Integrated in Vitro, in Vivo, Molecular Docking, and ADMET in Silico Assessment of β-Caryophyllene as a NorA Efflux Pump inhibitor in Resistant Staphylococcus Aureus (MRSA)

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Abstract
Efflux pumps are one of the main mechanisms used by bacteria for antimicrobial resistance. These protein structures are capable of extruding antibiotics and reducing their intracellular concentrations to non-toxic levels for the bacterial cell. Thus, due to the absence of clinically approved products capable of acting as efflux pump inhibitors (EPIs), this study aimed to investigate the potential of the sesquiterpene β-caryophyllene to act as an EPI of the NorA pump present in Staphylococcus aureus strains, through in vitro, in vivo, and in silico approaches. For this purpose, in in vitro assays, the terpene was combined at a sub-inhibitory concentration with norfloxacin or ethidium bromide. In in vivo tests using the Zebrafish model, the animals were intramuscularly infected and orally treated with the terpene combined with norfloxacin. Additionally, molecular docking was performed with the NorA protein to predict ligand interactions, as well as ADME/Tox predictions. Overall, the molecular docking assays revealed that β-caryophyllene shows good affinity for the active site of the NorA protein, promoting hydrophobic interactions and hydrogen bonds with key residues such as PHE86. This was supported by in vitro assays, in which the compound was able to reduce the minimum inhibitory concentration (MIC) of norfloxacin when combined with it. This activity was also corroborated in vivo, where the association between β-caryophyllene and norfloxacin significantly reduced the number of colony-forming units (CFUs) in infected muscle tissues. ADMET property predictions demonstrated good intestinal absorption, absence of interaction with cytochrome P450 enzymes, and low hepatic toxicity; however, potential immunotoxicity and adverse effects on the blood-brain barrier (BBB) were observed. Therefore, β-caryophyllene emerged as a promising candidate for inhibiting the NorA efflux pump.

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