Structural and Evolutionary Analysis Indicate That the SARS-CoV-2 Mpro Is a Challenging Target for Small-Molecule Inhibitor Design

The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mixed-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar severe acute respiratory syndrome (SARS) Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins showed major differences in both shape and size, indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site’s conformational changes during the simulation time indicated its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicated that the virus’ mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.

. Differences between SARS-CoV-2 and SARS-CoV Mpros proteins. The last column shows differences in total energies (in kcal/mol) calculated as differences in Gibbs free energy folding between SARS-CoV Mpro and introduced single-point mutations as they are in SARS-CoV-2 Mpro structure.  Figure S2. The example of time mode analysis of the maximal accessible volume (MAV) (blue mesh) of Mpro structures. The catalytic dyad is shown as red sticks. The last column shows the average location of water hot-spots (cyan spheres) during the simulation time. The position of the biggest hot-spot in each row reflects the position of the catalytic water molecule.
Supplementary Figure S4. Localisation of the global hot-spots of all analysed Mpros. SARS-CoV Mpro N3 and SARS-CoV Mpro. The structures of all analysed Mpro structures are superposed and the colour-coding is as follows: orange, red, yellow, green, pink and purple hot-spots are from the SARS-CoV-2 Mpro N3 , white hot-spots from the SARS-CoV-2 Mpro, black hot-spots from the SARS-CoV Mpro N3 , and grey hot-spots from the SARS-CoV Mpro structure. The active site residues are shown as red sticks, the differing residues of the SARS-CoV-2 Mpro as blue sticks, and the proteins' structures are shown in surface representation.
Supplementary Figure S5. Localisation of the global hot-spots identified in the binding site cavities in SARS-CoV-2 and SARS-CoV main proteases. Hot-spots of individual cosolvents are represented by spheres, and their size reflects the hot-spots density. The colour coding is as follows: purple -urea, green -dimethylsulfoxide, yellow -methanol, orange -acetonitrile, pink -phenol, red -benzene.
Supplementary Figure S6. Localisation of the local hot-spots of all analysed Mpros: COVID-19 Mpro, SARS-CoV Mpro and SARS-CoV Mpro-f. Hotspots for individual cosolvents are represented by spheres, and their size reflects the hot-spots density. The colour-coding is as follows: purpleurea, green -DMSO, yellow -methanol, orange -acetonitrile, pink -phenol, red -benzene. The active site residues are shown as red sticks, the N3 inhibitor structure as green lines, and the proteins' structures are shown in cartoons representation.
Supplementary Figure S7. Comparison of the space occupied by covalently bound fragments in the active site cavity (Diamond Light Source group) (A) with maximal accessible volume calculated by AQUA-DUCT software (B). It is noted, that part of the ligands extends beyond the protein surface.
Supplementary  Figure S8. Comparison of the rebuilded loop in 1q2w (gold), with loops from 6lu7 (blue) and 2h2z (red) structures. The white cartoon depicts original 1q2w pdb file. The H41 residue from active side dyad is shown in stick representation.