Absolute Binding Free Energy Calculation: We performed ABFE calculations to compute the non-covalent binding free energy contribution of the inhibitors to the M pro target. ABFE calculations, although computationally expensive, provide a theoretically rigorous framework for estimating ligand binding energies and have been shown to yield results that agree well with experimental binding energies. For performing the ABFE calculations, we followed the protocol reported in our recent publication 32 on estimating the total binding energy of peptidomimetic inhibitors of the SARS-CoV-2 M pro . Two sets of simulations for each model system were performed for the binding energy calculations, consisting of the ligand in bulk solution and in the protein-binding site. A set of restraints defined by one distance, two angles, and three dihedral harmonic potentials were imposed on the bound ligand to keep it in the protein-binding site following the decoupling steps of the free energy calculation (see Supporting Information for details). For the ligand in bulk solution, the restraint term following the decoupling event is computed analytically using the method proposed by Boresch et al. Similar to our previous protocol, 31 𝜆 windows were used for the ligand in bulk solution and 42 𝜆 windows were used for the protein-ligand simulation. For each 𝜆 window, a series of simulations consisting of 100,000-step steepest descent energy minimization, 1ns of NVT equilibration and 1 ns of NPT equilibration were performed. A timestep of 2 fs was used for the simulations with reference temperature set to 298.15 K. The LINCS constraint algorithm was used to constrain H-bonds. The Particle Mesh Ewald (PME) algorith was used to treat long-range electrostatic interactions and a cut-off distance of 12 Å was used for short-range electrostatics.