Discrete Dynamics of Warhead Modulation on Covalent Inhibition of Oxyr: A QM/MM Study

The bacterial transcriptional factor OxyR, a peroxide sensor conserved in bacterial virulence pathways, has the capability to exhibit exceptional reactivity toward hydrogen peroxide (H₂O₂). H₂O₂ is essential for oxidizing cysteine thiolates to maintain cellular redox homeostasis and is dispensable for bacterial growth that can potentially mitigate drug resistance, thus underlining OxyR as a valuable target. We employ quantum mechanics/molecular mechanics (QM/MM) umbrella sampling (US) simulations at the DFTB3/MM level of theory and propose a reaction mechanism with four potential covalent inhibitors. The potential of mean force reveals the direct role of intrinsic reactivity of inhibitors, for instance, benzothiophenes and modified experimental inhibitors with methyl oxo-enoate warhead-activated carbonyl samples in the first step of reaction, which shed light on the significance of proton transfer indispensable for full inhibition, whereas the nitrile inhibitor undergoes a stepwise mechanism with a small proton-transfer energy barrier and lower imaginary frequencies that materialize instantly after nucleophilic attack. To unveil the molecular determinants of respective binding affinities, transition states along the reaction path are optimized and characterized with B3LYP 6-31+G(d,p). Furthermore, the post-simulation analysis indicates the catalytic triad (His130/Cys199/Thr129), thermodynamically favored for inhibition, which restricts water molecules from acting as the potential source of protonation/deprotonation. This study thus serves as a preamble to add variation in the proposed structures and unveils the impact of functional groups lying in warheads that modulate the kinetics of proton transfer, which will certainly aid to design more selective and efficient irreversible inhibitors of OxyR.