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

Abstract

The catalytic reaction in SARS-CoV-2 main protease is activated by a proton transfer (PT) from Cys145 to His41. The same PT is likely also required for the covalent binding of some inhibitors. Here we use a multiscale computational approach to investigate the PT thermodynamics in the apo enzyme and in complex with two potent inhibitors, N3 and the α-ketoamide 13b . We show that with the inhibitors the free energy cost to reach the charge-separated state of the active-site dyad is lower, with N3 inducing the most significant reduction. We also show that a few key sites (including specific water molecules) significantly enhance or reduce the thermodynamic feasibility of the PT reaction, with selective desolvation of the active site playing a crucial role. The approach presented is a cost-effective procedure to identify the enzyme regions that control the activation of the catalytic reaction and is thus also useful to guide the design of inhibitors.

Characterization of the non-covalent interaction between the PF-07321332 inhibitor and the SARS-CoV-2 main protease

“…MD simulations were carried out for the Chymotrypsin-like catalytic domain I + II [ 22 , 41 ] of the two isoforms using, in turn, the Amber99sb-ildn [ 42 ] and the OPLS-AA/M FFs [ 32 ] with the GROMACS code [ 43 ] (version 2018.8). In the H–C isoform, H41 was assigned to the tautomer with the protonated N δ [ 25 ]. The potential parametrization for the PF-07321332 ligand was generated using two web interfaces: PrimadORAC [ 44 ] for AMBER FF and LigParGen [ 45 ] for OPLS-AA FF.…”

“…Most recently, a thorough MD study on the dimer [ 24 ] showed that the ion-pair configuration of the dyad is not compatible with a catalytically competent binding mode for peptide substrates. Substrate docking on the 3CL pro with the dyad in neutral form is hence supposed to favor the CYS to HIS PT [ 25 ] preceding the acylation step in the catalysis.…”

“…Recently, the catalytic mechanism in 3CL pro was investigated using hybrid QM/MM multi-scale hybrid methods [ 25 , 26 ]. Both these studies agree on the fact that in the apo (unligated) form, the neutral state for the dyad is significantly more stable than the zwitterionic state.…”

“…Regarding the role of the ligand in the holo form, however, these studies strikingly predict an opposite effect on the ion-pair state, stabilising in Ref. [ 25 ] and destabilising in Ref. [ 26 ] One weak point of these QM/MM approaches for the holo forms is that the latter are prepared [ [25] , [26] , [27] ] starting from the X-ray structures where the ligand is already covalently bound to CYS and that were presumably obtained from protein stock pre-incubated with the ligand.…”

“…[ 25 ] and destabilising in Ref. [ 26 ] One weak point of these QM/MM approaches for the holo forms is that the latter are prepared [ [25] , [26] , [27] ] starting from the X-ray structures where the ligand is already covalently bound to CYS and that were presumably obtained from protein stock pre-incubated with the ligand. The X-ray structure, on the other hand, is not necessarily similar to the actual initial non covalent bonding pose as the transition state involving the substrate and the binding site must be the result of a concerted process with a C145 to H41 PT transfer induced by the ligand non covalent docking, followed by a structural modification involving both the ligand and the nearby residues (including the oxyanion hole [ 28 ]) to arrive at the covalently bonded form seen in the ligand incubated samples.…”