Exploring the Mechanism of Covalent Inhibition: Simulating the Binding Free Energy of α-Ketoamide Inhibitors of the Main Protease of SARS-CoV-2

Abstract

The development of reliable ways of predicting the binding free energies of covalent inhibitors is a challenge for computer-aided drug design. Such development is important, for example, in the fight against the SARS-CoV-2 virus, in which covalent inhibitors can provide a promising tool for blocking M^pro, the main protease of the virus. This work develops a reliable and practical protocol for evaluating the binding free energy of covalent inhibitors. Our protocol presents a major advance over other approaches that do not consider the chemical contribution of the binding free energy. Our strategy combines the empirical valence bond method for evaluating the reaction energy profile and the PDLD/S-LRA/β method for evaluating the noncovalent part of the binding process. This protocol has been used in the calculations of the binding free energy of an α-ketoamide inhibitor of M^pro. Encouragingly, our approach reproduces the observed binding free energy. Our study of covalent inhibitors of cysteine proteases indicates that in the choice of an effective warhead it is crucial to focus on the exothermicity of the point on the free energy surface of a peptide cleavage that connects the acylation and deacylation steps. Overall, we believe that our approach should provide a powerful and effective method for in silico design of covalent drugs.

Figure 1.

Various mechanistic schemes of formation of a covalent complex between a peptidomimetic inhibitor and a Cysteine protease. The inhibitor is represented in generic form of RR’CO, where R and R’ can be any aliphatic or aromatic chain. The reacting molecular fragments are represented in square boxes. The green, red and blue colored arrows are used to represent schemes A, B and C, respectively. The difference among schemes A, B and C has been discussed in detail in the main text.

Figure 2.

Structures of dimeric SAR-CoV-2 M^pro and its α-ketoamide inhibitor 13b. (A) Protomer A and B are in orange and blue colored ribbon representations, respectively. The inhibitor and catalytic residues in protomer A are shown in purple and green stick representations. (B) The most electrophilic center of 13b, is highlighted with a black arrow.

Figure 3.

A general free energy profile that explains the formation of a covalent protein (P) inhibitor (I) complex. P···I, [P---I]^‡ and P---I denote the non-covalent, activated complex(s) during chemical reaction(s) and covalent binding state, respectively. ΔG_non-cov, ΔG_chem and ΔG_cov represent the non-covalent (P···I) binding energy, reaction free energy and total covalent (P---I) binding free energy, respectively.

Figure 4.

The Free energy surface (FES) of formation of the covalent protein ligand (SARS-CoV-2 M^pro 13b) complex. The square boxes near the surfaces represent the reaction coordinate at the corresponding position of the FES.