The monomer-dimer equilibrium modulation of Mpro induced by inhibitor binding.

(a) Crystal structure of SARS-CoV-2 Mpro dimer, monomer, and active site pockets of SARS-CoV-2 Mpro with subsites and two catalytic dyad residues (H41 and C145) highlighted. (b) Native mass spectra of (b) unbound Mpro, (c) Mpro-PF-07321332 complex, and (d) Mpro-ebselen complex.

Inhibitors against SARS-CoV-2 Mpro and their inhibitory properties

Mpro subunit exchange dynamics.

(a) The strategy for Mpro subunit exchange. (b) Monitoring of unbound Mpro subunit exchange by native mass spectrometry. Mpro subunit exchange is affected by (c) PF-07321332, (d) PF-00835231, (e) GC376, (f) boceprevir, and (g) ebselen. The red dashed lines indicate the heterodimers.

Conformational dynamics analysis of SARS-CoV-2 Mpro with PF-07321332.

(a) The deuterium uptake plot for all identified peptides from Mpro at 1, 10, and 60 min. (b) Deuterium uptake of PF-07321332-bound Mpro by global HDX-MS. (c) The heat map of Mpro upon binding with PF-07321332 at 10 minutes was overlaid onto the crystal model of Mpro (PDB entry 7ALI). (d) The uptake line plots in the PF-07321332-bound Mpro at 10-min HDX labelling.

A new allosteric binding mode of ebselen on Mpro.

(a) Deuterium uptake plot of ebselen-bound Mpro by global HDX-MS. (b) The heat map of Mpro upon binding with ebselen at 10 minutes was overlaid onto the crystal model of Mpro (PDB entry 7ALI). (c) The uptake line plots in the ebselen-bound Mpro at 10 minutes HDX labelling. (d) MD simulation of SARS-CoV-2 Mpro in the presence of ebselen on C300. Shifts in key intra-dimer interactions before and after covalent binding of ebselen at C300 on Mpro. Left panel: key amino acids involved in intra-dimer interactions are depicted on the crystal model of Mpro (PDB entry 7ALI). Right panel: the distances between R4 of one monomer and E290 of the other monomer Cα atoms, and residues S1 of one monomer and the other monomer E166 Cα atoms were measured, respectively.