Water Plays a Cocatalytic Role in Epoxide Ring Opening Reaction in Aspartate Proteases: A QM/MM Study

Abstract

Aspartate proteases are potential targets for various diseases, and many of their inhibitors are FDA-approved drugs. However, these peptidomimetic and reversibly bound drugs become ineffective upon prolonged use. Attempts have been made to design and synthesize various nonpeptidic epoxide-based irreversible inhibitors to combat the drug-resistance enigma.Here, we study the mechanism of epoxide ring opening in two widely studied aspartate proteases, HIV-1 protease and pepsin. Our results from QM/MM molecular dynamics show that the epoxide ring opening in aspartate proteases follow a two-step mechanism with the formation of an oxyanion intermediate, stabilized by a set of water molecules in the protein active site. These water molecules by virtue of "low-barrier hydrogen bonds" with the epoxide ring reduce the intrinsic reaction barrier while remaining structurally unperturbed, thus playing a cocatalytic role. We validated our results by reproducing the experimentally observed protease/pepsin−epoxide covalent complexes as end products. The observed stability of our oxyanion intermediate in a four-water-coordinated state is also consistent with the reported stable state of the hydroxide ion in water as OH − (H 2 O) 4 . Our study could pave the way for the design of new class "HIV protease irreversible inhibitors" from the acquired knowledge of the structures of intermediate and transition states traced during the explored reaction mechanism.

Mechanistic and thermodynamic characterization of oxathiazolones as potent and selective covalent immunoproteasome inhibitors

“…These shortcomings of the computed free energies can be attributed to the inaccuracies of the approximate DFTB3 method. Several studies have shown [48] , [49] , [50] that QM/MM free energy calculations using DFTB3 for the QM region can be efficiently corrected to obtain free energy profiles in accordance with experimental data.…”

Mechanisms of Proteolytic Enzymes and Their Inhibition in QM/MM Studies

“…A recent comparative study of HIV-1 protease and pepsin investigated the mechanism of the epoxide ring opening in an covalent inhibitor, employing the semi empirical selfconsistent charge density functional tight binding (SCC-DFTB, DFTB3) quantum method in a hybrid QM/MM approach [54]. The calculations suggested a two-step mechanism for the epoxide ring opening and an unexpected oxyanion intermediate, which was stabilized by four co-catalytic water molecules in the protein active site, which had been identified in a previous study on HIV protease inhibitor bonds, using an electron localization function (ELF) based on DFT [55].…”

Mechanism of darunavir binding to monomeric HIV-1 protease: a step forward in the rational design of dimerization inhibitors

“…To trace the binding pathway of DRV and to calculate the free energy of binding, we used umbrella sampling starting from an initial unbound state to attain the DRV bound mPR complex. 8,47 The initial unbound state was created by manually placing the ligand at an adequately far distance such that the contacts between the protein and ligand were zero. The RMSD of separated DRV and mPR with respect to our modelled DRV-mPR structure is considered as the reaction coordinate to generate the free energy profile (Fig.…”

“…We also calculated the energetics of DRV binding to the monomeric HIVPR through the potential of mean force (PMF) calculations using umbrella sampling. 8,47 The sampling was done by varying the DRV-mPR distance from 6 Å to 0 Å with a bin size of 0.1 Å and force constant of 50 kcal mol −1 Å −1 . Each window was simulated for 10 ns and the last 5 ns was used for data analysis.…”