Molecular dynamics simulation and binding free energy
To understand the stability, intermolecular interactions, and the binding energy of the three ligand-TMPRSS2 complexes, the molecular dynamics (MD) simulation has been performed using the OPLS3e force field [29] implemented in Desmond v5package[30]. Further, the system was built with the pre-defined TIP4P water model and orthorhombic periodic boundary conditions at the distances 10 Å. Then the counterions were used to neutralize the charge of these complexes with the balancing Na+/Cl ions. Further, the constructed system for each ligand-TMPRSS2 complex was energy minimized by heating and equilibrium processes before the MD simulation. For the minimization, the minimization and heating protocol was fixed based on the steepest descent method, annealing temperature at 0-300 K, and 2000 steps with the time steps of 0.001 ps. Further, the system was normalized in an equilibrium state at 1000 steps with a time step of 0.001 ps. Finally, the production step of the systems was continued up to 100 ns with the time steps of 0.001 ps, 300 K, 1 ATM pressure, and applied using the Nose-Hoover method with NPT ensemble[31]. Intermolecular interactions and conformation of each ligand-protein complex was analyzed from the final results of MD simulation. Among the 1000 fractions, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 ns fractions were used to determine the binding free energy (MM/GBSA) of the ligand-TMPRSS2 complexes by Primeapplication incorporated to Schrodinger software package[32].