Primin inhibits New Delhi metallo-β-lactamase-5 and restores meropenem activity in Escherichia coli.

Abstract

Background: New Delhi metallo-β-lactamase-5 (NDM-5)-producing Escherichia coli is a major driver of carbapenem resistance, and clinically effective metallo-β-lactamase inhibitors remain unavailable. Primin (PRI), a benzoquinone isolated from Primula obconica, is a plant-derived compound with potential bioactivity. Because meropenem (MEM) is a clinically important carbapenem that is readily hydrolyzed by NDM-5, it was selected as the partner antibiotic to determine whether PRI could restore its antibacterial activity.

Purpose: This study aimed to evaluate PRI as an NDM-5 inhibitor and to determine whether it could restore the activity of MEM.

Methods: Structure-based virtual screening was performed against NDM-5 using the crystal structure (Protein Data Bank entry 4TZE) and a natural compound library. Enzymatic inhibition assays, minimum inhibitory concentration determination, checkerboard assays for fractional inhibitory concentration index calculation, and time-kill assays were conducted using an NDM-5-producing E coli strain. Bio-layer interferometry, molecular docking, molecular dynamics simulations with molecular mechanics Poisson-Boltzmann surface area analysis, and site-directed mutagenesis (Met67Ala and Phe70Ala) were applied to characterize binding interactions. A murine infection model was used to evaluate in vivo efficacy.

Results: PRI inhibited NDM-5 activity in a dose-dependent manner (IC₅₀ = 16.8 ± 0.3 μM) and showed no significant antibacterial activity alone. In combination with MEM, PRI exhibited strong synergistic effects (fractional inhibitory concentration index = 0.125) and enhanced bactericidal activity. Binding analyses demonstrated that PRI interacts with the catalytic pocket of NDM-5, involving residues Met67 and Phe70. Mutations at these residues reduced binding affinity and inhibitory activity. In vivo, PRI combined with MEM reduced bacterial burden, attenuated inflammation, and alleviated tissue injury.

Conclusions: PRI, a plant-derived benzoquinone, inhibits NDM-5 and restores MEM activity against Escherichia coli. These findings suggest that PRI is a promising lead compound for developing metallo-β-lactamase inhibitors and antibiotic adjuvants.