3C-like protease inhibitors block coronavirus replication in vitro and improve survival in MERS-CoV-infected mice

Abstract

Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses.

Fig. 1. Synthesis scheme for compound series 6a to 6k and 7a to 7k.

Stepwise compound synthesis with intermediate compounds is shown for 3C-like protease (3CLpro) inhibitors of the 6a to 6k and 7a to 7k series. The alcohol inputs were reacted with (L) leucine isocyanate methyl ester or (L) cyclohexylalanine isocyanate methyl ester to yield products, which were then hydrolyzed to the corresponding acids with lithium hydroxide in aqueous tetrahydrofuran. Subsequent coupling of the acids to glutamine surrogate methyl ester “8” furnished compounds “4”. Lithium borohydride reduction yielded alcohols “5”, which were then oxidized to the corresponding aldehydes “6” with Dess-Martin periodinane reagent. The bisulfite adducts “7” were generated by treatment with sodium bisulfite in aqueous ethanol and ethyl acetate. ^aAmino acid methyl ester isocyanate/TEA/CH₃CN/reflux/2 hours; ^b1M LiOH/THF/RT/3 hours; ^cEDCI/HOBT/glutamine surrogate/DIPEA/DMF/RT/24 hours; ^d2M LiBH₄/THF/methanol/RT/12 hours; ^eDess-Martin periodinane/DCM/15° to 18°C/3 hours; ^fNaHSO₃/ethyl acetated/ethanol/H₂O/44° to 55°C. Full details are provided in Supplementary Materials and Methods.

Fig. 2. X-ray cocrystal structures of compounds with coronavirus 3CL proteases.

Shown are cocrystal structures of the Middle East respiratory syndrome coronavirus (MERS-CoV) 3CLpro with compound 6h (A to C) and MERS-CoV 3CLpro with compound 7j (D to F). Shown are cocrystal structures of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro (G to I) or SARS-CoV-2 3CLpro (J to L) with compound 7j. (A), (D), (G), and (J) show F_o-F_c omit maps (green mesh) contoured at 3σ. (B), (E), (H), and (K) show hydrogen bond interactions (dashed lines) between the inhibitor and the 3CL protease. (C), (F), (I), and (L) show electrostatic surface representation of the binding pocket occupied by the inhibitor. Neighboring residues are colored yellow (nonpolar), cyan (polar), and white (weakly polar).

Fig. 3. Effects of treating hDPP4-KI mice infected with MERS_MA-CoV with compound 6j or 6h.

(A) Shown is a dose-dependent curve for inhibition of MERS-CoV in cell culture by compound 6j. Serial dilutions of compound 6j were added to confluent Huh-7 cells, which were immediately infected with MERS-CoV at a multiplicity of infection (MOI) of 0.01. After incubation of the cells at 37°C for 48 hours, viral titers were determined using a plaque-forming assay, and 50% inhibitory concentration (EC₅₀) values were determined with GraphPad Prism software. (B and C) hDPP4-KI mice infected with mouse-adapted MERS-CoV (MERS_MA-CoV) (n = 6) were treated with compound 6j or 6h starting at 1 day post virus infection (dpi) for up to 10 days, and survival (B) and body weight (C) were monitored for 15 days. Control mice received vehicle only. (D and E) hDPP4-KI mice infected with MERS_MA-CoV were treated with compound 6j (n = 5) starting at 1, 2, or 3 dpi, and survival (D) and body weight (E) were monitored for 15 days. Untreated mice and vehicle-treated mice (n = 4) were included as controls. Data points represent the mean and the SEM for one experiment. The analysis of survival curves in groups was performed using a log-rank (Mantel-Cox) test and a Gehan-Breslow-Wilcoxon test.

Fig. 4. Lung virus titers and histopathology of 6j-treated hDPP4-KI mice infected with MERS_MA-CoV.

hDPP4-KI mice were infected with MERS_MA-CoV at 0 dpi and then were treated with vehicle as a control or with compound 6j starting at 1 dpi until euthanasia (n = 4 or 5 per group). (A) Lungs were collected, and virus titers were measured at 3 and 5 dpi. Lungs were examined for edema and for hyaline membrane formation (B), and lung sections were stained with hematoxylin and eosin stain for histopathology at 6 dpi (C to F). (B) Tissues were scored for edema and hyaline membrane formation using the scale: 0, none; 1, rare (<5 alveoli); 2, <33% of lung fields; 3, 34 to 66% lung fields, and 4, >66% lung fields (30). (C) to (F) show representative histopathology images for vehicle control in (C) and (E) and compound 6j treatment in (D) and (F) at 40× [(C) and (D)] or 100× [(E) and (F)]. Asterisks indicate P < 0.01 by multiple t tests.