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.

In vitro and in silico evidences about the inhibition of MepA efflux pump by coumarin derivatives.

The discovery of antibiotics has significantly transformed the outcomes of bacterial infections in the last decades. However, the development of antibiotic resistance mechanisms has allowed an increasing number of bacterial strains to overcome the action of antibiotics, decreasing their effectiveness against infections they were developed to treat. This study aimed to evaluate the antibacterial activity of synthetic coumarins Staphylococcus aureus in vitro and analyze their interaction with the MepA efflux pump in silico. The Minimum Inhibitory Concentration (MIC) determination showed that none of the test compounds have antibacterial activity. However, all coumarin derivatives decreased the MIC of the standard efflux inhibitor ethidium bromide, indicating antibacterial synergism. On the other hand, the C14 derivative potentiated the antibacterial activity of ciprofloxacin against the resistant strain. In silico analysis showed that C9, C11, and C13 coumarins showed the most favorable interaction with the MepA efflux pump. Nevertheless, due to the present in silico and in vitro investigation limitations, further experimental research is required to confirm the therapeutic potential of these compounds in vivo.

In vitro and in silico antibacterial evaluation of coumarin derivatives against MDR strains of Staphylococcus aureus and Escherichia coli.

The increase in antibiotic resistance rates has attracted the interest of researchers for antibacterial compounds capable of potentiating the activity of conventional antibiotics. Coumarin derivatives have been reported to develop effective antibacterials with possible new mechanisms of action for treating infectious diseases caused by bacteria with a profile of drug resistance. In this context, the aim of the present study we have now prepared one variety of new synthetic coumarins evaluating the pharmacokinetic and chemical similarity in silico, their antimicrobial activity against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), and potential for the modulation of antibiotic resistance against Staphylococcus aureus (SA10) and Escherichia coli (EC06) clinical isolate bacteria by in vitro assay. The antibacterial activity and antibiotic-enhancing properties were evaluated by the broth microdilution method and pharmacokinetically characterized according to the Lipinsk rule of 5 and had their similarity analyzed in databases such as ChemBL and CAS SciFinder. The results demonstrated that only compound C13 showed significant antibacterial activity (MIC ≤256 µg/mL), and all other coumarins did not display relevant antibacterial activity (MIC ≥1024 µg/mL). However, they did modulate the antibiotics activities to norfloxacin and gentamicin, except, compound C11 to norfloxacin against Staphylococcus aureus (SA10). The in silico properties prediction and drug-likeness results demonstrated that all coumarins presented a good drug-likeness score with no violations and promising in silico pharmacokinetic profiles showing that they have the potential to be developed into an oral drug. The results indicate that the coumarin derivatives showed good in vitro antibacterial activity. These new coumarin derivatives also demonstrated the capacity to modulate antibiotic resistance with potential synergy action for current antimicrobials assayed, as antibiotic adjuvants, to reduce the emergence of antimicrobial resistance.

Anti-inflammatory activities of the hydroalcoholic extracts from Erythrina velutina and E. mulungu in mice

This work studied the anti-inflammatory activities of the hydroalcoholic extracts (HAEs) from Erythrina velutina Willd. (Ev) and E. mulungu Mart. ex Benth. (Em) in the carrageenan- and dextran-induced mice hind paw edema models. These medicinal plants belonging to the Fabaceae family are used in some Brazilian communities to treat pain, inflammation, insomnia and disorders of the central nervous system. In the present work, the extracts were administered orally in male mice at the doses of 200 or 400 mg/kg. In the carrageenan-induced test, only Em showed anti-inflammatory activity, decreasing the paw edema, at the doses of 200 and 400 mg/kg. No effect was observed with Ev in this model. On the other hand, in the dextran model, Ev demonstrated anti-inflammatory effect, showing decrease of the paw edema at the 1, 2, 3, 4 and 24th h. Em (200 or 400 mg/kg) presented anti-inflammatory effect at the 2, 3 and 4th h after administration of dextran, as compared to control. In conclusion, the work showed that Ev and Em present anti-edematous actions, which possibly occurs by distinct mechanisms. While Ev seems to interfere especially in inflammatory processes in which mast cells have an important role, Em exerts greater activity in the inflammatory process that depends mainly on polymorphonuclear leucocytes. However, further studies are needed to determine the exact mechanism of action of the species investigated.

Plantas medicinais e seus constituintes bioativos: uma revisão da bioatividade e potenciais benefícios nos distúrbios da ansiedade em modelos animais: [revisão] / Medicinal plants and their bioactive constituents: a scientific review of bioactivity and potential benefits in the anxiety disorders in animal models: [revision]

A procura de novos agentes terapêuticos provenientes de plantas medicinais para doenças psiquiátricas tem progredido significativamente na última década. Isso reflete num grande número de preparações herbárias para as quais o potencial psicoterapêutico tem sido avaliado em diversos modelos animais. O intuito desta revisão é fornecer uma ampla visão das plantas medicinais que apresentam efeitos terapêuticos significantes em modelos animais de doenças psiquiátricas, especificamente os distúrbios da ansiedade. Um considerável número de constituintes herbários cujos efeitos comportamentais e ações farmacológicas têm sido bem caracterizados podem ser bons candidatos para futuras investigações que podem resultar em uso clínico, merecendo, portanto, uma maior atenção em estudos posteriores.

A search for novel pharmacotherapy from medicinal plants for psychiatric illnesses has progressed significantly in the past decade. This is reflected in the large number of herbal preparations for which psychotherapeutic potential has been evaluated in a variety of animal models. The aim of this review is to provide an overview of medicinal plants that have significant therapeutic effects in animal models of psychiatric illnesses, specifically anxiety disorders. A considerable number of herbal constituents whose behavioral effects and pharmacological actions have been well characterized may be good candidates for future investigations that may result in clinical use, thus deserving increased attention in future studies.

Expression of muscarinic and dopaminergic receptors and monoamine levels frontal cortex of epileptic rats.

Apart from stroke, epilepsy is the most common neurological disorder with 0.5% of prevalence. The present study was performed in order to determine the monoamine levels, (M(1)-like) muscarinic and (D(1)- and D(2)-like) dopaminergic receptor changes in frontal cortex of adult rats after pilocarpine-induced status epilepticus (SE). Male Wistar rats were treated with a single dose of pilocarpine (400 mg/kg, s.c.) and the control group received 0.9% saline (s.c.). Both groups were sacrificed 1 h after treatment. The frontal cortex was dissected for neurochemical assays. The results show a downregulation of 27% in M(1) muscarinic receptor density, but in the dissociation constant (K(d)) value remained unaltered. D(1) and D(2) dopaminergic receptor densities and their K(d) values remained unaltered. Monoamine and metabolites levels presented decreases of 44%, 27%, 30% and 42% in dopamine (DA), homovanilic acid (HVA), norepinephrine (NE) and 5-hydroxyindoleacetic acid (5-HIAA) contents, respectively. Moreover, in serotonin (5-HT) level remained unaltered and the 4-hydroxy-3-methoxy-phenylacetic acid (DOPAC) concentration was augmented by 34%. The results suggest that dopaminergic system in this area studied may not be directly involved in the seizures and status epilepticus, but different monoamines and metabolites can be modified in this cerebral area during seizure process. In conclusion, the neurochemical alterations that occur in frontal cortex of adult rats observed during the establishment of the status epilepticus induced by pilocarpine are decrease in M(1) receptor density concentration and a reduction in DA and NE levels.

Modifications in muscarinic, dopaminergic and serotonergic receptors concentrations in the hippocampus and striatum of epileptic rats.

The present study was undertaken in order to investigate the muscarinic (M(1)), dopaminergic (D(1) and D(2)) and serotonergic (5-HT(2)) receptors densities in hippocampus and striatum of Wistar rats after status epilepticus (SE) induced by pilocarpine. The control group was treated with 0.9% saline. An other group of rats received pilocarpine (400 mg/kg, s.c.) and both groups were sacrificed 1 h after treatment. The results have shown that pilocarpine administration and resulting SE produced a downregulation of M(1) receptor in hippocampus (41%) and striatum (51%) and an increase in the dissociation constant (K(d)) values in striatum (42%) alone. In both areas the 5-HT(2) receptor density remained unaltered, but a reduction (50%) and an increase (15%) in the K(d) values were detected in striatum and hippocampus, respectively. D(1) and D(2) receptor densities in hippocampus and striatum remained unaltered meanwhile K(d) values for D(1) receptor declined significantly, 33% in hippocampus and 26% in striatum. Similarly, K(d) values for D(2) decreased 55% in hippocampus and 52% in striatum. From the preceding results, it is clear that there is a possible relation between alterations in muscarinic receptor density and others systems studied as well as they suggest that changes in dissociation constant can be responsible for the establishment of pilocarpine-induced SE by altering the affinity of neurotransmitters such as acetylcholine, dopamine and serotonine.

Antioxidant effect of nimodipine in young rats after pilocarpine-induced seizures.

Nimodipine (ND) is a centrally active calcium antagonist that blocks the voltage-dependent L-type channels. Its antiepileptic properties have been proved in various animal models, including pilocarpine-induced seizures in adult rats. In order to investigate protective effects of the ND (10 (ND10) and 30 mg/kg (ND30), i.p.), young male rats (21-day-old) received ND injections before pilocarpine administration (400 mg/kg, s.c., pilocarpine group (P400)). The pretreatment with ND10 and ND30 prolonged the latencies of seizures and death on this seizure model. ND pretreatment in two doses decreased the levels of lipid peroxidation when compared to pilocarpine group. The P400 administration increased the striatal catalase activity. However, the administration of ND, in dose of 30 mg/kg, 30 min before pilocarpine, preserved catalase activity in normal levels. On the other hand, no change was detected in the animals treated with the dose of 10 mg/kg. Our results confirm the neuroprotective effect of ND on the seizures in young rats, suggesting that this drug acts positively on lipid peroxidation. Our observations shows that nimodipine cannot induces these effects via blockade of Ca(2+)-channel.

Oxidative stress in the hippocampus after pilocarpine-induced status epilepticus in Wistar rats.

The role of oxidative stress in pilocarpine-induced status epilepticus was investigated by measuring lipid peroxidation level, nitrite content, GSH concentration, and superoxide dismutase and catalase activities in the hippocampus of Wistar rats. The control group was subcutaneously injected with 0.9% saline. The experimental group received pilocarpine (400 mg.kg(-1), subcutaneous). Both groups were killed 24 h after treatment. After the induction of status epilepticus, there were significant increases (77% and 51%, respectively) in lipid peroxidation and nitrite concentration, but a 55% decrease in GSH content. Catalase activity was augmented 88%, but superoxide dismutase activity remained unaltered. These results show evidence of neuronal damage in the hippocampus due to a decrease in GSH concentration and an increase in lipid peroxidation and nitrite content. GSH and catalase activity are involved in mechanisms responsible for eliminating oxygen free radicals during the establishment of status epilepticus in the hippocampus. In contrast, no correlations between superoxide dismutase and catalase activities were observed. Our results suggest that GSH and catalase activity play an antioxidant role in the hippocampus during status epilepticus.

Effects of haloperidol on rat behavior and density of dopaminergic D2-like receptors.

The present work shows the effects of a typical neuroleptic drug (haloperidol, HAL) on rat behavior (catalepsy and locomotor activity) and dopaminergic D2-like receptor densities in the hippocampus and striatum. Male Wistar rats (2-3 months old) were treated daily for 30 days with HAL (0.2 or 1mg/kg, intraperitoneally (i.p.)). At the end of treatment and 1h or 1, 3, 7 and 15 days after drug withdrawal, animals were subjected to behavioral tests and sacrificed afterwards for binding assays. The results showed that behavioral effects with both doses were significant only 1h and 1 day after withdrawal, and similar to controls at the third day. An up-regulation of D2 receptors was observed in the striatum (28% increase) but not in the hippocampus after 24h HAL (1mg/kg) withdrawal. However, an up-regulation was seen in both areas (1mg/kg) 3 days after drug withdrawal (58 and 42% increases in the hippocampus and striatum, respectively), and continued after 7 days of withdrawal only in the striatum (43 and 49% for the doses of 0.2 and 1mg/kg, respectively), suggesting the influence of dose, age, and time of drug withdrawal on these parameters. The up-regulation disappeared after 15 days of haloperidol withdrawal. Increases (72 and 140%) in constant dissociation values (K(d)) values were also observed 7 days after withdrawal. Results show differences on a time-basis between behavioral alterations and dopaminergic D2 receptors up-regulation.