Identification and evaluation of potent Middle East respiratory syndrome coronavirus (MERS-CoV) 3CLPro inhibitors

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness with fever, cough and shortness of breath. Up to date, it has resulted in 1826 human infections, including 649 deaths. Analogous to picornavirus 3C protease (3C^pro), 3C-like protease (3CL^pro) is critical for initiation of the MERS-CoV replication cycle and is thus regarded as a validated drug target. As presented here, our peptidomimetic inhibitors of enterovirus 3C^pro (6b, 6c and 6d) inhibited 3CL^pro of MERS-CoV and severe acute respiratory syndrome coronavirus (SARS-CoV) with IC₅₀ values ranging from 1.7 to 4.7 μM and from 0.2 to 0.7 μM, respectively. In MERS-CoV-infected cells, the inhibitors showed antiviral activity with EC₅₀ values ranging from 0.6 to 1.4 μM, by downregulating the viral protein production in cells as well as reducing secretion of infectious viral particles into culture supernatants. They also suppressed other α- and β-CoVs from human and feline origin. These compounds exhibited good selectivity index (over 70 against MERS-CoV) and could lead to the development of broad-spectrum antiviral drugs against emerging CoVs and picornaviruses.

Keywords: 3C-like protease; Coronavirus; MERS-CoV; Peptidomimetic inhibitor; Picornavirus; SARS-CoV.

Graphical abstract

Fig. 1

Antiviral activity of 6b, 6c and 6d against MERS-CoV in Huh-7 cells. Huh-7 cells in 96-well plates were mock-infected or infected with MERS-CoV at an MOI of 0.1 for 1 h at 37 °C. After washing with PBS, cells were treated with 3-fold serial dilutions of test compounds (6a, 6b, 6c and 6d) or a control compound (GEM). On day 2 p.i., cell lysates were harvested for measuring cell viability. The data represent the means ± standard deviations from three independent experiments.

Fig. 2

Inhibition of MERS-CoV NP production by 6b, 6c and 6d in a dose-dependent manner. Huh-7 cells in 6-well plates were infected with MERS-CoV at an MOI of 0.02 for 1 h at 37 °C.The virus-infected cells were treated with increasing concentrations (0.1, 1 and 10 μM) of each compound. Co-treatment of interferon-alpha 2A (IFN; 50 ng/ml) and ribavirin (RBV; 100 μM) was used as a positive control. On day 1 p.i., cells were harvested and loaded to 10% SDS-PAGE (30 μg per well). Immunoblotting was performed using rabbit anti-NP antibody and HRP-conjugated goat anti-rabbit secondary antibody. β-Actin was used as a loading control.

Fig. 3

Downregulation of MERS-CoV progeny generation by 3CL^proinhibitors, 6b, 6c and 6d. MERS-CoV-infected Huh-7 cells in 6-well plates were treated with 1 μM 6b, 6c and 6d for 1 day. Culture supernatants were harvested and serially diluted by 10-fold in DMEM (10⁻¹ to 10⁻³). Fresh Vero cells in 6-well plates were infected with the diluted cell culture inoculum for 1 h. And the number of infectious viral particles was counted by addition of the overlay medium for 3 days and by neutral red staining.

Fig. 4

Docking of inhibitor 6d with MERS-CoV 3CL^pro. Cys148 of the protease makes a covalent bond with the carbonyl carbon of the inhibitor aldehyde, forming a stable tetrahedral species (the inset), and the resulting oxyanion being stabilized by His 41. The protease is shown in a charge-potential surface. The putative substrate-binding subsites S1′, S1, S2, S3 and S4 are indicated. Moreover, the possible hydrogen bonds of 6d to the protease are further drawn with dashed lines.