Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir

Abstract

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir not only inhibits the main protease (M^pro) and unexpectedly the RNA-dependent RNA polymerase (RdRp) but also modulates host immune responses. Our results thus reveal the possible anti-SARS-CoV-2 mechanism of simeprevir and highlight the translational potential of optimizing simeprevir as a therapeutic agent for managing COVID-19 and future outbreaks of CoV.

Figure 1

Repurposing FDA-approved drugs for SARS-CoV-2 through cellular screening. (A) Summary of methodology used in this paper. (B) Screening for FDA-approved small molecule therapeutics for activities in suppressing SARS-CoV-2 replication in Vero E6 cells. Dose–response curves in the suppression of SARS-CoV-2 replication in Vero E6 cells and cytotoxicity for simeprevir (C) and remdesivir (D) are shown. Data points in all plots represent mean ± S.E.M. For all data points, n = 3 replicates.

Figure 2

(A) Viral replication–suppression efficacies of different combinations of simeprevir and remdesivir concentrations. The numbers after S (simeprevir) and R (remdesivir) indicate the respective drug concentrations in μM. Data points in all plots represent mean ± S.E.M. For all data points, n = 3 replicates. (B) Bliss score analyses of synergism. (left) Diagram showing 12 combinations of simeprevir and remdesivir and their respective percentage inhibition (% inhibition, color-coded) of SARS-CoV-2 replication in Vero E6 cells compared to DMSO controls. (right) Excess over Bliss score (ΔBliss, color-coded) of different drug combinations. A positive and negative number indicates a likely synergistic and antagonistic effect, respectively, while a zero value indicates independence of action.

Figure 3

Simeprevir weakly inhibits M^pro and RdRp. Assay scheme and enzyme activity of main protease (M^pro), papain-like protease (PL^pro), and RNA-dependent RNA polymerase (RdRp) under various concentrations of simeprevir. (A) For M^pro, a CFP-YFP conjugate with a M^pro cleavage site linker is utilized, where relative activity is determined by the residual FRET efficiency after cleavage. (B) For PL^pro, rhodamine-conjugated ISG15 is used as a substrate for the enzyme, whose relative activity is determined by release of the fluorophore. (C) For RdRp, an extension assay based on the dsRNA-binding property of the intercalating agent Picogreen was established. Data points in all plots represent mean ± S.E.M. For all data points, n = 3 replicates.

Figure 4

RNA-seq analysis and validation of simeprevir-mediated host response and antiviral activity. (A) Schematic representation of RNA-seq sample preparation. Treatment sequence and incubation time of simeprevir and SARS-CoV-2 was indicated with arrows and legends. (B) Venn diagrams showing differentially expressed genes (DEGs) comparing simeprevir-treated (1.1 or 3.3 μM), infected, and mock-infected samples. (C) Bubble plot of top 20 hits of positively enriched reactome gene sets under simeprevir treatment using gene set analysis (GSEA). Enriched gene sets were filtered with criteria false discovery rate (FDR) q-value < 0.25 and nominal p-value < 0.05 before ranked with their normalized enrichment scores (NES). (D) Enrichment plots of GSEA results using gene ontology (GO) gene sets. (E) Clustered heatmap showing the row-normalized expression level of genes belonging to GO term “response to type I interferon”. (F) Viral titration assay using A549-ACE2 cells treated with 4 μM simeprevir and/or 1 μM JAK inhibitor I. (G) Relative RNA level of ISG15 in uninfected or infected A549-ACE2 cells, treated with 4 μM simeprevir and/or 1 μM JAK inhibitor I. Data points in all plots represent mean ± S.D. For all data points, n = 3 replicates; * p-value < 0.05, *** p-value < 0.005.