Clofoctol-derived amphiphilic peptidomimetics exhibit broadened antimicrobial activity via multiple contributing mechanisms.

INTRODUCTION: Antimicrobial resistance (AMR) poses an escalating global health threat, yet the antibiotic discovery pipeline has stagnated over recent decades. Multi-mechanistic antibacterial action may offer a promising strategy to address this challenge. OBJECTIVES: This study aimed to use clofoctol, a clinically used antibacterial agent, as the starting scaffold for cationic amphiphilic modification to generate derivatives with improved aqueous solubility, broadened antibacterial spectrum, and enhanced selectivity, followed by mechanistic characterization of the lead compound. METHODS: Minimum inhibitory concentration (MIC) and hemolysis assays were performed to assess structure-activity relationship of clofoctol derivatives. The lead compound was then systematically evaluated for bactericidal kinetics, resistance development, antibiofilm activity, salt tolerance, mammalian cytotoxicity, and therapeutic efficacy in a murine keratitis model. To investigate the mechanisms, we evaluated membrane perturbation, topoisomerase IV-mediated DNA decatenation, intracellular reactive oxygen species (ROS) and adenosine triphosphate (ATP) quantification, transcriptomic analysis, and RT-qPCR validation. RESULTS: 30 was identified as the lead candidate, displaying improved apparent aqueous solubility, broadened activity against Gram-positive and Gram-negative bacteria (MICs = 0.39-3.125 µg/mL), favorable selectivity index and therapeutic index (TI = 272.5) relative to clofoctol. Furthermore, 30 exhibited rapid bactericidal kinetics, good salt tolerance, and low propensity for resistance development. In a murine keratitis model, 30 reduced bacterial burden by 4.65 and 3.81 log CFU in S. aureus ATCC29213- and P. aeruginosa ATCC9027- induced infections, respectively. These reductions were comparable to those achieved by vancomycin and gatifloxacin under the same conditions, and no observable ocular toxicity was detected. Mechanistic investigation supported membrane perturbation as the best-supported antibacterial component. Compound 30 exposure was also associated with intracellular ATP depletion, ROS accumulation, and in vitro inhibition of topoisomerase IV-mediated DNA decatenation. CONCLUSION: These results identify 30 as a promising antimicrobial agent with broadened antibacterial activity, favorable preliminary safety profiles, and multiple contributing antibacterial mechanisms.