Proton-Coupled Conformational Activation of SARS Coronavirus Main Proteases and Opportunity for Designing Small-Molecule Broad-Spectrum Targeted Covalent Inhibitors

Abstract

The SARS coronavirus 2 (SARS-CoV-2) main protease (Mpro) is an attractive broad-spectrum antiviral drug target. Despite the enormous progress in structure elucidation, the Mpro's structure-function relationship remains poorly understood. Recently, a peptidomimetic inhibitor has entered clinical trial; however, small-molecule orally available antiviral drugs have yet to be developed. Intrigued by a long-standing controversy regarding the existence of an inactive state, we explored the proton-coupled dynamics of the Mpros of SARS-CoV-2 and the closely related SARS-CoV using a newly developed continuous constant pH molecular dynamics (MD) method and microsecond fixed-charge all-atom MD simulations. Our data supports a general base mechanism for Mpro's proteolytic function. The simulations revealed that protonation of His172 alters a conserved interaction network that upholds the oxyanion loop, leading to a partial collapse of the conserved S1 pocket, consistent with the first and controversial crystal structure of SARS-CoV Mpro determined at pH 6. Interestingly, a natural flavonoid binds SARS-CoV-2 Mpro in the close proximity to a conserved cysteine (Cys44), which is hyper-reactive according to the CpHMD titration. This finding offers an exciting new opportunity for small-molecule targeted covalent inhibitor design. Our work represents a first step toward the mechanistic understanding of the proton-coupled structure-dynamics-function relationship of CoV Mpros; the proposed strategy of designing small-molecule covalent inhibitors may help accelerate the development of orally available broad-spectrum antiviral drugs to stop the current pandemic and prevent future outbreaks.

Figure 1.. Crystal structure and pH-dependent activity for SARS-CoV-2/CoV Mpros.

a) X-ray crystal structure of SARS-Cov-2 Mpro dimer (PDB: 6y2g). Protomer A is colored gray, while domains I, II, and III in protomer B are colored red, green, and cyan, respectively. The oxyanion loop L1 (residues 138–145) is colored magenta and the N-finger loop (residues 1–10) as well as G11 are colored yellow. b) pH-activity profile of SARS-CoV Mpro determined by the HPLC-based peptide cleavage assay. c) A zoomed-in view of the S1 specificity pocket in protomer A and the N-finger loop in protomer B. Residues involved in the dimer interface interactions are explicitly shown (those in protomer B denoted with an asterisk). The conserved S1 pocket residues Phe140, His163, Glu166, His172, and the catalytic dyad Cys145 and His41 are explicitly shown.

Figure 2.. Conformational changes of the S1 pocket in SARS-CoV-2 Mpro is coupled to the switch of His172 protonation state.

a), b) Probability distribution of the minimum distance between the carboxylate oxygens of Glu166 and the imidazole nitrogens of His172 (a) and His163 (b). d) Distribution of the χ₁ angle of Glu166. e) Distribution of the distance between the center of mass of Glu166 (carboxylate oxygens) and the oxyanion loop (Cα atoms of residues 138–145). g) Distribution of the distance between the center of mass of the aromatic rings of His163 and Phe140. h) Distribution of the χ₁ angle of Phe140. Data for the protomer with neutral and charged His172 are colored green and orange, respectively. All plots were based on simulation run 1 (run 2 data given in Fig. S6). The black and red dashed lines indicate the corresponding values from the protomer A of the SARS-CoV-2 Mpro structure (PDB: 6y2g), and the inactive protomer B of the SARS-CoV Mpro structure (PDB: 1uj1). c), f) and i) Snapshots showing the conformational differences between the the protomers with neutral (green) and charged (orange) His172. The oxyanion loop is colored magenta.

Figure 3.. Interactions between the S1 pocket residues and Ser1 of the opposite protomer in SARS-CoV-2 Mpro are disrupted by the protonation of His172.

a), b), c) Probability distributions of the Glu166^A/Phe140^A/His172^A–Ser1^B distances are colored green; distributions of the Glu166^B/Phe140^B/His172^B–Ser1^A distances are colored orange. His172 is neutral in protomer A and charged in protomer B. The Glu166/Phe140–Ser1 distance refers to that between the N-terminal amino nitrogen and the nearest carboxyl oxygen of Glu166 or the carbonyl oxygen of Phe140. The His172–Ser1 distance is refers that between the backbone carbonyl oxygen of Ser1 and the nearest imidazole nitrogen of His172. d) Zoomed-in view of the interactions between the S1 pocket (protomer A) and Ser1 (protomer B) in the X-ray structure of SARS-CoV-2 Mpro (PDB: 6y2g). Hydrogen bonds are shown in dashed lines.

Figure 4.. A natural flavonoid compound offers a starting point for designing small-molecule broad-spectrum targeted covalent inhibitors.

X-ray structure of SARS-CoV-2 Mpro in complex with galangin (PDB: 6m2n) shows that it is in the proximity of both Cys44 and Cys145.