre-treated Vero cells with clinical SARS-Cov-2 isolates. Furthermore, SensoLyte^® 520 SARS-CoV-2 papain-like protease and SensoLyte^® 520 SARS-CoV-2 M^PRO activity assay kits were used to evaluate the inhibitory activity of the drugs on the respective enzymes.

Results: Erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin dose-dependently inhibit SARS-CoV-2-induced CPE in Vero cells, with inhibitory concentration-50 (IC₅₀) values of 3.27 µM, 4.23 µM, 9.29 µM, 3.19 µM, and 84.31 µM, respectively. Furthermore, erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin dose-dependently inhibited SARS-CoV-2 papain-like protease with IC₅₀ values of 0.94 µM, 0.88 µM, 1.14 µM, 1.07 µM, and 1.51 µM, respectively, and inhibited the main protease (M^PRO) with IC₅₀ values of 1.35 µM, 1.25 µM, 7.36 µM, 1.15 µM, and 2.44 µM, respectively.

Conclusion: The IC₅₀ for all the drugs, except ivermectin, was at the clinically achievable plasma concentration in humans, which supports a possible role for the drugs in the management of COVID-19. The lack of inhibition of CPE by ivermectin at clinical concentrations could be part of the explanation for its lack of effectiveness in clinical trials.

Keywords: COVID-19; SARS-CoV-2; erythromycin; folic acid; ivermectin; pyridoxine; retapamulin.

Figure 1

Inhibition of SARS-CoV-2-induced CPE. For erythromycin, retapamulin, folic acid, and ivermectin, high dose, medium dose, and low dose respectively represent 10 µM, 7.5 µM, and 5 µM, but for pyridoxine, the doses were 20 µM, 15 µM, and 10 µM. The doses were selected based on achievable C_max in human subjects at standard doses of the drugs. All concentrations of test drugs were found nontoxic to Vero cells. Analyses were performed with outliers because biological factors probably dictated differences (Mowbray et al., 2019).

Figure 2

Inhibition of SARS-CoV-2 papain-like protease. For erythromycin, retapamulin, folic acid, and ivermectin, high dose, medium dose, and low dose respectively represent 10 µM, 5 µM, and 2.5 µM, but for pyridoxine, the doses were 20 µM, 10 µM, and 5 µM. The doses were selected based on achievable C_max in human subjects at standard doses of the drugs and to input the dose doubling escalation method. All concentrations of test drugs were found nontoxic to Vero cells. Analyses were performed with outliers substituted by averaging or neighborhood method because precision rather than biological factors probably dictated differences (Mowbray et al., 2019).

Figure 3

Inhibition of SARS-CoV-2 main protease (MPRO or 3CL protease). For erythromycin, retapamulin, folic acid, and ivermectin, high dose, medium dose, and low dose respectively represent 10 µM, 5 µM, and 2.5 µM, but for pyridoxine, the doses were 20 µM, 10 µM, and 5 µM. The doses were selected based on achievable C_max in human subjects at standard doses of the drugs and to input the dose doubling escalation method. All concentrations of test drugs were found nontoxic to Vero cells. Analyses were performed with outliers substituted by averaging or neighborhood method because precisions rather than biological factors probably dictated differences (Mowbray et al., 2019).