## 3 Results and Discussion
Particularly, a strongly destabilized IP (9.62 kcal mol\({}^{-1}\)) was located on the PES, from which the proton dislocated on HIP might be shuttled to Cys145, leading back to the RC. In addition, different pathways might be suitable for the nucleophilic Cys' attack to the Se atom of EbSeS. First, the deprotonated Cys might attack the Se-S bond of EbSeS, leading to a TCI that has been previously identified in vacuo and in water in model molecular systems [53]. In our case, the TCI is located only 1.25 kcal mol\({}^{-1}\) above the RC. Wiberg bond indexes analysis [54] of this intermediate in the gas phase (Table 1) revealed a behaviour similar to that computed for the small molecular model presenting a -S-Se-S bond, whose coordinates were taken from [PERSON] et al [53]. Figure 2: Energy profile for the inhibition mechanism of MP\({}^{\text{pro}}\) by EbSe. Activation energies of TS1 and TS2 are relative to the RC and TCI, respectively. Energies are reported in Table S2 (Supplementary Materials). Level of theory: SMD-B3 LYP-D3(B)/6-311G(d,p), cc\(\triangleright\)VTZ//B3 LYP-D3(B)/6-311G(d,p), cc\(\triangleright\)VTZ. For our TCI, the indexes are consistent with two asymmetric S-Se bonds, as is reasonable given the different sizes of the two thiolates bonded to Se, ethanethiolate and an enzymatic cysteine, with respect to the two symmetric thiolates of [53]. Further re-optimization of the TCI in diethyl ether (see Computational Methods) does not significantly affect the geometry, confirming the stability of this intermediate in a non-polar environment, such as the enzymatic pocket. Such a structure might spontaneously loose ethanethiolate (EtS'), leading to the desired thiolate exchange and the EbSe-S-Cys(M\({}^{\text{pro}}\)) adduct. However, this process is computed to be endergonic (+5.20 kcal mol\({}^{-1}\) with respect to the RC), and the back proton transfer from HIP to EtS' is required for the overall inhibition process to become exergonic (\(-1.78\) kcal mol\({}^{-1}\) with respect to the RC). Whether such a back proton transfer happens directly from the IP (leading to the concerted breaking of the EbSe Se-S bond and to the formation of Cys-Se bond, bypassing the formation of a TCI) or at a later mechanistic stage (e.g., at the TCI itself) was not investigated, since such analysis is expected to be strongly influenced by the nature of the thiolate, by cavity rearrangements and by the presence of explicit water molecules, which can assist with the thiol exchange mechanism. Thus, we require further molecular dynamics investigation to understand how the evolution of the binding site can affect ebeselen inhibition. However, the overall thermodynamic feasibility of the inhibition process seems to be less favorable with respect to the same process for EbSe (\(-1.78\) and \(-6.86\) kcal mol\({}^{-1}\), respectively). Since the chalcogenolate exchange between a thiolate and a diselenide was previously computed to be less thermodynamically favorable \begin{table}
\begin{tabular}{c c c} \hline \hline  & EbS(e)S & **CH3S(S/SeCH3)SCH3 2** \\ \hline S1–S2 & 0.190  \\ S2–S3 & 0.833  \\ S1–Se & 0.325 & 0.535 \\ Se–S3 & 0.703 & 0.537 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Wiberg indexes for S1-S2-S3 1 and S1-Se-S 3 1 TCIs, with EbSeS and a sulfur analogue, and for two previously described minimal models. Level of theory: B3 LYP-D3(B)/6-311G(d,p), cc-pVTZ. Figure 3: (**A**) Docking simulations between M\({}^{\text{pro}}\) and EbSeS. The carboxyl and amino moieties of EbSe-Cys were replaced with H atoms to create the ethanthiolate (EBSeS), as a general model of EbSe-thiol adduct/metabolite. For better visualization, only the side chain of the main residues is shown. (**B**) Optimized M\({}^{\text{pro}}\) cluster, with EbSeS docked inside. For clarity, only C\({}^{\text{a}}\), H\({}^{\text{a}}\) and amino acid side chains are displayed, Met165 is not shown and the cluster is oriented for optimal view. Level of theory: B3 LYP-D3(B)/6-311G(d,p), cc-pVTZ.