Remdesivir and Cyclosporine Synergistically Inhibit the Human Coronaviruses OC43 and SARS-CoV-2

Abstract

Remdesivir, a prodrug targeting RNA-dependent-RNA-polymerase, and cyclosporine, a calcineurin inhibitor, individually exerted inhibitory activity against human coronavirus OC43 (HCoV-OC43) in HCT-8 and MRC-5 cells at EC₅₀ values of 96 ± 34 ∼ 85 ± 23 nM and 2,920 ± 364 ∼ 4,419 ± 490 nM, respectively. When combined, these two drugs synergistically inhibited HCoV-OC43 in both HCT-8 and MRC-5 cells assayed by immunofluorescence assay (IFA). Remdesivir and cyclosporine also separately reduced IL-6 production induced by HCoV-OC43 in human lung fibroblasts MRC-5 cells with EC₅₀ values of 224 ± 53 nM and 1,292 ± 352 nM, respectively; and synergistically reduced it when combined. Similar trends were observed for SARS-CoV-2, which were 1) separately inhibited by remdesivir and cyclosporine with respective EC₅₀ values of 3,962 ± 303 nM and 7,213 ± 143 nM by IFA, and 291 ± 91 nM and 6,767 ± 1,827 nM by a plaque-formation assay; and 2) synergistically inhibited by their combination, again by IFA and plaque-formation assay. Collectively, these results suggest that the combination of remdesivir and cyclosporine merits further study as a possible treatment for COVID-19 complexed with a cytokine storm.

Keywords: COVID-19; IL-6; IL-8; OC43; SARS-CoV-2; cyclosporine; remdesivir; synergistic.

FIGURE 1

Single or combined treatment of remdesivir and cyclosporine profoundly reduced HCoV-OC43 infection as assayed by immunofluorescence (IFAs) in human HCT-8 colorectal carcinoma cells. (A) Time course of HCoV-OC43 N protein expression in infected HCT-8 cells. Western analysis and IFA were performed against an antibody against HCoV-OC43 N protein (Mab9013) with the samples at the indicated time points. (B) Single treatments of remdesivir and cyclosporine reduced HCoV-OC43 infection in HCT-8 cells in a dose dependent manner. (C–E) Combined treatments of remdesivir and cyclosporine synergistically reduced the HCoV-OC43 infection in HCT-8 cells. IFAs were performed with an antibody against N protein (green) of HCoV-OC43 in HCoV-OC43 (0.05 MOI) infected HCT-8 cells at 30 h.p.i. treated with vehicle (0.5% DMSO) or compounds as indicated. Nuclei (blue) were counter stained with Hoechst dye and used to determine the relative cell viability by using the number of nuclei in vehicle control as 100% (Supplementary Figure S1). The fluorescent signal was normalized with cell viability to calculate the infection rate that no compound treatment was set at 100%. HCT-8 cells were seeded the day before compound treatment or HCoV-OC43 infection. The tested compounds were added to the wells 1 h prior to the addition of HCoV-OC43 at an MOI of 0.05 and the resulting cultures incubated for an additional 30 h at 37°C. AVE ±SD of three independent experiments are shown (A, B, D) The statistical significance was evaluated using one-way ANOVA followed by Tukey’s multiple comparison test. * and ** denote statistical significance of p < 0.05, and p < 0.01 respectively. IFA images shown are representative of three independent experiments (C). Shown inhibition % (AVE) and synergy scores (AVE ± SD) are from three independent experiments (E) analyzed via SynergyFinder (https://synergyfinder.fimm.fi/).

FIGURE 2

Single or combined treatments of remdesivir and cyclosporine profoundly reduced HCoV-OC43 infection in human fetal lung fibroblast MRC-5 cells assayed by IFA. (A) Single treatments of remdesivir and cyclosporine reduced HCoV-OC43 infection in MRC-5 cells in a dose dependent manner. (B–D) Combined treatments of remdesivir and cyclosporine synergistically reduced the HCoV-OC43 infection in MRC-5 cells. IFAs were performed with an antibody against N protein (green) of HCoV-OC43 in HCoV-OC43 (0.05 MOI) infected MRC-5 cells at 30 h.p.i. treated with vehicle (0.5% DMSO) or compounds as indicated. Nuclei (blue) were counter stained with Hoechst dye and used to determine the relative cell viability by using the number of nuclei in vehicle control as 100% (Supplementary Figure S2). The fluorescent signal of IFA was normalized with cell viability to calculate the infection rate that no compound treatment was set at 100%. MRC-5 cells were seeded the day before compound treatment or HCoV-OC43 infection. Tested compounds were added to the wells 1 h prior to the addition of HCoV-OC43 at an MOI of 0.05. The resulting cultures were then incubated for an additional 30 h at 37°C. AVE ± SD of three independent experiments are shown (A and C). The statistical significance was evaluated using one-way ANOVA followed by Tukey’s multiple comparison test. * and ** denote statistical significance of p < 0.05, and p < 0.01 respectively. IFA images shown are representative of three independent experiments (B). Shown inhibition % (AVE) and synergy scores (AVE ± SD) are from three independent experiments (D) analyzed via SynergyFinder (https://synergyfinder.fimm.fi/).

FIGURE 3

Reduction of IL-6 levels by single and combined treatments of remdesivir and cyclosporine in HCoV-OC43 infected human MRC-5 fetal lung fibroblast cells. (A) IL-6 production induced by HCoV-OC43 infection in MRC-5 cells over 3 days. (B) Dose dependent reduction of IL-6 levels by remdesivir and cyclosporine treatments in HCoV-OC43 infected MRC-5 cells at 30 h.p.i. Shown are the AVE ± SD from three independent experiments (A–C) The two-tailed unpaired Student’s t test was used evaluated the dose effect of single drug treatment on IL-6 production. (C and D) Synergistic reduction of IL-6 levels produced by HCoV-OC43 infected MRC-5 cells treatment with the combination of remdesivir and cyclosporine. MRC-5 cells were seeded the day before compound treatment or HCoV-OC43 infection. The tested compounds were added to the wells 1 h prior to the addition of HCoV-OC43 at an MOI of 0.05. The resulting cultures were then incubated for an additional 30 h (B–D), or as indicated (A), at 37°C. Subsequently, the culture supernatants were subjected to detection and quantitation of IL-6 by ELISA. AVE ± SD from three independent experiments (A–C) are shown; The statistical significance was evaluated using one-way ANOVA followed by Tukey’s multiple comparison test. * and ** denote statistical significance of p < 0.05, and p < 0.01 respectively (B and C). Shown inhibition % (AVE) and synergy scores (AVE ± SD) are from three independent experiments (D) analyzed via SynergyFinder (https://synergyfinder.fimm.fi/).

FIGURE 4

Single or combined treatments of remdesivir and cyclosporine profoundly reduced SARS-CoV-2 infection of Vero E6 cells assayed by IFA. (A) Single treatments of remdesivir and cyclosporine reduced SARS-CoV-2 infection of Vero E6 cells in a dose dependent manner. (B–D) Combined treatments of remdesivir and cyclosporine synergistically reduced SARS-CoV-2 infection in Vero E6 cells. IFAs were performed with antibody against SARS-CoV-2 N (green) and DAPI staining (blue) for the Vero E6 host live cells. Vero E6 cells were treated with each compound at the indicated concentrations for 1 h at 37°C. The cells were adsorbed with SARS-CoV-2 (TCDC#4) at MOI = 0.01 for 1 h at 37°C. After virus adsorption, the cells were washed with PBS and fresh medium with each compound added at the indicated concentrations and then incubated for 1 day. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. The cells were stained with anti-SARS-CoV-2 N protein antibody and anti-human IgG-Alexa Fluor 488 (green). Nuclei were counter stained with DAPI (blue) and used to determine the relative cell viability by using the number of nuclei in vehicle control as 100% (Supplementary Figure S3). N protein expression was measured using a high-content image analysis system (Molecular Devices). The fluorescent signal was normalized with cell viability to calculate the infection rate that no compound treatment was set at 100%. EC₅₀ and CC₅₀ values were calculated by Prism software. Shown are the AVE ± SD from three independent experiments (A and C) * and ** denote statistical significance of p < 0.05, and p < 0.01 respectively. Shown results of IFA are representative of three independent experiments (B). Shown inhibition % (AVE) and synergy scores (AVE ± SD) are from three independent experiments (D) analyzed via SynergyFinder (https://synergyfinder.fimm.fi/).

FIGURE 5

Single or combined treatment with remdesivir and cyclosporine synergistically reduced infectious SARS-CoV-2 viral loads as determined by plaque formation assays. (A) Single treatment of SARS-CoV-2-infected Vero E6 cells with remdesivir and cyclosporine reduced the number of plaque-forming units in a dose dependent manner. (B) Combined treatment of SARS-CoV-2-infected Vero E6 cells with remdesivir and cyclosporine significantly decreased plaque formation caused by SARS-CoV-2. (C and D) Combined treatment of SARS-CoV-2-infected Vero E6 cells with remdesivir and cyclosporine synergistically reduced SARS-CoV-2 viral loads. Plaque assays were performed in triplicate using 24-well tissue culture plates. Vero E6 cells were seeded in DMEM with 10% FBS and antibiotics 1 day before infection. SARS-CoV-2 was added to the cell monolayer for 1 h at 37°C. Subsequently, viruses were removed and the cell monolayer was washed once with PBS before covering with overlay media containing 1% methylcellulose (Sigma, cat#M0387) and the test compounds at indicated concentrations for 5–7 days. The cells were fixed with 10% formaldehyde solution (Marcon^TM Chemicals, cat #H121-08) overnight. After removal of overlay media, the cells were stained with crystal violet and the plaques were counted. The percentage of inhibition was calculated as [1–(VD/VC)] × 100%, where VD and VC refer to the virus titer in the presence and absence of the test compound, respectively. The inhibition in plaque formation results shown are representative of three independent experiments, each in triplicate (B). Cell viability was determined as described (Kuo et al., 2021) by measurement of relative alkaline phosphatase activity; viability in the absence of compound treatment was set as 100%. AVE ± SD of three independent experiments were shown (A and C); The statistical significance was evaluated using one-way ANOVA followed by Tukey’s multiple comparison test. * and ** were used to denote the statistical significance for p < 0.05, and p < 0.01 respectively (C). Shown inhibition % (AVE) and synergy scores (AVE ± SD) are from three independent experiments (D) analyzed via SynergyFinder (https://synergyfinder.fimm.fi/).