Integrated computational and experimental evaluation of natural products targeting PGM/AlgC and HisIE in multidrug-resistant respiratory pathogens.

Background: The increasing prevalence of multidrug-resistant (MDR) Gram-negative respiratory pathogens has reduced the effectiveness of conventional antimicrobial therapies and created a need for antibacterial strategies targeting bacterial metabolic and virulence-associated functions. AlgC, a phosphomannomutase/phosphoglucomutase involved in cell-envelope and exopolysaccharide biosynthesis, and the bifunctional histidine-biosynthesis enzyme HisIE represent potentially relevant bacterial targets. Objective: This study evaluated the antibacterial and enzyme-inhibitory activities of three natural products, 5-hydroxysophoranone, 10'-desmethoxystreptonigrin, and isoforsythiaside-and investigated their predicted interactions with AlgC and HisIE using an integrated computational and Methods: Molecular docking was performed to examine the interactions of the selected compounds with the predicted catalytic or ligand-binding regions of AlgC and HisIE. Two lead protein-ligand complexes were subsequently evaluated using 500-ns molecular dynamics simulations and MM/GBSA analysis. Antibacterial activity against MDR respiratory isolates was assessed using broth dilution and disc-diffusion assays. Cytotoxicity was examined in A549 and BEAS-2B epithelial cells using the MTT assay. Cell-associated antibacterial activity, recombinant-enzyme inhibition, Western blotting, and quantitative real-time PCR were used to assess antibacterial effects and changes in selected metabolic and virulence-associated markers. Results: The selected natural products showed favorable predicted interactions with the investigated AlgC and HisIE binding regions. Among the tested compounds, 10'-desmethoxystreptonigrin exhibited the strongest predicted interaction with AlgC and showed the lowest minimum inhibitory concentrations, the greatest reduction in cell-associated bacterial burden, and the strongest inhibition of the recombinant enzymes. The two simulated lead complexes retained generally stable protein-ligand interactions during the molecular dynamics trajectories. Treatment with the compounds was also associated with reduced expression of selected metabolic and virulence-associated markers, including pgm/algC, hisIE, lasR, pslA, and ompA. Isoforsythiaside demonstrated comparatively greater epithelial-cell compatibility, whereas 10'-desmethoxystreptonigrin showed greater antibacterial potency but lower host-cell tolerance. Conclusion: The combined computational and experimental findings identify 10'-desmethoxystreptonigrin and 5-hydroxysophoranone as candidate natural-product scaffolds with antibacterial and recombinant AlgC/HisIE-inhibitory activities. The observed enzyme inhibition and changes in selected molecular markers are consistent with possible interference in AlgC- and HisIE-associated processes; however, they do not by themselves establish direct intracellular target engagement or pathway-specific causality. Further studies using target-engagement assays, genetically defined bacterial strains, broader clinical-isolate panels, biofilm and quorum-sensing assays, and