Potential anti-COVID-19 agents, cepharanthine and nelfinavir, and their usage for combination treatment

Abstract

Antiviral treatments targeting the coronavirus disease 2019 are urgently required. We screened a panel of already approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new agents having higher antiviral potentials than the drug candidates such as remdesivir and chroloquine in VeroE6/TMPRSS2 cells: the anti-inflammatory drug cepharanthine and human immunodeficiency virus protease inhibitor nelfinavir. Cepharanthine inhibited SARS-CoV-2 entry through the blocking of viral binding to target cells, while nelfinavir suppressed viral replication partly by protease inhibition. Consistent with their different modes of action, synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation was highlighted. Mathematical modeling in vitro antiviral activity coupled with the calculated total drug concentrations in the lung predicts that nelfinavir will shorten the period until viral clearance by 4.9 days and the combining cepharanthine/nelfinavir enhanced their predicted efficacy. These results warrant further evaluation of the potential anti-SARS-CoV-2 activity of cepharanthine and nelfinavir.

Keywords: Medical Substance; Pharmaceutical Preparation; Virology.

Graphical abstract

Figure 1

Cepharanthine (CEP) and nelfinavir (NFV) inhibit SARS-CoV-2 infection (A) Schematic of the SARS-CoV-2 infection assay. VeroE6/TMPRSS2 cells were inoculated with SARS-CoV-2 in the presence of compounds. After washing out unbound virus, the cells were incubated with compounds for 24-48 hr. Cells were harvested for immunofluorescence (IFA) or immunoblot analyses of viral N protein at 24 hr, and cytopathic effects (CPEs) were observed at 48 hr after infection. Solid and dashed boxes indicate the periods with and without treatment, respectively. (B–E) (B) Virus-induced CPE following drug treatment was recorded at 48 hr after infection. The quantified survival cell numbers (relative percentage to the control) are also shown at the bottom. Immunofluorescence (C) and immunoblot (D) detection of viral N protein expression in the infected cells at 24 hr after infection, and the red and blue signals show N and DAPI, respectively. (E) Immunofluorescent detection of viral N protein in human lung epithelial-derived cell line, Calu-3 cells. Dimethyl sulfoxide (DMSO), 0.4%; lopinavir (LPV), 16 μM; chloroquine (CLQ), 16 μM; favipiravir (FPV), 32 μM; remdesivir (RDV), 20 μM (C and D) and 10 μM (B and E); CEP, 8 μM; NFV, 4 μM (B-D) and 8 μM (E). These data were from three independent experiments.

Figure 2

Dose-response curves for the antiviral activity of CEP and NFV (A and B) Dose-response curves for compounds. In (A), secreted viral RNA at 24 hr after inoculation was quantified and plotted against drug concentration and chemical structures shown below each graph (for CEP, the structure of the major component is shown). In (B), viability of cells treated with the compounds was quantified by MTT assay. IC₅₀, IC₉₀, and CC₅₀ values were estimated by median effect model and are shown. These data were from three independent experiments.

Figure 3

Antiviral mechanism of action for CEP and NFV (A and B) Time-of-addition analysis to examine steps in SARS-CoV-2 life cycle. (A) shows the schematic of the time-of-addition analysis. Compounds were added at different times (a, whole; b, entry; or c, post-entry): (A) presentation during the 1h virus inoculation step and maintained throughout the 24 hr infection period (whole life cycle); (B) present during the 1 hr virus inoculation step and for an additional 2 hr and then removed (entry); or (C) added after the inoculation step and present for the remaining 22 hr of infection (post-entry). Solid and dashed boxes indicate the periods with and without treatment, respectively. In (B), the antiviral activities of each compound under the various protocols are estimated by quantifying the levels of secreted viral RNA at 24 hr after inoculation (B; mean ± SD). RDV, 15 μM; CLQ, 15 μM; CEP, 8.2 μM; NFV, 4 μM. These data were from three independent experiments. ∗∗P < 0.01; N.S., not significant (Student's t-test)

Figure 4

CEP inhibits SARS-CoV-2 cell binding (A) Predicted binding of CEP molecule to SARS-CoV-2 spike protein. Spike protein, CEP molecule, and protein binding site residues around CEP within 4 Å are shown in cartoon representation colored in orange, green stick, and surface representation, respectively. An overlapping view of the ACE2 with CEP is shown in semi-transparent cartoon representation colored in cyan. (B) Virus-cell binding assay. VeroE6/TMPRSS2 cells were incubated with virus (MOI = 0.001) in the presence of the indicated compounds for 30 min at 4°C to allow virus-cell binding. After extensive washing, cell-bound viral RNA was quantified, where the background depicts residual viral inocula in the absence of cells (B; mean ± SD). These data were from three independent experiments. ∗∗p < 0.01; N.S., not significant (Student's t-test)

Figure 5

NFV potentially targets SARS-CoV-2 main protease (A) Predicted binding of NFV to SARS-CoV-2 main protease. Representation of SARS-CoV-2 main protease (green), NFV molecule (cyan stick), and protease binding site residues around NFV within 4 Å (surface representation) is shown. (B) Dose-dependent inhibition curves for NFV on the catalytic activity of the SARS-CoV-2 main protease. The IC₅₀ is also shown. (B; mean ± SD)

Figure 6

Combination treatment with CEP and NFV (A) Dose-response curve of CEP/NFV co-treatment in the infection experiment (MOI = 0.001). Extracellular viral RNA levels at 24 hr after infection were quantified and plotted against concentrations of CEP (0.78, 1.25, 2.00, 3.20, and 5.12 μM: 1.6-fold serial dilution) and NFV (1.08, 1.30, 1.56, 1.87, and 2.24 μM: 1.2-fold serial dilution). (B) Cell viability upon co-treatment with compounds. (C) The three-dimensional interaction landscapes of CEP and NFV were evaluated based on the Bliss independence model. Red and blue colors on the contour plot indicate synergy and antagonism, respectively. These data were from three independent experiments (A, B; mean ± SD).

Figure 7

Mathematical prediction of the impact of CEP and NFV therapy on viral dynamics (A) The time-dependent antiviral effects of NFV (500 mg, TID, oral) and CEP [25 mg, intravenous drip or 10 mg, oral] predicted by pharmacokinetics/pharmacodynamics (PK/PD) model are shown, with enlarged views of the gray zones in upper panels. (B) Viral load dynamics in the presence or absence of NFV (oral), CEP (intravenous), CEP (oral), and NFV (oral)/CEP (intravenous) combined therapies predicted by pharmacokinetics/pharmacodynamics/viral dynamics (PK/PD/VD) models are shown. (C) The cumulative antiviral load [area under the curve in (B)] (upper) and the reduction time (days) for virus elimination (lower) with drug treatments [NFV (oral), CEP (intravenous), and the NFV (oral)/CEP (intravenous) combination] are shown.