Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication

Abstract

The main protease, M^pro (or 3CL^pro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the M^pro from both SARS-CoV and SARS-CoV-2 with IC₅₀ values in the nanomolar range. Crystal structures of SARS-CoV-2 M^pro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.

Fig. 1. M^pro of SARS-CoV-2 and SARS-CoV are inhibited in vitro by GC373 and GC376.

a Schematic representation of inhibitor prodrug GC376, used to treat cats of FIP, and GC373, the actual protease inhibitor. b IC₅₀ values for GC373 and GC376 for SARS-CoV-2 M^pro and c SARS-CoV M^pro cleavage of Abz-SVTLQSG-Y(NO2)-R. N = 3, values are represented as mean ± SE.

Fig. 2. The crystal structure of SARS-CoV-2 M^pro in complex with GC373 (converted from GC376).

a Apo-SARS-CoV-2 M^pro forms a dimer (6WTM.pdb). b Prodrug GC376, when incubated with SARS-CoV-2 M^pro, converts to GC373 which forms a covalent bond with C145. Surface representation reveals the active site pocket in complex with GC373 (6WTJ.pdb). c Ribbon representation of one SARS-CoV-2 protomer in complex with inhibitor GC373 binding in domain II. d GC373 interacts covalently with the active site cysteine of SARS-CoV-2 M^pro. Electron density at 1σ is shown in gray mesh.

Fig. 3. GC373 binds in the active site pocket of SARS-CoV-2 M^pro.

a GC373 forms a covalent bond with C145, and the oxyanion is stabilized by backbone H-bonds with G143, S144, and C145. b The P2 position of GC373 is stabilized by a hydrophobic pocket by the general base H41, and residues M49, and M165. c, d H-bonds are established with GC373 and side chains of H163 and E166, as well as backbone of residue E166. SARS-CoV-2 M^pro is represented in cartoon representation with the inhibitor in pink. P1, P2, and P3 of the peptidyl inhibitor are indicated. PDB Code:6WTJ.

Fig. 4. GC373 and GC376 potently inhibit SARS-CoV-2.

a, b SARS-CoV-2 growth in Vero E6 cells was determined by plaque assays 48 h after infection in the presence of various concentrations of drug. Cytotoxicity was measured using the CellTiter-Glo assay. a Percent inhibition of SARS-CoV-2 by GC373 in Vero E6 cells (blue open circles) and cytotoxicity in Vero E6 (blue closed circles) and A549 cells (orange). b Percent inhibition of SARS-CoV-2 by GC376 in Vero E6 cells (blue open circles) and cytotoxicity in Vero E6 (blue, closed circles) and A549 cells (orange). c, d To measure the reduction in virus yield, Vero E6 cells were infected with MOI = 0.01 of SARS-CoV-2 in triplicate without or with various concentration of GC373 c or GC376 d for 24 h and the supernatants harvested, RNA isolated and quantified by qRT-PCR. In the EC₅₀ curves, values are represented as mean ± SD of at least two independent experiments. For toxicity assay, values are represented as mean ± SD of 12 independent experiments. RNA data are presented as mean ± SD, with n = 3 independent experiments and individual data points shown.