Discovery of 2-thiobenzimidazoles as noncovalent inhibitors of SARS-CoV-2 main protease

Abstract

The discovery of antiviral agents against SARS-CoV-2 is an important step toward ending the COVID-19 pandemic and to tackle future outbreaks. In this context, the main protease (M^pro) represents an ideal target for developing coronavirus antivirals, being conserved among different strains and essential for survival. In this work, using in silico tools, we created and validated a docking protocol able to predict binders to the catalytic site of M^pro. The following structure-based virtual screening of a subset of the ZINC library (over 4.3 million unique structures), led to the identification of a hit compound having a 2-thiobenzimidazole scaffold. The inhibitory activity was confirmed using a FRET-based proteolytic assay against recombinant M^pro. Structure-activity relationships were obtained with the synthesis of a small library of analogs, guided by the analysis of the docking pose. Our efforts led to the identification of a micromolar M^pro inhibitor (IC₅₀ = 14.9 µM) with an original scaffold possessing ideal drug-like properties (predicted using the QikProp function) and representing a promising lead for the development of a novel class of coronavirus antivirals.

Graphical abstract

Fig. 1

Structure of M^pro and its inhibitors. (A) X-ray crystal structure of the SARS-CoV-2 M^pro homodimer highlighting the different domains, the active site and the catalytic dyad. (B) Structures of the most advanced inhibitors, covalent and noncovalent, discovered to date.

Fig. 2

X-ray co-crystal structure of X77 in complex with M^pro (PDB entry 6 W63). (A) Key residues are shown as cyan sticks, H-bond interactions and π-π stackings are depicted as red and green dashes, respectively. (B) Surface rendering noting the binding pockets and the overlapped poses of X77 in the crystal structure (orange sticks) and in the re-docking experiment (purple sticks). The calculated RMSD between the two poses is 0.559.

Fig. 3

(A) Workflow for the virtual screening leading to the identification of 15 virtual hits. The number of structures filtered during each step is given in the left bars. (B) Screening of the virtual hits against SARS-CoV-2 M^pro using the FRET assays. Proteolysis was monitored by cleaved substrate fluorescence following a 15 min incubation of 50 µM compound with 15 nM M^pro. Residual percent enzyme activity was calculated after normalization of the initial velocity to the DMSO negative control. Results are the average standard deviation (error bars) of three repeats. Boceprevir was used as positive control. (C) Concentration-response curves of compound 8. Values were obtained from linear regression analysis of initial velocities and IC₅₀ curves were plotted using the software Prism. The assay was performed also in the presence of 0.02 % of Triton-X (blue values) to rule out aggregation-based inhibitory effects. Values are average of three independent experiments. The structure of 8 is also shown.

Fig. 4

(A) Docking pose of compound 8 in the active site of SARS-CoV-2 M^pro. Key residues are shown as cyan sticks, H-bond interactions are depicted as red dashes. (B) Surface rendering of the docking pose noting the binding pockets. (C) Overview of the synthetic modification carried out for the hit expansion.

Scheme 1

Synthetic procedures for the synthesis of α-substituted chloroacetyl imidazolidinones and oxazolidinones.

Scheme 2

Synthetic pathway for the preparation of the 2-thiobenzimidazole building blocks and for the final compounds. Structural data for 16–38 are given in Table 1.