Abelson Kinase Inhibitors Are Potent Inhibitors of Severe Acute Respiratory Syndrome Coronavirus and Middle East Respiratory Syndrome Coronavirus Fusion

Abstract

The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) cause significant morbidity and morality. There is currently no approved therapeutic for highly pathogenic coronaviruses, even as MERS-CoV is spreading throughout the Middle East. We previously screened a library of FDA-approved drugs for inhibitors of coronavirus replication in which we identified Abelson (Abl) kinase inhibitors, including the anticancer drug imatinib, as inhibitors of both SARS-CoV and MERS-CoV in vitro Here we show that the anti-CoV activity of imatinib occurs at the early stages of infection, after internalization and endosomal trafficking, by inhibiting fusion of the virions at the endosomal membrane. We specifically identified the imatinib target, Abelson tyrosine-protein kinase 2 (Abl2), as required for efficient SARS-CoV and MERS-CoV replication in vitro These data demonstrate that specific approved drugs can be characterized in vitro for their anticoronavirus activity and used to identify host proteins required for coronavirus replication. This type of study is an important step in the repurposing of approved drugs for treatment of emerging coronaviruses.

Importance: Both SARS-CoV and MERS-CoV are zoonotic infections, with bats as the primary source. The 2003 SARS-CoV outbreak began in Guangdong Province in China and spread to humans via civet cats and raccoon dogs in the wet markets before spreading to 37 countries. The virus caused 8,096 confirmed cases of SARS and 774 deaths (a case fatality rate of ∼10%). The MERS-CoV outbreak began in Saudi Arabia and has spread to 27 countries. MERS-CoV is believed to have emerged from bats and passed into humans via camels. The ongoing outbreak of MERS-CoV has resulted in 1,791 cases of MERS and 640 deaths (a case fatality rate of 36%). The emergence of SARS-CoV and MERS-CoV provides evidence that coronaviruses are currently spreading from zoonotic sources and can be highly pathogenic, causing serious morbidity and mortality in humans. Treatment of SARS-CoV and MERS-CoV infection is limited to providing supportive therapy consistent with any serious lung disease, as no specific drugs have been approved as therapeutics. Highly pathogenic coronaviruses are rare and appear to emerge and disappear within just a few years. Currently, MERS-CoV is still spreading, as new infections continue to be reported. The outbreaks of SARS-CoV and MERS-CoV and the continuing diagnosis of new MERS cases highlight the need for finding therapeutics for these diseases and potential future coronavirus outbreaks. Screening FDA-approved drugs streamlines the pipeline for this process, as these drugs have already been tested for safety in humans.

FIG 1

Imatinib inhibits SARS-CoV and MERS-CoV replication. Cells were treated with imatinib for the first 4 h (squares) or at 5 h postinfection (triangles) in all cases. Imatinib significantly inhibits SARS-CoV in Vero E6 (A) and Calu-3 (B) cells and MERS-CoV in Vero E6 (C) and MRC5 (D) cells when added for the first 4 h of infection but has a significantly diminished effect when added at 5 h postinfection. Data are presented as the mean ± standard deviation from 3 independent experiments. *, P < 0.05; **, P < 0.01.

FIG 2

Imatinib inhibits SARS-CoV and MERS-CoV RNA production. Cells were treated with imatinib at the start of infection (squares) or at 4 h postinfection (triangles) with either SARS-CoV in Vero E6 (A) and Calu-3 (B) cells or MERS-CoV in Vero E6 (C) and MRC5 (D) cells. Imatinib does not significantly inhibit RNA production when added at 4 h postinfection. Data are presented as the mean ± standard deviation from 3 independent experiments. *, P < 0.05; **, P < 0.01.

FIG 3

Imatinib does not interfere with trafficking of MERS-CoV or SARS-CoV pseudovirions to early or late endosomes, respectively. BSC1 or Huh7 cells were infected with PS/SARS-S (A) or PS/MERS-S (B), respectively, with or without a 10 μM imatinib 1-h pretreatment. For imaging colocalization of PS/MERS-S with EEA1, cells were fixed at 1 h postinfection. For imaging colocalization of PS/SARS-S with LAMP1, cells were fixed at 2 h postinfection. Graphs represent percent colocalization of virions to endosomes for 100 cells. Error bars represent mean ± standard deviation. ns, not significant.

FIG 4

Imatinib does not interfere with trafficking of live MERS-CoV or SARS-CoV to early or late endosomes, respectively. Vero E6 cells were infected with live SARS-CoV (A) or MERS-CoV (B), with or without a 1-h 10 μM imatinib pretreatment. For imaging colocalization of SARS-CoV S with EEA1 or LAMP1, cells were fixed at 1 h and 2 h postinfection, respectively. For imaging colocalization of MERS-CoV S with EEA1, cells were fixed at 1 h postinfection. Graphs represent percent colocalization of virions to endosomes for 100 cells. Error bars represent mean ± standard deviation. ns, not statistically significant.

FIG 5

Imatinib inhibits SARS-CoV and MERS-CoV virion fusion with endosomal membranes. BSC1 or Huh7 cells were infected with PS/SARS-S or PS/MERS-S, respectively, with or without a 1-h 10 μM imatinib pretreatment. Cells were incubated for 3 h and then incubated in a solution containing CCF2. (A) Flow cytometry of pseudovirus entry from one representative experiment. (B) The graph shows quantification of pseudovirus entry from 3 independent experiments. Error bars represent mean ± standard deviation. ***, P < 0.001.

FIG 6

Specific knockdown of Abl2, and not Abl1, significantly inhibits MERS-CoV and SARS-CoV replication. (A and B) Specific knockdown of ABl1 (A) and Abl2 (B) was achieved in Vero E6 cells. Numbers under bands are the percentage of each knocked down protein relative to scrambled siRNA, normalized for tubulin levels. (C and D) Knockdown of Abl2, and not Abl1, specifically inhibits production of infectious SARS-CoV (C) and MERS-CoV (D) from transfected cells. Data in panels C and D are presented as mean ± standard deviation from 3 independent experiments. **, P < 0.01.