As previously proposed from X-ray diffraction studies, and later supported by computational studies using different approaches, the chemical reaction leading to M pro inactivation requires the imidazole group of H41 to activate the SH group of C145 to generate a highly nucleophilic thiolate (CysS -) that would readily react with the inhibitor. According to the recent literature, the equilibrium between the neutral dyad and the CysS -/HisH + ion pair appears to be tipped in favour of the neutral pair by ligand binding, but it may depend on the stereoelectronic properties of the ligand itself. Thus, while some studies report a neutral dyad significantly more stable than the ion pair (by ca. 11 kcal•mol -1 ) and even not being an stable state, others suggest the ion pair is not so destabilized with respect the initial neutral dyad, or even slightly more stable than the initial neutral dyad (i.e. our previous study with B1). Our previous study on the proteolysis reaction of SARS-CoV-2 M pro using the polypeptide Ac-Val-Lys-Leu-Gln-ACC as a substrate (with ACC being a fluorescent 7-amino-4carbamoylmethylcoumarin tag) suggests that the mechanism of action of this enzyme slightly differs from other cysteine proteases. First of all, the enzyme:substrate initial complex is the neutral C145/H41 dyad (E:I) instead of the ion pair dyad C145 -/H41 + (E (+/-) :I). This result is in agreement with studies carried out by us, and others, using different inhibitors and substrates, but in contrast with the protonation state of the catalytic dyad suggested from the ligand-free SARS-CoV-M pro recently solved by neutron crystallography at pH 6.6. Nevertheless, as already pointed out, questions remain about how pH or the presence of an inhibitor or substrate influence the protonation state of the dyad in SARS-CoV M pro . The acylation reaction consists of a proton transfer from C145 to the H41 concomitant, or not, with the nucleophilic attack on the carbonyl carbon atom of the peptide bond by the sulfur atom of C145, leading to a pseudo-stable intermediate. Then, the cleavage of the peptide bond by M pro is assisted by proton transfer from the protonated H41 + to the substrate, forming a stable acyl-enzyme covalent intermediate. Thus, while modelling of the proteolysis reaction showed that the formation of the ion pair and the attack of the Cys145 to the carbonyl carbon of the substrate took place in a single step, in the inhibition reaction by N3, or our designed B1 and B2 MA compounds, or the simulation with M pro -substrate peptide models, the reaction appears to proceed in a stepwise manner: in the first step, Cys145 is activated by His41, forming the ion pair E (+/- ) :I, followed in the second step by attack of the sulfur atom of Cys145 on the Cβ atom of the inhibitor and proton transfer from His41 to the Cα atom of the inhibitor, leading to a stable covalent E-I intermediate. The rate-limiting step of the process, in all three cases, was enzyme-inhibitor covalent bond formation, with activation free energies ranging from 11.8 to 9.8 kcal•mol -1 . From the thermodynamic point of view, the exergonic process obtained with N3 (reaction energy -17.9 kcal•mol -1 ) was consistent with its experimentally observed stability (e.g. revealed by X-ray crystallographic structures) , while the inactivation reactions of M pro with B1 and B2 were also exergonic but very different from each other (-27.9 and -11.4 kcal•mol -1 , respectively), predicting that compound B1 would be an irreversible inhibitor, but that compound B2 would have a more reversible character. Analysis of the QM-MM interaction energies between the different residues located in the substrate-binding pockets of M pro and the peptide (in the study of the proteolysis reaction) or the inhibitor (in the case of the inhibitors) confirms the predictions assumed during the design of B1 and B2; i.e. the interactions between the protein and the inhibitors are dominated by those in the P1:::S1 site. Thus, our previous results indicate that a low barrier Cys145 covalent modification can be obtained by modulating either the recognition portion or the warhead. Interactions between the recognition moiety and M pro active site affect the chemical step because they dictate the pose of the inhibitor in the active site of the enzyme. Consequently, to design an efficient inhibitor, the presence of a reactive warhead and the interactions between the recognition moiety of the inhibitor and the different sub-sites of the binding pocket of the protein must be taken into account. The results from previous studies on this and related cysteine proteases can be used to guide the design and QM/MM simulations can provide a useful tool to investigate the reactivity of designed covalent inhibitors within their protein targets. Here, we propose and investigate the inhibition of the SARS-CoV-2 M pro by two potential covalent (peptidyl) inhibitors endowed with two chemically diverse warheads. Building upon the findings on information derived from our previous studies on the proteolysis of M pro , and on the reaction of the inhibition with several peptidyl irreversible inhibitors, two compounds B3 and B4 are proposed (Scheme 1). A methyl oxo-enoate was used in B3, inspired by dimethylfumarate structure, while a hydroxymethylketone (HMK) was used as warhead in B4 compound. This reactive group also present in the structure of PF-00835231 M pro inhibitor, now in clinical trial . The recognition part possessed by both B3 and B4 compounds, was selected on the based on QM/MM results obtained with previously proposed inhibitors B1 and B2 as well as by suggestions emerged, from QM-MM protein-substrate interactions. S2 appears to be a