Unlike Chloroquine, Mefloquine Inhibits SARS-CoV-2 Infection in Physiologically Relevant Cells

Abstract

Despite the development of specific therapies against severe acute respiratory coronavirus 2 (SARS-CoV-2), the continuous investigation of the mechanism of action of clinically approved drugs could provide new information on the druggable steps of virus-host interaction. For example, chloroquine (CQ)/hydroxychloroquine (HCQ) lacks in vitro activity against SARS-CoV-2 in TMPRSS2-expressing cells, such as human pneumocyte cell line Calu-3, and likewise, failed to show clinical benefit in the Solidarity and Recovery clinical trials. Another antimalarial drug, mefloquine, which is not a 4-aminoquinoline like CQ/HCQ, has emerged as a potential anti-SARS-CoV-2 antiviral in vitro and has also been previously repurposed for respiratory diseases. Here, we investigated the anti-SARS-CoV-2 mechanism of action of mefloquine in cells relevant for the physiopathology of COVID-19, such as Calu-3 cells (that recapitulate type II pneumocytes) and monocytes. Molecular pathways modulated by mefloquine were assessed by differential expression analysis, and confirmed by biological assays. A PBPK model was developed to assess mefloquine's optimal doses for achieving therapeutic concentrations. Mefloquine inhibited SARS-CoV-2 replication in Calu-3, with an EC₅₀ of 1.2 µM and EC₉₀ of 5.3 µM. It reduced SARS-CoV-2 RNA levels in monocytes and prevented virus-induced enhancement of IL-6 and TNF-α. Mefloquine reduced SARS-CoV-2 entry and synergized with Remdesivir. Mefloquine's pharmacological parameters are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points suggesting that mefloquine may accumulate in the lungs. Altogether, our data indicate that mefloquine's chemical structure could represent an orally available host-acting agent to inhibit virus entry.

Keywords: COVID-19; SARS-CoV-2; antimalarial drug; antiviral; mefloquine.

Figure 1

The antiviral effect of mefloquine against SARS-CoV-2. Calu-3 (A,B,E) or Vero E6 (C,D) were infected with SARS-CoV-2 D614G (A–D) or gamma strain € at MOI 0.01 (Vero) or 0.1 (Calu-3), for 1 h at 37 °C. Inoculum was removed and cells were incubated with fresh DMEM containing the indicated concentrations of mefloquine (MQ), chloroquine (CQ), or remdesivir (RDV). Virus titers were measured by PFU/mL in the culture supernatants at 24 h post infection (hpi) for Vero (C,D) or 48 hpi for Calu-3 (A,B,E) cells. Results are displayed as virus titers (A,C,E) or percentage of inhibition (B,D). The data represent the means ± SEM of three independent experiments. * p < 0.05.

Figure 2

Mefloquine reduces the viral load and viral-induced enhancement of IL-6 and TNF-α in SARS-CoV-2-infected monocytes. Primary human monocytes isolated from health donors were infected with SARS-CoV-2 at an MOI of 0.01 for 1 h at 37 °C. Inoculum was removed and cells were treated with the indicated concentrations of mefloquine (MQ), chloroquine (CQ), or remdesivir (RDV). Virus replication (A) and LDH (B), IL-6 (C,D), or TNF-α (E,F) levels were assessed in the culture supernatants at 24 h post-infection. The data represent the means ± SEM of monocytes from at least 5 healthy donors. Ns: not significant, * p < 0.05, ^# p < 0.05 and ** p < 0.01.

Figure 3

GSEA enrichment plots of the six endocytosis-related pathways upregulated by mefloquine. We ran GSEA on the mefloquine gene expression signature obtained from CMAP to identify KEGG pathways enriched in differentially expressed genes. The six endocytosis-related pathways with a significant enrichment score (FDR < 0.05) are shown here. Subplots contain an identical colored bar, representing the complete gene list ordered by changes in the expression levels induced by mefloquine, with red indicating the highest z-scores (upregulated genes), and blue the lowest ones (downregulated genes). Each subplot corresponds to a pathway and black vertical lines indicate the position of its genes in the ordered list. Note that for all six pathways, these lines tend to be located towards the top of list (upregulated genes). The GSEA enrichment score is computed iteratively across the ordered list, as shown in green. The final GSEA enrichment score of each pathway (reported on Table S1) is the highest deviation from zero. The bottom portion of the plot shows the z-score (y-axis) at each position of the list (x-axis).

Figure 4

Effects of mefloquine on SARS-CoV-2 endocytosis-mediated entry. (A) To initially evaluate mefloquine’s effect on virus entry, Calu-3 cells or SARS-CoV-2 virus particles were pre-incubated with 1 µM of mefloquine for 1 h at 37 °C and then infected at an MOI of 0.1. After 24 hpi, culture supernatants were harvested, and SARS-CoV-2 replication was measured using the plaque assay. Results are displayed as virus titers (PFU/mL). (B,C) Representative images (from 4 independent experiments) of ultrastructural analysis by transmission electron microscopy of Vero E6 cell infected with an MOI of 1 of SARS-CoV-2 (B) and treated with 5 µM of mefloquine for 4 h (C). Cell endosomes with spherical SARS-CoV-2 virus particles (white arrows). (D) SARS-CoV-2 virus particles were counted inside the endosomes of Vero E6 cells treated with mefloquine or not (nil). * p < 0.05 and *** p < 0.01.

Figure 5

Mefloquine enhances the anti-SARS-CoV-2 activity of remdesivir. Calu-3 cells were infected with SARS-CoV-2 at an MOI 0.1 for 1 h at 37 °C. Inoculum was removed and cells were treated with 1 or 2 µM of mefloquine combined with the indicated concentrations of remdesivir (RDV). Virus titers were measured by PFU/mL in the culture supernatants after 48 hpi. Results are displayed as the percentage of inhibition.

Figure 6

Mefloquine’s PBPK model predicted the plasma concentration for different dosages. Predicted mefloquine plasma concentration for 450 mg TID (A) or 350 mg QID (B) doses for 3 days. The dotted and the solid lines represent the EC₉₀ values of mefloquine for SARS-CoV-2 in Vero E6 and Calu-3 cell types, respectively.