Reduction mechanism of XOR-mediated nitroaromatics hypoxia targeted AGT inhibitors: Molecular docking, MD simulation and ONIOM (QM/MM) investigation

Hypoxia-activated nitroaromatic compounds represent a promising strategy for developing tumor-targeted O⁶-alkylguanine-DNA alkyltransferase (AGT) inhibitors, crucial for enhancing alkylating agent efficacy in cancer therapy. In this study, we investigated the reduction mechanisms of two hypoxia-activated AGT inhibitors, 2-nitro-6-benzyloxypurine (2-NBP) and O⁶-(3-nitro)benzylguanine (3-NBG), mediated by xanthine oxidoreductase (XOR) using molecular docking, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations. Docking revealed that both inhibitors bind XOR's active site, with their nitroaromatic rings stacking over the flavin's isoalloxazine ring, mainly through hydrophobic interactions. MD simulations revealed 2-NBP bound XOR more favorably than 3-NBG. QM/MM calculations elucidated the individual reduction mechanisms of both inhibitors, involving six 1e⁻/1H⁺ transfers. The key differences lay in the lower energy barriers for 2-NBP in the first, third, and sixth steps compared to 3-NBG. Combined with MD simulation results, the QM/MM computations demonstrated that 2-NBP was more readily reduced by XOR than 3-NBG, despite the rate-limiting step (the fifth 1e⁻/1H⁺ transfer) of 3-NBG reduction exhibiting slightly more favorable in kinetics and thermodynamics than 2-NBP. Additionally, a water molecule in the active site was found to facilitate the second 1e⁻/1H⁺ transfer, reducing the energy barrier significantly. This work provided a theoretical basis for designing tumor-targeted AGT inhibitors.