Saracatinib Inhibits Middle East Respiratory Syndrome-Coronavirus Replication In Vitro

Abstract

The Middle East respiratory syndrome-coronavirus (MERS-CoV), first identified in Saudi Arabia, is an emerging zoonotic pathogen that causes severe acute respiratory illness in humans with a high fatality rate. Since its emergence, MERS-CoV continues to spread to countries outside of the Arabian Peninsula and gives rise to sporadic human infections following the entry of infected individuals to other countries, which can precipitate outbreaks similar to the one that occurred in South Korea in 2015. Current therapeutics against MERS-CoV infection have primarily been adapted from previous drugs used for the treatment of severe acute respiratory syndrome. In search of new potential drug candidates, we screened a library composed of 2334 clinically approved drugs and pharmacologically active compounds. The drug saracatinib, a potent inhibitor of Src-family of tyrosine kinases (SFK), was identified as an inhibitor of MERS-CoV replication in vitro. Our results suggest that saracatinib potently inhibits MERS-CoV at the early stages of the viral life cycle in Huh-7 cells, possibly through the suppression of SFK signaling pathways. Furthermore, saracatinib exhibited a synergistic effect with gemcitabine, an anticancer drug with antiviral activity against several RNA viruses. These data indicate that saracatinib alone or in combination with gemcitabine can provide a new therapeutic option for the treatment of MERS-CoV infection.

Keywords: MERS-CoV; Middle East Respiratory Syndrome; Src-family kinase inhibitor; gemcitabine; saracatinib.

Figure 1

Identification of saracatinib as an anti-MERS-CoV inhibitor from screening of bioactive compound libraries. (A) The percentage inhibition of MERS-CoV induced-CPE from each compound in the primary screening. Each dot represents the individual compound tested. The hit identification was based on the calculation of average % inhibition ± 3 standard deviation (mean ± 3SD) cut-off, which in this study was 47% inhibition of the virus induced-CPE. (B) The structure of saracatinib. (C,D) Dose-response curve (DRC) analyses of the inhibition of MERS-CoV by saracatinib. Huh-7 cells were (C) mock-infected (grey circle) or infected with MERS-CoV (black square); (D) rMERS-CoV (black square) and rMERS-CoV-S2 (open circle) in the presence of various concentrations of saracatinib. At 72 h p.i., cell viability was measured using MTS-based CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay. The data represent means (±SD) of at least two independent experiments performed in duplicate.

Figure 2

In vitro antiviral activity of saracatinib against MERS-CoV. Antiviral efficacy of saracatinib against MERS-CoV in Huh-7 cells. MERS-CoV infected Huh-7 cells were treated with saracatinib at indicated concentrations for 24 h, after which culture supernatant and cell lysates were collected. (A) Amount of infectious viral particles released to culture supernatants was determined by plaque assay. (B) MERS-CoV nucleocapsid (N) protein levels in lysates of infected cells were determined by Western blot analysis. Immunoblot detection of β-actin is shown as a loading control. (C,D) Quantification of intracellular MERS-CoV RNAs by RT-qPCR assay. Total RNA was isolated from lysates of infected cells for quantification of intracellular MERS-CoV RNA levels (ORF1a and upE) and results were normalized to GAPDH mRNA. Data represent means (±SD) of at least two independent experiments performed in duplicate. Significant differences are indicated by * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Saracatinib inhibits the early stages of MERS-CoV life cycle. (A) Schematic representation of time-of-addition/removal experiment. (B) Huh-7 cells were treated with 10 µM saracatinib for 1 h prior to virus infection (pre) or 0.5% DMSO (virus control, VC), for 1 h during infection (co), at 1 h post-infection (post 1–24), and at 4 h post-infection (post 4–24). After 24 h, the amount of infectious viral particles released to culture supernatants was determined by plaque assay. All values represent means ± SD of two independent experiments performed in duplicate. Significant differences are indicated by * p < 0.05. (C) Schematic representation of time-of-addition experiment. Huh-7 cells were inoculated with MERS-CoV at 4 °C to allow attachment/binding. After 1 h incubation, plates were shifted to 37 °C to allow synchronous entry and infection. Saracatinib (10 µM) was treated during the 4 °C incubation only or added at indicated time points during the 37 °C incubation and remained present until sample collection. After 24 h p.i.; (D) cell culture supernatants were collected for virus titration using plaque assay. (E,F) Total RNA isolated from infected lysates was used for analyses of intracellular MERS-CoV genomic RNA (ORF1a) and mRNA (upE) by qRT-qPCR. Data represent the means ± SD of two independent experiments performed in duplicate. Significant differences are indicated by * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

MERS-CoV is sensitive to RNA-mediated depletion of Fyn and Lyn kinases. (A) Specific knockdown of Fyn, Lyn, Src, and Yes kinases. Huh-7 cells were transfected with 100 nM nontargeting (NT) siRNA or siRNAs targeting Fyn, Lyn, Src, and Yes mRNA. At 48 h post-transfection, the cells were infected with MERS-CoV at an MOI of 0.02. After 12 h of infection, lysates of infected cells were collected and subjected to Western blot analyses. The β-actin was used as the loading control. The result of a representative experiment out of two repeats is shown. (B) Specific knockdowns of Fyn and Lyn interfere with the production of infectious MERS-CoV from siRNA transfected cells. Data presented is from means ± SD of 3 independent experiments. Significant differences are indicated by * p < 0.05, ** p < 0.01.

Figure 5

In vitro antiviral activity of gemcitabine against MERS-CoV. (A) The structure of gemcitabine. (B) Antiviral potency of gemcitabine against MERS-CoV. The antiviral EC₅₀ (black squares) and CC₅₀ (grey circles) of gemcitabine were determined by dose-response curve (DRC) analyses as described in Figure 2. (C,D) Antiviral efficacy of gemcitabine against MERS-CoV in Huh-7 cells. MERS-CoV infected Huh-7 cells were treated with gemcitabine at indicated concentrations for 24 h, after which the culture supernatant and cell lysates were collected. (C) Amount of infectious viral particles released to culture supernatants was determined by plaque assay. (D) MERS-CoV N protein levels in lysates of infected cells were determined by Western blot analysis. The immunoblot detection of β-actin is shown as a loading control. Data represent means (±SD) of at least two independent experiments performed in duplicate. Significant differences are indicated by * p < 0.05.

Figure 6

Cytotoxicity of gemcitabine and saracatinib, alone or in combination, in Huh-7 cells. Cells were treated with increasing concentrations of gemcitabine or saracatinib alone, or in combination for 48 h and cell viability was measured using the CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay. Data represent means (±SD) of at least two independent experiments performed in duplicate.