Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors.     

Structure-activity relationships of 2-aryl-1H-indole inhibitors of the NorA efflux pump in Staphylococcus aureus

The synthesis of 22 2-aryl-1H-indoles, including 12 new compounds, has been achieved via Pd- or Rh-mediated methodologies, or selective electrophilic substitution. All three methods were based on elaborations from simple indole precursors. SAR studies on these indoles and 2-phenyl-1H-indole in Staphylococcus aureus as NorA efflux pump inhibitors indicated 5-nitro-2-(3-methoxycarbonyl)phenyl-1H-indole was a slightly more potent inhibitor than the lead INF55. A promising new antibacterial lead compound against S. aureus (2-phenyl-1H-indol-5-yl)-methanol, was also found.     

Effect of Carvacrol and Thymol on NorA efflux pump inhibition in multidrug-resistant (MDR) Staphylococcus aureus strains

Undue exposure to antimicrobials has led to the acquisition and development of sophisticated bacterial resistance mechanisms, such as efflux pumps, which are able to expel or reduce the intracellular concentration of various antibiotics, making them ineffective. Therefore, inhibiting this mechanism is a promising way to minimize the phenomenon of resistance in bacteria. In this sense, the present study sought to evaluate the activity of the Carvacrol (CAR) and Thymol (THY) terpenes as possible Efflux Pump Inhibitors (EPIs), by determining the Minimum Inhibitory Concentration (MIC) and the association of these compounds in subinhibitory concentrations with the antibiotic Norfloxacin and with Ethidium Bromide (EtBr) against strains SA-1199 (wild-type) and SA-1199B (overexpresses NorA) of Staphylococcus aureus. In order to verify the interaction of the terpenes with the NorA efflux protein, an in silico molecular modeling study was carried out. The assays used to obtain the MIC of CAR and THY were performed by broth microdilution, while the Efflux Pump inhibitory test was performed by the MIC modification method of the antibiotic Norfloxacin and EtBr. docking was performed using the Molegro Virtual Docker (MVD) program. The results of the study revealed that CAR and THY have moderate bacterial activity and are capable of reducing the MIC of Norfloxacin antibiotic and EtBr in strains of S. aureus carrying the NorA efflux pump. The docking results showed that these terpenes act as possible competitive NorA inhibitors and can be investigated as adjuvants in combined therapies aimed at reducing antibiotic resistance.

     

Thiazolidinedione and thiazole derivatives potentiate norfloxacin activity against NorA efflux pump over expression in Staphylococcus aureus 1199B strains

Thiazol and thiazolidinedione derivatives are known in the literature for presenting several biological activities, such as anti-diabetic, anti-inflammatory, antiparasitic, antifungal and antimicrobial activity. With this in mind, this study reports on the synthesis and antibacterial activity of thiazole (NJ) and thiazolidinedione (NW) derivatives, as well as their effects in association with norfloxacin, against NorA efflux pumps in the Staphylococcus aureus 1199B (SA-1199B) strain. Among the 14 compounds evaluated, 9 were found to potentiate norfloxacin activity, with 4 compounds from the NJ series promoting a threefold norfloxacin MIC reduction. Molecular docking assays were used to confirm the binding mode of most active compounds. In the in silico study, the efficiency of the interaction of NJ series compounds with the NorA pump were evaluated. Derivatives from both series did not show considerable intrinsic antibacterial activity (MIC > 1024 μg/mL) against any of the tested strains. However, the NJ16 and NJ17 compounds, when associated with norfloxacin, reduced the MIC of this drug threefold and inhibited NorA pumps in the 1199B strain. Moreover, some NW (05, 10, 18, 19 and 21) and NJ compounds (16, 17, 18 and 20) presented low to moderate cytotoxicity against normal cells. Molecular docking studies supported the potent in vitro inhibitory activity of NJ16 and NJ17, which showed NJ16 and NJ17 possessed more favorable binding energies of −9.03 Kcal/mol and −9.34 Kcal/mol, respectively. In addition, NJ16 showed different types of interactions involved in complex stabilization. In conclusion, NJ16 and NJ17, in combination with norfloxacin, were able to completely restore the antibacterial activity of norfloxacin against S. aureus SA-1199B, the norA-overexpressing strain, with low cytotoxicity in normal cells.     

NorA functions as a multidrug efflux protein in both cytoplasmic membrane vesicles and reconstituted proteoliposomes

Overexpression of NorA, an endogenous efflux transporter of Staphylococcus aureus, confers resistance to certain fluoroquinolone antimicrobials and diverse other substrates. The norA gene was amplified by PCR and cloned in the expression vector pTrcHis2. Histidine-tagged NorA (NorA-His) was overexpressed in Escherichia coli cells to prepare two experimental systems, everted membrane vesicles enriched with NorA-His and proteoliposomes reconstituted with purified NorA-His. In membrane vesicles, NorA-His actively transported Hoechst 33342, a dye that is strongly fluorescent in the membrane but has low fluorescence in an aqueous environment. Transport was activated by the addition of ATP or lactate and reversed by the addition of nigericin, with the addition of K(+)-valinomycin having little effect. Transport of Hoechst 33342 was inhibited competitively by verapamil, a known inhibitor of NorA, and by other NorA substrates, including tetraphenyl phosphonium and the fluoroquinolones norfloxacin and ciprofloxacin. In contrast, sparfloxacin, a fluoroquinolone whose antimicrobial activity is not affected by NorA expression, exhibited noncompetitive inhibition. NorA induction and overexpression yielded 0.5 to 1 mg of a largely homogeneous 40- to 43-kDa protein per liter of culture. NorA-His incorporated into proteoliposomes retained the ability to transport Hoechst 33342 in response to an artificial proton gradient, and transport was blocked by nigericin and verapamil. These data provide the first experimental evidence of NorA functioning as a self-sufficient multidrug transporter.     

Cadmium and manganese transport in Staphylococcus aureus membrane vesicles.

The presence of plasmid gene cadB did not affect Cd2+ accumulation, whereas plasmid gene cadA reduced Cd2+ accumulation by whole cells but not by membrane vesicles. Membrane vesicle studies indicated that Cd2+ uptake occurred via the Mn2+ transport system which was energized by the membrane electrical potential. Mn2+ and Cd2+ were competitive inhibitors of each other's transport, with Km's of 0.95 microM Mn2+ and 0.2 microM Cd2+. The kinetic parameters were nearly identical with vesicles prepared from sensitive and resistant cells, indicating that the cadA-encoded Cd2+ efflux system was inoperative in membrane vesicle preparations. Experiments with energy-inhibited cells indicated that the cadB gene product may bind Cd2+.     

Staphylococcus aureus MDR efflux pump inhibitors from a Berberis and a Mahonia (sensu strictu) species

Bioactive fractionation, based on multi-drug resistance (MDR) pump inhibition in Staphylococcus aureus, resulted in the isolation of the active inhibitors 5′-methoxyhydnocarpin-D from leaves of Berberis (formerly Mahonia) trifoliolata and pheophorbide a from Berberis fendleri. The hydnocarpin derivative was not found in the latter species. Pheophytin a (the phytol derivative of pheophorbide a) was identified from both species, but it proved to have no MDR pump inhibitory activity. The somewhat uncommon, and inactive, flavonoid tricin was identified from B. trifoliolata. The occurrence of a flavonolignan in Mahonia-tpe species and its absence in Berberis sensu strictu may provide a chemical differentiation between the two groups which are now recombined on the basis of DNA studies. The strong bacterial efflux pump inhibition of pheophorbide a could be of importance as a plant defense against natural pathogens.     

Synthesis of functionalized 2-aryl-5-nitro-1H-indoles and their activity as bacterial NorA efflux pump inhibitors

In order to develop structure-activity relationships and to provide access to antibacterial agents for dual action studies, a variety of aryl group-substituted 2-aryl-5-nitro-1H-indoles were synthesized and the activity of the compounds assessed as inhibitors of the NorA multidrug resistance pump in the bacterium Staphylococcus aureus. The NorA protein from the major facilitator superfamily of efflux pumps confers resistance to a variety of structurally dissimilar antimicrobials such as norfloxacin, ethidium bromide, berberine and acriflavin. The compound [4-benzyloxy-2-(5-nitro-1H-2-yl)-phenyl]-methanol was the most potent pump inhibitor.     

Synthesis and evaluation of PSSRI-based inhibitors of Staphylococcus aureus multidrug efflux pumps

Phenylpiperidine selective serotonin reuptake inhibitors (PSSRIs) block the function of selected multidrug efflux pumps of Staphylococcus aureus. In this study PSSRI-based piperidine derivatives were prepared, evaluated for inhibition of two multidrug resistance (MDR)-conferring efflux pump systems, and tested for potentiation of antimicrobial activity of antibacterial efflux pump substrates. It is demonstrated that the 4-phenyl moiety of PSSRI-based efflux pump inhibitors (EPIs) is not an absolute structural requirement for inhibiting the NorA and MepA MDR efflux pumps. Potency of efflux inhibition is maintained or enhanced by replacing the aryloxymethyl substituent at position-3 of PSSRIs with arylalkene and arylthioether moieties. Novel 3-aryl piperidine EPIs that significantly increase substrate antibiotic activity against strains of S. aureus expressing NorA and MepA are described.     

The membrane potential in everted vesicles of Escherichia coli.

Ion-selective electrodes were used to measure the equilibration of thiocyanate across the membrane of everted (“inside-out”) vesicles of Escherichia coli W1485. Membrane potentials, vesicle interior positive, generated by the oxidation of NADH, succinate, and d-lactate, or by the hydrolysis of ATP, fell in the range of 100–150 mV depending on the carbon source for cell growth and the substrate used to energize the membranes. There was no relationship between the rate of oxidation of different substrates and the membrane potential they generated. The membrane potential generated by oxidation of NADH was relatively constant between pH 7.0 and 8.5. Somewhat lower values obtained at pH 5.5 to 6.5 were attributed to the effect of pH on substrate oxidation.     

On the challenges of detecting whole Staphylococcus aureus cells with biosensors

Due to the increasing number of nosocomial infections and multidrug‐resistant bacterial strains, Staphylococcus aureus is now a major worldwide concern. Rapid detection and characterization of this bacterium has become an important issue for biomedical applications. Biosensors are increasingly appearing as low‐cost, easy‐to‐operate and fast alternatives for rapid detection. In this review, we will introduce the main characteristics of S. aureus and will focus on the interest of biosensors for a faster detection of whole S. aureus cells. In particular, we will review the most promising strategies in the choice of ligand for the design of selective and efficient biosensors. Their specific characteristics as well as their advantages and/or disadvantages will also be commented.     

鹰嘴豆芽素A对耐甲氧西林金黄色葡萄球菌外排系统的抑制作用

【目的】MRSA外排系统是其对多种药物耐药性的主要原因,为此研究鹰嘴豆芽素A对MRSA外排系统的抑制作用。【方法】以MRSA41577为供试菌株,通过二倍稀释法、双平板法和荧光分光光度法测定鹰嘴豆芽素A对MRSA41577的抑制作用及其对外排系统的影响;通过RT-PCR检测加药前后MRSA41577外排蛋白norA的表达量;SDS-PAGE检测加药前后与MRSA41577外排相关蛋白的变化,并用液相色谱质谱仪进行鉴定确认。【结果】鹰嘴豆芽素A本身无抑菌活性,但鹰嘴豆芽素A和环丙沙星联合作用后,MRSA41577对环丙沙星的敏感性显著提高,其中40μg/mL的鹰嘴豆芽素A能使环丙沙星对MRSA41577的MIC从64μg/mL降低到8μg/mL,且作用强度与药物浓度呈正相关。经鹰嘴豆芽素A处理MRSA41577后,菌体内环丙沙星的蓄积量随着药物处理时间的增加而增加,在处理15min时与对照组相比,菌体内环丙沙星蓄积量增加了83%(P﹤0.01),与阳性对照利血平的作用效果相当。鹰嘴豆芽素A能降低MRSA41577的norA表达量,与对照组相比,鹰嘴豆芽素A和环丙沙星作用MRSA41577 16h后,norA的相对表达量降低了65%,其抑制效果优于阳性对照利血平的48%。SDS-PAGE电泳结果显示,鹰嘴豆芽素A作用MRSA4157716h后,与对照组相比菌体的蛋白谱带发生了明显的变化,其中与外排系统相关的蛋白除norA蛋白明显减少外,ABC转运体ATP结合蛋白也明显减少。【结论】鹰嘴豆芽素A是MRSA41577的外排泵抑制剂,其作用机制是通过降低MRSA41577菌体内norA基因的表达量,进而减少norA蛋白的表达量,来抑制MRSA41577对环丙沙星等药物的排出而恢复其对药物的敏感性。     

Formation of the transmembrane electrochemical potential on Staphylococcus cells and membrane vesicles

The generation of transmembrane difference of electrochemical potentials was registered on the intact cells and ultrasonication-obtained membrane vesicles of Staphylococcus aureus with the application of transmembrane electrophoresis of permeant anions, potassium transport in the presence of valinomycin and 8-anilinonaphthalene-1-sulphonate fluorescence. The membrane potential is formed when the chain of electron transfer or H+-ATPase functions or when the pH gradient varies (the nonenzymic pathway). M-chlorinecarbonylcyanidephenylhydrazonium, a protonophore uncoupler potassium cyanide, an inhibitor of the respiratory chain, N',N-dicyclohexylcarbodiimide, an inhibitor of ATPase, cause the membrane potential dissipation. The orientation of the transmembrane electric field is as follows: "minus" inside cells and "plus" inside membrane vesicles.     

Amino acid transport in whole cells and membrane vesicles of Arthrobacter pyridinolis

Arginine, and several other amino acids, can only support growth of Arthrobacter pyridinolis if malate is also present in the medium. Arginine is transported by a high affinity lysine-arginine-ornithine-type transport system which is stimulated by malate in both whole cells and vesicles, is respiration-coupled, and appears to depend upon a respiration-generated membrane potential but not on a ΔpH. Arginine is also transported by a low-affinity system which transports canavanine. Studies of an arginine auxotroph suggest that the lysine-arginine-ornithine system may be the system of major physiological significance for arginine transport. Phenylalanine is one of a few amino acids which can act as sole source of carbon for A. pyridinolis. Transport of phenylalanine occurs by two kinetically distinct systems. Both of these transport systems are respiration-coupled, are not appreciably stimulated by malate either in cells or vesicles, but are markedly stimulated by ascorbate-phenazine methosulfate. Studies with inhibitors indicate that the transport systems for phenylalanine utilize both a ΔpH and a membrane potential.     

Synthesis and evaluation of new arylbenzo[b]thiophene and diarylthiophene derivatives as inhibitors of the NorA multidrug transporter of Staphylococcus aureus

The synthesis based on palladium catalytic coupling of 38 new-arylated benzo[b]thiophenes or thiophenes is described in a few steps. We also report the direct arylation of the position 3 of the benzo[b]thiophenic structure, a 'one pot' 2,5-heterodiarylation of thiophenes as well as the synthesis of precursors of amino-acids with a 2-arylated benzo[b]thiophene core. These compounds were evaluated on bacteria strains: most of them did not exhibit any antibiotic activity but were found to selectively inhibit the NorA multidrug transporter of Staphylococcus aureus. As such, they restored the activity of the NorA substrates ciprofloxacin against a resistant S. aureus strain in which this efflux pump is over-expressed.