Expanding antimicrobial chemotypes: indole-based DNA gyrase inhibitors with potential dual mechanism against multidrug-resistant bacteria.

The escalating threat of multidrug-resistant (MDR) pathogens necessitates the development of antibacterial agents with novel mechanisms of action. In this study, indole-chalcones and their 4,5-dihydroisoxazole derivatives were synthesized and characterized using various analytical and spectral techniques. Single-crystal X-ray diffraction (SC-XRD) confirmed the structural integrity of the optimized compounds. Antibacterial screening against MDR-ESKAPE pathogens identified two compounds, 24 (E-3-(1-cyclopentyl-1H-indol-3-yl)-1-(2,4-dichlorophenyl)prop-2-en-1-one) and 27 (E-3-(1-cyclopentyl-1H-indol-3-yl)-1-(4-hydroxyphenyl)prop-2-en-1-one) as the most potent candidates. Notably, compound 24 exhibited the lowest minimum inhibitory concentration (MIC) of 19.53 μg/mL against both bacterial strains, demonstrating strong activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) pathogens. To elucidate the mechanistic basis of their antibacterial action, flow cytometry was conducted, revealing that compound 24 induced extensive membrane damage, resulting in over 89.8 % bacterial cell death, thereby demonstrating its potent bactericidal properties. Molecular docking analyses of compound 24 and 27 against S. aureus and P. aeruginosa DNA gyrase B revealed strong, stable interactions in comparison to Novobiocin taken as positive control, suggesting DNA gyrase inhibition. Additionally, molecular dynamics simulations supported the stability of these protein-ligand complexes, with RMSD values below 2.0 Å and consistent hydrogen-bonding patterns throughout the simulation. These in-silico predictions were experimentally validated through an in-vitro DNA supercoiling assay, which confirmed the significant suppression of gyrase activity. The combination of extensive membrane disruption and confirmed DNA gyrase inhibition establishes a dual mechanism of action for these derivatives, particularly for compound 24, which is crucial for overcoming conventional resistance pathways. Hemolytic assays indicated no cytotoxicity, positioning indole-chalcone derivatives as promising broad-spectrum antibacterial agents.