Molecular dynamics (MD) simulations of the enzyme-ligand complexes

Complexes of the best 10 ligands docked to the allosteric groove binding site of the SARS-CoV-2 3CLpro in the cluster 1 and cluster 2 representative conformations were subjected to 150 ns MD simulations (a total of 20 simulations of 150 ns in duration). The systems were prepared as described in the “Molecular dynamics (MD) simulations of the free enzyme” section, with only difference being the temperature of the simulations, which was set to 310 K, and the number of Na⁺ and Cl^- ions (Na⁺ and Cl^- ions were added according to Machado and Pantano (2020) which were set to achieve a neutral environment with salt concentration of 0.15 M). This was done to better simulate the conditions inside the human body.

The binding energy, ΔG_bind, of the simulated complexes was calculated using the MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) protocol (Genheden & Ryde, 2015; Hou et al., 2011), available as a part of AmberTools16 (Case et al., 2016). MM/GBSA is a method for the calculation of ΔG_bind from snapshots of MD trajectory (Ferenczy, 2015) with an estimated standard error of 1–3 kcal/mol (Genheden & Ryde, 2015). ΔG_bind is calculated in the following manner:

where the symbol < > represents the average value over 100 snapshots collected from a 30 ns part of the corresponding MD trajectories. For all enzyme-ligand complexes, the whole trajectory was divided into 5 parts of 30 ns length and ΔG_bind was calculated for all 5 parts of the simulations and reported as mean ± standard deviation. The calculated MM/GBSA binding free energies were decomposed into specific residue contribution on a per-residue basis according to established procedures. This protocol calculates the contributions to ΔG_bind arising from each amino acid side chains and identifies the nature of the energy change in terms of interaction and solvation energies, or entropic contributions (Gohlke et al., 2003; Rastelli et al., 2010). In this case, the entropy term was not calculated.