## 4 Conclusions  
In this work, we investigated the mechanistic details of S-Se bond formation between EbSe and the catalytic Cys of M\({}^{\text{pro}}\), and a comparison was made with EbSeS. To the best of our knowledge, our investigation provides, for the first time, the stationary points for the covalent binding mechanism of Ebselen and one of its possible metabolites to an enzymatic Cys, i.e., M\({}^{\text{pro}}\) Cys145. Overall, the energetics computed for the metabolite suggest its weaker inhibition potential with respect to EbSe, in agreement with the recent studies on the activity of EbSe upon reduction to diselenide or in the presence of DTT/GSH. However, we must stress that the nature of the leaving thiol might exert an important effect on the thermodynamics of the whole reaction, especially since EbSe might reach the M\({}^{\text{pro}}\) bonded to an extremely bulky group, such as a whole protein. This prompts further studies based on molecular dynamics with suitable accurate force-fields for the selenides, as well as a fine experimental investigation of EbSe antiviral activity in biologically plausible conditions. Moreover, we provide thermodynamic support to the hypothesis of Se-C bond-breaking within M\({}^{\text{pro}}\)[20], and we suggest a detailed mechanistic investigation to understand if and how the direct participation of His residues of the catalytic pocket lowers the activation energy of the process. Conversely to EbSeS, EbSe mechanism is straightforwardly similar to the canonical mechanism of Cys proteases, and the Cys145 attack on the Se atom appears to proceed without an appreciable activation energy. In addition, differently from the RC with EbSeS, EbSe exhibits an almost linear arrangement of S-Se-N atoms of Cys145 and EbSe (157\({}^{\circ}\)), making it suitable for the S\({}_{\text{N}}\)2 process to occur without large structural rearrangements. Notably, since SARS-CoV-2 PL\({}^{\text{pro}}\) employs a catalytic triad that also includes reactive Cys and His residues, a similar mechanism is readily extendable to this and other viral Cysproteases, both for EbSe and EbSeS. Thus, efforts are prompted to design selenium-based systems able to reach the target protein with the N-Se bond still intact. This requires a fine-tuning of the N-Se bond, designing ad hoc molecular features [58] to prevent ring-opening in the presence of free cysteines such as those present in the serum, preserving the ability of N-Se bond activation in the target proteins. The following are available online at [[https://www.mdpi.com/article/10.390/app11146291/s1](https://www.mdpi.com/article/10.390/app11146291/s1)]([https://www.mdpi.com/article/10.390/app11146291/s1](https://www.mdpi.com/article/10.390/app11146291/s1)), energies, Cartesian coordinates and figures of all the investigated structures, Table S1: Coordinates (A) energies (E, Hartree) and number of imaginary frequencies (Nimag) of stationary points as computed at B3 LYP-D3(BJ)/6-311G(d,p), cc-pVTZ level of theory, Table S2: Electronic (\(\Delta\)E) and Gibbs (\(\Delta\)Gsolv) free energies (kcal mol\({}^{-1}\)) relative to the RC for Ebselen (EbSe) mechanism, Table S3: Electronic (\(\Delta\)E) and Gibbs (\(\Delta\)Gsolv) free energies (kcal mol\({}^{-1}\)) relative to the RC for Ebselen ethanethiolate (EbSeS) mechanism. Conceptualization, supervision, validation, and project administration, L.O. and J.B.T.R. ; methodology, L.O. and J.B.T.R. ; formal analysis, investigation, and data curation, A.M., P.A.N. and F.B.O. ; writing--original draft preparation, A.M., P.A.N. and L.O. ; writing--review and editing, L.O. and J.B.T.R. All authors have read and agreed to the published version of the manuscript. This research was funded by Universita degli Studi di Padova, thanks to the P-DiSC (BIRD2018-UNIPD) project MAD\({}^{3}\)S (Modeling Antioxidant Drugs: Design and Development of computer-aided molecular systems; P.I. L.O.). P.A.N. and J.B.T.R. were funded by CAPES (Ediatal 09-88887.505377/2020-00 and 88887.511828/2020-00) and F.B.O.