Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease

Abstract

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nu-cleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored Nδ (HD) and Nϵ (HE) protonation of His41 and His164, respectively, the α-ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

Direct Observation of Protonation State Modulation in SARS-CoV-2 Main Protease upon Inhibitor Binding with Neutron Crystallography

“…Mutual exclusivity of telaprevir’s hemithioketal and His41 Nε2 states reduce this value by 2 to 52 in the telaprevir complex, but combinational possibilities would multiply if, unlike telaprevir, an inhibitor has additional chemical groups ionizable at physiological pH. Furthermore, we note that the exact protonation state combinations of the ionizable residues that we observed in the active-site cavities of ligand-free 46 and ketoamide inhibitor-bound SARS-CoV-2 M pro have not been predicted by molecular simulations, 56 emphasizing the critical importance of experimentally determining the locations of H atoms. As a result, the design of covalent inhibitors against SARS-CoV-2 M pro should consider the observed protonation state changes of the ionizable residues in the active-site cavity triggered by inhibitor binding.…”

“…Knowledge of where H atoms relocate due to inhibitor binding can provide critical information on how protonation states and thus electric charges are modulated in the protein active-site cavity, improving rational drug design. For example, molecular dynamics simulations have predicted that protonation states in the SARS-CoV-2 M pro active-site cavity may be altered when an inhibitor binds, 56 but experimental evidence has been lacking.…”

Covalent and non-covalent binding free energy calculations for peptidomimetic inhibitors of SARS-CoV-2 main protease

“…[32][33][34][35] The availability of these structures have enabled structure-based modeling approaches in an effort to understand key inhibitor-receptor interactions, conformational dynamics, and structural stability of the binding domain of the M pro . [36][37][38][39][40][41][42][43][44] A principal goal of computational drug design is the ability to accurately and reliably predict the binding mode, selectivity, and affinity of a drug candidate towards a target. Alchemical absolute binding free energy (ABFE) calculations are suitable in this regard and provide a theoretically rigorous way of computing ligand binding energies of disease targets.…”

Tuning Proton Transfer Thermodynamics in SARS-CoV-2 Main Protease: Implications for Catalysis and Inhibitor Design

“…Information on the catalytic dyad configurations and interactions is provided in the SI , and a more extensive structural analysis of the MD simulations can be found in our previous work. 32 Then, by applying the MD-PMM approach, we computed at each MD frame for each simulation ensemble the time evolution of the energy change upon PT that provides the reaction free energy Δ G 0 (see eq 6 in the SI ).…”