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 (3CL pro ) of SARS-CoV-2. The core structures of 3CL pro 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 3CL pro 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 3CL pro inhibition, which may constitute possible lead compounds for the development of antiviral agents in Covid-19 treatment.