Discovery of M Protease Inhibitors Encoded by SARS-CoV-2

Abstract

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (M^pro, also called 3C-like protease [3CL^pro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting M^pro in the picornavirus-like supercluster, is a potent inhibitor for the M^pro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC₅₀) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC₅₀) of 0.91 ± 0.03 μM. Only a small portion of SARS-CoV-2 M^pro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 M^pro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

Keywords: COVID-19; GC376; M protease; Mpro; SARS-CoV-2; antiviral research.

FIG 1

Structure of GC376 and the IC₅₀ and the inhibitory constant (K_i) of recombinant M^pro of SARS-CoV-2. (A) GC376 is a peptidomimetic antiviral drug. The IC₅₀ (B) and the K_i (C) of M^pro of SARS-CoV-2 are shown. The proteolytic activity of M^pro was determined by the FRET protease assay, as described in the text. RFU, relative fluorescence units.

FIG 2

GC376 inhibited SARS-CoV-2 virus replication in Vero E6 cells. (A) Inhibition of SARS-CoV-2-induced CPE by GC376. In a 96-well plate, Vero E6 cells were infected with SARS-CoV-2 virus (100 TCID₅₀ per well), and cells were treated with various concentrations of GC376. At 120 h postinfection (hpi), cells were examined with a microscope (magnification, ×100). The cell control (CC) column refers to cells without compound treatment and virus infection. Vero E6 cells were all lysed at 120 h after SARS-CoV-2 infection as shown in the virus control (VC) column. GC376 was added to SARS-CoV-2-infected cells in twofold serial dilutions starting from a concentration of 20 μM in the left-hand column. The drug control (DC) column refers to cells in the absence or presence of GC376 without virus infection. Cells were fixed with formaldehyde and stained with 0.1% crystal violet. Results from one representative plate of two are shown. (B) Effects of GC376 on SARS-CoV-2-induced CPE or cell proliferation were generated using a sigmoidal dose-response curve model (GraphPad Prism 6 software) from which the IC₅₀ values were derived. The effect of GC376 on cell proliferation was determined by MTT assay.

FIG 3

Docked conformations of GC376 in SARS-CoV-2 and FIPV M^pro proteases. (A) GC376 docking to the SARS-CoV-2 M^pro protein X-ray structure (NCBI Protein Data Bank accession no. 6LU7). (B) GC376 docking to the FLIP 3C-like protease protein X-ray structure (accession no. 4ZRO). (C) Structure alignment of FIPV (accession no. 4ZRO, purple) and SARS-CoV-2 (accession no. 6LU7, white). The docking result of GC376 in FIPV displays as cyan, and the docking result of GC376 displays as orange. The red box shows the different residues in the FIPV and SARS-CoV-2 binding sites.