In Silico Substrate-Binding Profiling for SARS-CoV-2 Main Protease (M^pro) Using Hexapeptide Substrates

The SARS-CoV-2 main protease (M^pro) is essential for the life cycle of the COVID-19 virus. It cleaves the two polyproteins at 11 positions to generate mature proteins for virion formation. The cleavage site on these polyproteins is known to be Leu-Gln↓(Ser/Ala/Gly). A range of hexapeptides that follow the known sequence for recognition and cleavage was constructed using RDKit libraries and complexed with the crystal structure of M^pro (PDB ID 6XHM) through extensive molecular docking calculations. A subset of 131 of these complexes underwent 20 ns molecular dynamics simulations. The analyses of the trajectories from molecular dynamics included principal component analysis (PCA), and a method to compare PCA plots from separate trajectories was developed in terms of encoding PCA progression during the simulations. The hexapeptides formed stable complexes as expected, with reproducible molecular docking of the substrates given the extensiveness of the procedure. Only Lys-Leu-Gln*** (KLQ***) sequence complexes were studied for molecular dynamics. In this subset of complexes, the PCA analysis identified four classifications of protein motions across these sequences. KLQ*** complexes illustrated the effect of changes in substrate on the active site, with implications for understanding the substrate recognition of M^pro and informing the development of small molecule inhibitors.