Carla Araya-Cloutier

Background: The overexpression of the NorA efflux pump is known to be an important factor in the antimicrobial resistance mechanism of Staphylococcus aureus . Therefore, NorA inhibition can help disarm this pathogen and tackle antimicrobial resistance. In this study, we aim to unravel the molecular properties of prenylated (iso)flavonoids from Fabaceae as potential NorA inhibitors and to propose leading compounds for future research. Methods: A collection of 37 prenylated isoflavonoids and flavonoids (obtained by purification, chemically synthesized, or commercially purchased), collectively referred to as (iso)flavonoids, was evaluated for its activity against norA -overexpressing Staphylococcus aureus using the checkerboard assay with ciprofloxacin (NorA substrate) and erythromycin (primarily non-NorA substrate), in combination with ethidium accumulation assays. Moreover, a norA -knockout Staphylococcus aureus strain was used to corroborate the specificity of the observed effects. Subsequently, in silico binary QSAR and pharmacophore models were developed to elucidate the key molecular properties for potential NorA inhibition. Results: Seven prenylated (iso)flavonoids, namely, 8-prenylnaringenin, 6- C ,7- O -diprenylnaringenin, glabrene, neobavaisoflavone, wighteone, licoisoflavone A, and glycyrrhisoflavone, potentiated ciprofloxacin up to 8-fold in norA- overexpressing and up to 2-fold in norA- knockout strains at 10 μM, without any membrane permeabilization effects. Moreover, prenylated (iso)flavonoids potentiated erythromycin in norA- overexpressing Staphylococcus aureus only up to 2-fold. Binary QSAR models were generated using datasets from the checkerboard and ethidium accumulation assays with a total prediction accuracy of up to 90% for active and 88% for inactive compounds. Based on QSAR models, the polar surface area, the balance of hydrophobicity and hydrophilicity, and the overall hydrophobicity were correlated with antibiotic potentiation and efflux inhibition of prenylated (iso)flavonoids. Moreover, in our study, we revealed that fractional negative polar surface area and formal (negative) charges are key properties that differentiate prenylated (iso)flavonoids with antimicrobial activity from those that act as potential NorA inhibitors. A pharmacophore model provided the basis for further optimization of prenylated (iso)flavonoids, mainly neobavaisoflavone and wighteone, as potential NorA inhibitors. Conclusion: In our study, we provide, for the first time, predictive QSAR models of prenylated (iso)flavonoids as potential NorA inhibitors and propose two potential leads based on this family of plant-derived compounds. Future research on the specificity and validation of prenylated (iso)flavonoids as NorA inhibitors is required.

Prenylated phenolics are plant-derived compounds with antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), acting by targeting membranes resulting in fast permeabilization. Studies quantifying their membrane permeabilization capacity are lacking, limiting our understanding of the structural properties driving this effect. This study evaluated antimicrobial activity and permeabilization efficacy of 36 C- and O-prenylated phenolics, including 11 C- and O-prenylated phenolics chemically synthesized for this study. Minimum inhibitory concentrations (MICs) were obtained using the broth microdilution assay. Membrane permeabilization was measured by propidium iodide uptake using fluorescence spectrometry and microscopy. The most potent MRSA permeabilizers were luteone (29) and neobavaisoflavone (22), with EC₁₀ of 27 ± 7 and 28 ± 8 μg mL^-1, respectively. Diprenylated phenolics showed a strong negative correlation between permeabilization and their hydrophobic-to-polar surface area ratio (r_pearson = 0.88). For monoprenylated phenolics, prenyl configuration (chain) and molecular shape (globular) were important for effective permeabilization. Interestingly, potency of antimicrobial prenylated phenolics (MIC ≤ 50 μg mL^-1) was not correlated to permeabilization potency, suggesting other mechanisms of action in addition to membrane permeabilization. These quantitative findings on membrane permeabilization by prenylated phenolics contribute to our mechanistic understanding of how these compounds can inhibit microbial growth.

Given the negative side effects and increased resistance associated with conventional oral antimicrobials, prenylated phenolics are promising alternatives to combat the oral pathogen Streptococcus mutans. A total of 40 prenylated phenolics were evaluated for their antibacterial activity against S. mutans. Eleven prenylated phenolics were purified from licorice (Glycyrrhiza spp.) spent, including the newly discovered double prenylated 2-arylbenzofuran, isokanzonol V. Twenty-eight compounds showed similar potency as chlorhexidine. Activity was correlated with structural properties and novel structure–activity relationships were established: (i) Hydrophobic volume was the main driver associated to activity (double prenylated phenolics were consistently more active than single prenylated phenolics); (ii) the conversion of flavanones to chalcones by C-ring opening increases molecular length and planarity as well as antibacterial activity, and (iii) active single prenylated isoflavones have balanced molecular hydrophilicity. Hence, double or single prenylated phenolics featuring planarity, an extended configuration, and balanced hydrophilicity hold promise for S. mutans control.

Prenylated isoflavonoids have shown remarkable antimicrobial activity. Previous studies showed that the antimicrobial compounds glabridin and wighteone induced membrane permeabilization in microbial cells. Other compounds, such as lupiwighteone, showed no antimicrobial activity. In this study, the permeabilization efficacy and interaction with lipid bilayers of glabridin, wighteone, and lupiwighteone were assessed in vitro and in silico using model membranes. Permeabilization of liposomes by glabridin and wighteone confirmed the lipid bilayer as the primary target. Notably, lupiwighteone did not induce the permeabilization of liposomes. Molecular dynamics (MD) simulations were used to study the interaction of these compounds with phospholipid membranes. The calculated potential of mean force profiles for the three molecules correlated with liposome permeabilization, indicating a favorable intercalation inside the lipid bilayer for wighteone, followed by glabridin, and an unfavorable intercalation for lupiwighteone. Additionally, MD simulations indicated that the location of glabridin and wighteone in the membrane was just below the head groups. Furthermore, this study underscored the importance of partitioning between polar and hydrophobic areas for prenylated isoflavonoids, which conceivably determines the membrane insertion and, subsequently, the antimicrobial activity. Overall, this study showed that interactions with and permeabilization of the lipid bilayer are key factors for the antimicrobial activity of these compounds.

Overexpression of NorA efflux pumps plays a pivotal role in the multidrug-resistance mechanism in S. aureus. Here, we investigated the activities of prenylated isoflavonoids, present in the legume plant family (Fabaceae), as natural efflux pump inhibitors (EPIs) in fluoroquinolone-resistant S. aureus. We found that four prenylated isoflavonoids, namely neobavaisoflavone, glabrene, glyceollin I, and glyceollin III, showed efflux pump inhibition in the norA overexpressing S. aureus. At sub-inhibitory concentrations, neobavaisoflavone (6.25 µg/mL, 19 µM) and glabrene (12.5 µg/mL, 39 µM), showed up to 6 times more Eth accumulation in norA overexpressing S. aureus than in the control. In addition, these two compounds boosted the MIC of fluoroquinolones up to eightfold. No fluoroquinolone potentiation was observed with these isoflavonoids in the norA knockout strain, indicating NorA as the main target of these potential EPIs. In comparison to the reported NorA EPI reserpine, neobavaisoflavone showed similar potentiation of fluoroquinolone activity at 10 µM, higher Eth accumulation, and less cytotoxicity. Neobavaisoflavone and glabrene did not exhibit membrane permeabilization effects or cytotoxicity on Caco-2 cells. In conclusion, our findings suggest that the prenylated isoflavonoids neobavaisoflavone and glabrene are promising phytochemicals that could be developed as antimicrobials and resistance-modifying agents to treat fluoroquinolone-resistant S. aureus strains.

A novel 3D-apical-out mouse enteroid model to investigate effects on the intestinal epithelium, including cytotoxicity, cell viability, and biotransformation. Evaluating the impact of prenylated phenolics from licorice (Glycyrrhiza) roots.