The growing spread of NDM-1–producing bacteria represents a critical threat to the clinical efficacy of β-lactam antibiotics, including carbapenems, which are considered last-resort drugs. The continuous emergence of resistant strains underscores the urgent need to identify novel inhibitors capable of restoring the effectiveness of these antibiotics. In this study, a comprehensive computational protocol was applied to evaluate the inhibitory potential of four candidate compounds (M1, M2, P3, and P4) against the metallo-β-lactamase NDM-1. The workflow included 100 ns molecular dynamics simulations to explore the conformational stability of each complex, complemented by structural stability analyses (RMSD, RMSF), residue–ligand distance monitoring, and assessment of hydrogen bond evolution. Binding free energy calculations using the MM/GBSA approach were further conducted to provide thermodynamic insights into ligand affinity and complex stability.

The results demonstrated that compounds M2 and P4 exhibited superior dynamic behavior, maintaining stronger and more persistent interactions with catalytically relevant residues of the enzyme. These findings were reinforced by more favorable mean ΔG_bind values (–13.56 and –16.72 kcal/mol, respectively), suggesting robust binding affinity. Additionally, structural superimpositions over simulation time revealed distinct adaptive capacities of the ligands to the catalytic pocket, highlighting potential differences in their inhibitory mechanisms. Overall, the study positions M2 and P4 as promising scaffolds for the rational development of next-generation NDM-1 inhibitors. Moreover, the findings validate the utility of multivariate in silico approaches that integrate molecular dynamics, free energy calculations, and interaction mapping as powerful tools to prioritize candidates for experimental testing, ultimately supporting innovative strategies to counteract bacterial resistance.

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