The results show that for each inhibitor-M pro pair, both equilibrium approaches yield relatively similar results for the binding free energy estimates. However, the uncertainty in the computed binding free energy estimates is larger for the non-enhanced equilibrium ABFE approach (no H-REMD) relative to the enhanced sampling approach (H-REMD). This result is consistent with previous studies that have showed that the inclusion of the replica exchange algorithm in alchemical free energy simulations greatly improves the conformational sampling and convergence of free energy estimates. Table . Summary of the non-covalent binding free energy (∆𝐆 𝐧𝐨𝐧-𝐜𝐨𝐯𝐚𝐥𝐞𝐧𝐭 𝐨 ) results of the inhibitor-bound M pro complexes studied in this work. All energies are reported in kcal mol -1 . Our binding free energy results indicate that the non-covalent binding free energy contributions of the inhibitors to the M pro target is greatest for the PF-231 inhibitor (Table ). This is followed by the MI-30 inhibitor, which has a predicted binding energy of ~7.5 kcal mol -1 to the M pro . The inhibitors 11a and GC373 were predicted to have lower binding affinities in comparison to PF-231 and MI-30. Importantly, the predicted binding free energy results we report represent the non-covalent interaction energies between the inhibitors and the target protein. We were unable to find experimental inhibition constants (Ki) for all the inhibitors to directly compare with our predicted binding free energy results. However, comparison to experimental IC50 values for the inhibitors suggest that our predicted non-covalent binding free energy results correspond well with the relative potency rankings of the inhibitors to the M pro (Figure ),- although we note that IC50's in general are not suitable descriptors for estimating the biochemical potency of covalent inhibitors. This is because IC50's for covalent inhibitors will vary depending on the incubation time of the assay and do not provide any information about the affinity or chemical reactivity of a covalent inhibitor.