Virtual Double-System Single-Box: A Nonequilibrium Alchemical Technique for Absolute Binding Free Energy Calculations: Application to Ligands of the SARS-CoV‑2 Main Protease

In the context of drug–receptor binding affinity calculations
using molecular dynamics techniques, we implemented a combination
of Hamiltonian replica exchange (HREM) and a novel nonequilibrium
alchemical methodology, called virtual double-system single-box, with
increased accuracy, precision, and efficiency with respect to the
standard nonequilibrium approaches. The method has been applied for
the determination of absolute binding free energies of 16 newly designed
noncovalent ligands of the main protease (3CLpro) of SARS-CoV-2.
The core structures of 3CLpro ligands were previously identified
using a multimodal structure-based ligand design in combination with
docking techniques. The calculated binding free energies for four
additional ligands with known activity (either for SARS-CoV or SARS-CoV-2
main protease) are also reported. The nature of binding in the 3CLpro active site and the involved residues besides the CYS–HYS
catalytic dyad have been thoroughly characterized by enhanced sampling
simulations of the bound state. We have identified several noncongeneric
compounds with predicted low micromolar activity for 3CLpro inhibition, which may constitute possible lead compounds for the
development of antiviral agents in Covid-19 treatment.