Unlocking bacterial defense: Exploring the potent inhibition of NorA efflux pump by coumarin derivatives in Staphylococcus aureus.

The occurrence of bacterial resistance has been increasing, compromising the treatment of various infections. The high virulence of Staphylococcus aureus allows for the maintenance of the infectious process, causing many deaths and hospitalizations. The MepA and NorA efflux pumps are transporter proteins responsible for expelling antimicrobial agents such as fluoroquinolones from the bacterial cell. Coumarins are phenolic compounds that have been studied for their diverse biological actions, including against bacteria. A pharmacokinetic in silico characterization of compounds C10, C11, C13, and C14 was carried out according to the principles of Lipinski's Rule of Five, in addition to searching for similarity in ChemBL and subsequent search for publications in CAS SciFinder. All compounds were evaluated for their in vitro antibacterial and modulatory activity against standard and multidrug-resistant Gram-positive and Gram-negative strains. The effect of coumarins C9, C10, C11, C13, and C14 as efflux pump inhibitors in Staphylococcus aureus strains was evaluated using the microdilution method (MepA or NorA) and fluorimetry (NorA). The behavior of coumarins regarding the efflux pump was determined from their interaction properties with the membrane and coumarin-protein using molecular docking and molecular dynamics simulations. Only the isolated coumarin compound C13 showed antibacterial activity against standard strains of Staphylococcus aureus and Escherichia coli. However, the other tested coumarins showed modulatory capacity for fluoroquinolone and aminoglycoside antibacterials. Compounds C10, C13, and C14 were effective in reducing the MIC of both antibiotics for both multidrug-resistant strains, while C11 potentiated the effect of norfloxacin and gentamicin for Gram-positive and Gram-negative bacteria and only norfloxacin for Gram-negative. Only coumarin C14 produced synergistic effects when associated with ciprofloxacin in MepA-carrying strains. All tested coumarins have the ability to inhibit the NorA efflux pump present in Staphylococcus aureus, both in reducing the MIC and inducing increased ethidium bromide fluorescence emission in fluorimetry. The findings of this study offer an atomistic perspective on the potential of coumarins as active inhibitors of the NorA pump, highlighting their specific mode of action mainly targeting protein inhibition. In molecular docking, it was observed that coumarins are capable of interacting with various amino acid residues of the NorA pump. The simulation showed that coumarin C10 can cross the bilayer; however, the other coumarins interacted with the membrane but were unable to cross it. Coumarins demonstrated their potentiating role in the effect of norfloxacin through a dual mechanism: efflux pump inhibition through direct interaction with the protein (C9, C10, C11, and C13) and increased interaction with the membrane (C10 and C13). In the context of pharmacokinetic prediction studies, the studied structures have a suitable chemical profile for possible oral use. We suggest that coumarin derivatives may be an interesting alternative in the future for the treatment of resistant bacterial infections, with the possibility of a synergistic effect with other antibacterials, although further studies are needed to characterize their therapeutic effects and toxicity.

Meldrum's acid derivates are MepA efflux pump inhibitors: In vitro and in silico essays.

Efflux pumps are proteins capable of expelling antibiotics from bacterial cells, have emerged as a major mechanism of bacterial resistance. In the ongoing pursuit to overcome and reduce bacterial resistance, novel substances are being explored as potential efflux pump inhibitors. Meldrum's acid, a synthetic molecule widely studied for its role in synthesizing bioactive compounds, holds promise in this regard. Therefore, the objective of this study is to evaluate the antibacterial activity of three derivatives of Meldrum's acid and assess their ability to inhibit efflux mechanisms, employing both in silico and in vitro approaches. The antibacterial activity of the derivatives was assessed using a broth microdilution testing method. Surprisingly, the derivatives did not exhibit direct antibacterial activity on their own. However, they displayed a significant effect in enhancing the efficacy of antibiotics, suggesting a potential role in potentiating their effects. Furthermore, fluorescence emission assays using ethidium bromide indicated that the derivatives could potentially block efflux pumps, as they exhibited fluorescence levels comparable to the positive control. To further investigate their inhibitory capacity, molecular docking studies were conducted in silico, revealing binding interactions similar to ciprofloxacin and carbonyl cyanide 3-chlorophenylhydrazone, known efflux pump inhibitors. These findings highlight the potential of Meldrum's acid derivatives as effective inhibitors of efflux pumps. By targeting these mechanisms, the derivatives offer a promising avenue to enhance the effectiveness of antibiotics and combat bacterial resistance. This study underscores the importance of exploring novel strategies in the fight against bacterial resistance and provides valuable insights into the potential of Meldrum's acid derivatives as efflux pump inhibitors. Further research and exploration in this field are warranted to fully exploit their therapeutic potential.

Chemical composition, antibacterial potential, and toxicity of the extracts from the stem bark of Hancornia speciosa Gomes (Apocynaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Hancornia speciosa is a medicinal plant popularly used to treat different medical issues, including infectious diseases. Exploring the therapeutic potentialities of the extracts from medicinal plants combined with conventional antibiotic drugs is a promising horizon, especially considering the rising microbial resistance. AIM OF THE STUDY: This study aimed to characterize the chemical composition of the ethereal (EEHS) and methanolic (MEHS) extracts of the stem bark of H. speciosa, and also evaluate their antibacterial and drug-modifying activity, and toxicity. MATERIALS AND METHODS: The extracts were characterized by gas chromatography coupled to mass spectrometry (GC-MS). Additionally, total phenol and flavonoid contents were determined. The antibacterial and antibiotic-modifying activity was evaluated against strains of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa using the serial microdilution method, obtaining the minimum inhibitory concentration (MIC). The toxicity assay was carried out using the Drosophila melanogaster model. RESULTS: Thirty compounds were identified in the extracts of the stem bark of H. speciosa, with triterpenoids being predominant in both extracts. Additionally, fatty alcohols, carbohydrates, fatty acids, phenolic acids, and phytosterols were identified in both extracts. EEHS and MEHS extracts had considerable phenol contents (346.4 and 340.0 mg GAE/g, respectively). Flavonoids were detected in a lower proportion (7.6 and 6.9 mg QE/g, respectively). H. speciosa extracts did not display intrinsic antibacterial activity against the bacterial strains evaluated, however, they were capable of modifying the activity of gentamicin, erythromycin, and norfloxacin. EEHS increased the efficacy of norfloxacin against E. coli and S. aureus, reducing MIC values by 50%. MEHS potentiated the action of gentamicin against all bacterial strains, especially against E. coli. The extracts did not display toxicity at clinically relevant concentrations against D. melanogaster. CONCLUSION: The stem bark of H. speciosa was considered a rich source of bioactive compounds. Our findings evidenced the therapeutic potential of H. speciosa extracts for the development of new pharmaceutical therapeutics against bacteria. Although the extracts did not exhibit intrinsic antibacterial activity, they enhanced the efficacy of commercial antibiotic drugs and were non-toxic at clinically relevant concentrations. Future studies are needed to elucidate the mechanisms of action of these extracts, ensuring their safety and efficacy.