A Novel Frameshifting Inhibitor Having Antiviral Activity against Zoonotic Coronaviruses

Abstract

Recent outbreaks of zoonotic coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused tremendous casualties and great economic shock. Although some repurposed drugs have shown potential therapeutic efficacy in clinical trials, specific therapeutic agents targeting coronaviruses have not yet been developed. During coronavirus replication, a replicase gene cluster, including RNA-dependent RNA polymerase (RdRp), is alternatively translated via a process called -1 programmed ribosomal frameshift (-1 PRF) by an RNA pseudoknot structure encoded in viral RNAs. The coronavirus frameshifting has been identified previously as a target for antiviral therapy. In this study, the frameshifting efficiencies of MERS-CoV, SARS-CoV and SARS-CoV-2 were determined using an in vitro -1 PRF assay system. Our group has searched approximately 9689 small molecules to identify potential -1 PRF inhibitors. Herein, we found that a novel compound, 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline (KCB261770), inhibits the frameshifting of MERS-CoV and effectively suppresses viral propagation in MERS-CoV-infected cells. The inhibitory effects of 87 derivatives of furo[2,3-b]quinolines were also examined showing less prominent inhibitory effect when compared to compound KCB261770. We demonstrated that KCB261770 inhibits the frameshifting without suppressing cap-dependent translation. Furthermore, this compound was able to inhibit the frameshifting, to some extent, of SARS-CoV and SARS-CoV-2. Therefore, the novel compound 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline may serve as a promising drug candidate to interfere with pan-coronavirus frameshifting.

Keywords: MERS-CoV; SARS-CoV-2; coronavirus; frameshifting; inhibitor.

Figure 1

Frameshifting ratio of MERS-CoV, SARS-CoV-1, and SARS-CoV-2. (A) Schematic diagram of the genomic organization of MERS-CoV. (B) Alignment of the full sequences of −1 PRF signals of MERS-CoV, SARS-CoV, SARS-CoV-2, HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43. Slippery sequences (UUUAAAC) are indicated above the aligned sequences. (C) Schematic diagram of a reporter system. (D) Frameshifting efficiencies of MERS-CoV, SARS-CoV, and SARS-CoV-2. Positive control expressing Renilla and firefly luciferases fused without −1 PRF signal, was set to 100% and negative control only expressing Renilla luciferase to 0%. Pos, positive control; Neg, negative control. **** p < 0.0001 vs. positive control (one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test).

Figure 2

A novel compound efficiently inhibiting MERS-CoV frameshifting. (A) Screening of compounds inhibiting the MERS-CoV −1 PRF frameshifting. Chemical compounds with inhibitory effects over 70% are indicated with red dots. (B) Chemical structure of the selected compound, 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline (KCB261770). (C) The relative frameshifting ratio of MERS-CoV −1 PRF (solid line, left y-axis) and cell viability (dashed line, right y-axis) were tested with increasing concentrations of KCB261770. (D) Cell lysates harboring MERS-CoV −1 PRF plasmids were incubated with compound KCB261770 for 60 min. The firefly and Renilla luciferase activities relative to the initial time points were monitored. The luciferase activities of the samples, including the compound-treated samples, were compared to those of the luciferase activities of the DMSO-treated samples at 0 time, which was set to 100%. DMSO was used as a negative control. ns; not significant (two-way ANOVA with Sidak’s multiple comparisons test).

Figure 3

Inhibitory effects of furo[2,3-b]quinoline derivatives on MERS-CoV frameshifting. (A) Chemical structure of furo[2,3-b]quinoline derivatives. (B) The inhibition rate of frameshifting among a total of 88 derivatives, including compound KCB261770 indicated in red, were determined. (C) Inhibition rate of frameshifting was compared between the initial library (blue dots) and furo[2,3-b]quinoline derivatives (red rectangles). The thick bars in the center represent the median values. **** p < 0.0001 (unpaired t-test with Welch’s correction).

Figure 4

Frameshifting inhibition by compound KCB261770 without decreasing cap-dependent translation. (A–C) Relative firefly and Renilla luciferase activities were measured in the cells harboring MERS-CoV −1 PRF plasmids treated with increasing concentrations of 6-thioguanine (A), cycloheximide (CHX; B), and compound KCB261770 (C). Circles with solid lines indicate firefly luciferase activities and triangles with dashed lines Renilla luciferase activities. * p < 0.05, ns; not significant (two-way ANOVA with Sidak’s multiple comparisons test).

Figure 5

Comparison of the inhibitory effects of compound KCB261770 on −1 PRFs of SARS-CoV, SARS-CoV-2, and MERS-CoV. (A,B) Frameshifting ratio relative to the untreated control was measured with or without compound KCB261770 (5 μM) in the indicated −1 PRFs. Frameshifting ratio was measured in the control cells, which does not have a PRF signal (A) or in the cells harboring coronavirus −1 PRFs (B). Frameshifting ratio of the each indicated PRF without treatment was set to 100%. MERS; MERS-CoV PRF, SARS-1; SARS-CoV PRF, SARS-2; SARS-CoV-2 PRF. **** p < 0.001, ns; not significant vs. control (one-way ANOVA with Tukey’s multiple comparisons test).

Figure 6

Suppression of MERS-CoV propagation by compound KCB261770. (A) Huh7 cells infected with 0.02 MOI of MERS-CoV were treated with increasing concentrations of compound KCB261770. At 2 dpi, genome copy number of MERS-CoV RNA in the media were determined by qRT-PCR using primers targeting MERS-CoV N gene. ND; not detected. (B) Cell viability was measured at 2 dpi with or without treatment of compound KCB261770 (5 μM). * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. control (MERS-CoV-infected cells without treatment) (one-way ANOVA with Dunnett’s multiple comparisons test).