DataSheet_1_Erythromycin, retapamulin, pyridoxine, folic acid, and ivermectin inhibit cytopathic effect, papain-like protease, and MPRO enzymes of SARS-CoV-2.pdf

BackgroundAlthough 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.AimThis 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 (MPRO) enzymes.MethodsNeutral 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 MPRO activity assay kits were used to evaluate the inhibitory activity of the drugs on the respective enzymes.ResultsErythromycin, 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 (MPRO) with IC50 values of 1.35 µM, 1.25 µM, 7.36 µM, 1.15 µM, and 2.44 µM, respectively.ConclusionThe 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.</p

DNA fragmentation of MDA-MB 231 breast cancer cells analysed in 1% agarose gel after 24 hours incubation with different concentration of Artonin E.

<p>Lanes 1–3 represents 3, 10 and 30 μM of Artonin E, lane 4 is untreated cancer cells, lane 5 is positive control well treated with 4μg/mL camptothecin and lane 6 is a 1kilobase DNA ladder.</p

Acridine orange and propidium iodide double staining of MDA-MB 231 breast cancer cells after 24 hour exposure.

<p>(A) Control, (B) 3 μM Artonin E. (C) 10 μM Artonin E, (D) 30 μM Artonin E and (E) Quantification of apoptotic morphology at 24 and 48 hours. Each result is presented as mean ± SD of three replicates. * indicates significant difference from the control of each phase (p<0.05). VC = Viable cells; BL = Cell membrane blebbing; CC = chromatin condensation; EA = Early apoptosis; LA = late apoptosis; MN = marginated nuclear chromatin; SN = secondary necrosis. Magnification X200.</p

Western blot analysis of Artonin E (3, 10, and 30 μM) treated MDA-MB231 breast cancer cells after 24 hours.

<p>(A) and the levels of protein expression quantified from the western blotting analysis of Artonin E treated MDA-MB 231 cells using Bio-rad Image Lab software (B).</p

The accession number and sequence of the primers used in the gene expression studies.

<p>The accession number and sequence of the primers used in the gene expression studies.</p

MDA-MB 231 breast cancer cell cycle regulation after 12 hours treatment with (B) 3 μM, (C) 10 μM, and (D) 30 μM Artonin E.

<p>(A) is untreated control. (E) Analysis of cell population in the cycle phases. Values are mean ± standard deviation of three replicates. *Means in each cell cycle phase significantly (p<0.05) different from the control.</p

Mechanism of action of Artonin E in MDA-MB 231 breast cancer cells.

<p>Mechanism of action of Artonin E in MDA-MB 231 breast cancer cells.</p

The percentage of MDA-MB-231 cell line viability after treatment with Artonin E.

<p>The experiment was done in triplicate at 24, 48, and 72 hours and each point are presented as mean ± SD.</p

Apoptosis pathway- and cell cycle-related genes and protein expression in Artonin E treated MDA-MB 231 cells.

<p>Apoptosis pathway- and cell cycle-related genes and protein expression in Artonin E treated MDA-MB 231 cells.</p