Efeito do p-cumarato de 4-clorobenzila sobre bactérias multirresistentes: atividade sobre biofilme, modo de ação, inibição de bombas de efluxo, modulação de resistência bacteriana e avaliação toxicológica

Abstract

Bacterial infections, particularly those caused by multidrug-resistant strains, represent a major global public health challenge, demanding the continuous development of novel therapeutic strategies and remaining a priority focus in scientific research. Therefore, the present study aimed to evaluate the antibacterial effect of 4-chlorobenzyl p-coumarate, its toxicity, as well as to investigate its mode of action. The compound was tested for activity against bacterial strains in both planktonic and biofilm forms, toxicity through in silico approaches, in vitro assay on human gingival fibroblasts and in vivo assay in Galleria mellonella larvae, its mechanism of action and properties related to efflux pump inhibition and modulation of antibiotic activity. Pharmacokinetic predictions were estimated through computational analyses, and potential molecular targets, as well as interactions with efflux mechanisms, were verified by molecular docking and molecular dynamics simulations. Following an initial screening against three Gram-positive and five

Gram-negative strains, the compound exhibited antibacterial activity against Gram- positive bacteria, including methicillin-resistant Staphylococcus aureus (MIC = 8

μg/mL; 27.77 μM) and Enterococcus faecalis (MIC = 16 μg/mL; 55.54 μM), leading to bacterial elimination within 24 h of exposure. Toxicological evaluations yielded promising results, showing low cytotoxicity up to 16 μg/mL (55.54 μM) in experimental models and low systemic toxicity at all tested concentrations. Pharmacokinetic predictions suggested a favorable profile, including suitability for oral administration. The compound reduced bacterial biofilm biomass by 71% at 4xMIC and penetrated the extracellular matrix to reach cells within the structure, compromising its architecture. The molecule demonstrated higher binding affinity and complex stability with the membrane-associated enzyme phosphatidylglycerol phosphate synthase (−113.343 kJ·mol 1), in addition to exhibiting lower residue − fluctuations and greater structural flexibility with NAD -dependent DNA ligase (<0.3 + nm). Molecular interactions were also observed with the efflux proteins MepA and NorA, and experimentally, the compound inhibited these pumps (ethidium bromide – MepA: MIC from 32 to 0.62 μg/mL; NorA: MIC from 64 to 0.50 μg/mL), enhancing antibiotic activity (ciprofloxacin MIC from 32 to 0.70 μg/mL; norfloxacin MIC from 128 to 8 μg/mL). The compound induced morphological alterations on both the surface and the interior of bacterial cells, culminating in cell lysis, and reduced intracellular DNA content. Collectively, these findings support the identification of a novel compound with potential to inhibit multidrug-resistant Gram-positive bacteria, including biofilm-related effects. They also suggest that the molecule may exert a dual mechanism of action by interfering with both phosphatidylglycerol synthesis and DNA replication, while additionally displaying inhibitory and modulatory effects on efflux pumps. Furthermore, 4-chlorobenzyl p-coumarate exhibited favorable toxicological parameters, supporting its consideration in translational research as a

promising pharmacological alternative to combat infections caused by multidrug- resistant Gram-positive bacteria.