Tuncay Karakurt, Neslihan Özbek
The emergence of carbapenem-resistant Klebsiella pneumoniae highlights the need to evaluate candidate compounds in terms of both KPC-2 recognition and behavior at the Gram-negative outer membrane barrier. Here, the clinically used cyclic boronate inhibitor vaborbactam and two newly synthesized piperazine-hydrazone derivatives, M1 and M2, were compared using molecular docking, replicate atomistic molecular dynamics simulations, MM/PBSA analysis, umbrella-sampling-derived potential of mean force profiles and preliminary phenotypic antibacterial screening. Docking suggested that M2 adopted an initial KPC-2 binding orientation more similar to that of vaborbactam than did M1, whereas replicate MD simulations revealed substantial trajectory-dependent variability and did not support a definitive stability ranking. MM/PBSA analysis showed a more favorable mean enthalpy-like contribution for M2 than for M1; the mean value for M2 approached that obtained for vaborbactam but exhibited greater energetic heterogeneity. In the porin-free membrane model, vaborbactam, M1 and M2 showed ligand-specific inward PMF increases of approximately 14.2, 33.4 and 52.25 kcal/mol, respectively, although these independently referenced profiles should not be interpreted as strictly endpoint-matched permeation barriers. M1 retained substantial hydration and predominantly sampled Lipid A/core contacts, whereas M2 showed pronounced reorientation and a larger inner-leaflet contact population during the externally driven pathway. In preliminary antibacterial screening against K. pneumoniae ATCC 13883, M2 produced a marginally larger inhibition zone than did M1, while both compounds showed identical MIC endpoints. Because KPC-2 production was not established and direct enzyme-inhibition or meropenem-combination assays were not performed, the experimental data do not demonstrate KPC-2 inhibition or superior whole-cell activity. M2 is therefore prioritized for mechanistic follow-up based on its comparatively favorable computational KPC-2 interaction profile rather than on its status as a validated antibacterial candidate.