Crystal Structure of SARS-CoV-2 Main Protease in Complex with the Non-Covalent Inhibitor ML188

Abstract

Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (M^pro) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 M^pro, and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 M^pro at 2.5 µM, which is more potent than against SAR-CoV-1 M^pro. We determined the crystal structure of ML188 in complex with SARS-CoV-2 M^pro to 2.39 Å resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19.

Keywords: Covid-19; ML188; Mpro; SARS-CoV-2; crystal structure; direct-acting antivirals; main protease; protease inhibitor; structure-based drug design.

Figure 1

(a) Chemical structure of ML188. (b) Dose-response curves and IC₅₀ values of compound ML188 against SARS2-M^Pro and SARS1-M^Pro.

Figure 2

(A) SARS2-M^pro dimer and single subunit shown in surface representation (rotated on the plane of the page). (B) SARS2-M^pro active site with subsites labeled and catalytic residues colored yellow. (C) ML188 in the SARS2-M^pro active site with catalytic residues labeled. (D) Electron density around ML188 and catalytic residues. The 2F_o-F_c direct maps are depicted as grey mesh contoured at 1.0 σ while the F_o-F_c difference maps have positive density depicted as green mesh contoured at 3.0 σ and negative density as red mesh contoured at −3.0 σ. (E) Alignment of ML188 in SARS2-M^pro (teal protein and green ligand; PDB: 7L0D) and SARS1-M^pro (magenta protein and orange ligand; PDB: 3V3M).

Figure 3

(A) The twelve amino acid differences between SARS2 and SARS1-M^pro shown as a dimer, single subunit, and active site. SARS2 (in cyan) and SARS1 (in magenta) are shown with transparent surfaces and amino acid differences shown as sticks. The dimer subunits (in orange) and the catalytic residues (in yellow) are included. (B) Stereo pair of SARS2-M^pro-ML188 complex active site. (C) Stereo pair of SARS1-M^pro-ML188 complex active site.