Development of dilipid polymyxins: Investigation on the effect of hydrophobicity through its fatty acyl component

Continuous development of new antibacterial agents is necessary to counter the problem of antimicrobial resistance. Polymyxins are considered as drugs of last resort to combat multidrug-resistant Gram-negative pathogens. Structural optimization of polymyxins requires an in-depth understanding of its structure and how it relates to its antibacterial activity. Herein, the effect of hydrophobicity was explored by adding a secondary fatty acyl component of varying length onto the polymyxin structure at the amine side-chain of l-diaminobutyric acid at position 1, resulting to the development of dilipid polymyxins. The incorporation of an additional lipid was found to confer polymyxin activity against Gram-positive bacteria, to which polymyxins are inherently inactive against. The dilipid polymyxins showed selective antibacterial activity against Pseudomonas aeruginosa. Moreover, dilipid polymyxin 1 that consists of four carbon-long aliphatic lipids displayed the ability to enhance the antibacterial potency of other antibiotics in combination against P. aeruginosa, resembling the adjuvant activity of the well-known outer membrane permeabilizer polymyxin B nonapeptide (PMBN). Interestingly, our data revealed that dilipid polymyxin 1 and PMBN are substrates for the MexAB-OprM efflux system, and therefore are affected by efflux. In contrast, dilipid polymyxin analogs that consist of longer lipids and colistin were not affected by efflux, suggesting that the lipid component of polymyxin plays an important role in resisting active efflux. Our work described herein provides an understanding to the polymyxin structure that may be used to usher the development of enhanced polymyxin analogs.     

Kinetic parameters in the compartmental analysis of lithium transport in Lemna gibba using the stable isotopes, 6Li and 7Li, as tracers

Due to the lack of a convenient radiotracer of Li, we have used the stable isotopes, ⁶Li and ⁷Li, for unidirectional flux measurements in Lemna gibba L. (GI). Detection was performed using an ionic microanalyser. The conventional method of compartmental analysis has been improved in several ways. Growth of the plant samples has been taken into consideration. The possible non-uniqueness of the kinetic solution fitting the experimental points has been considered by numerical calculation. The derivatives and not only the direct functions have been used in order to detect whether a constant term had to be added to the first-order terms. The validity of the estimates of the characteristic parameters of transport has been checked by using efflux and influx data simultaneously. The "manipulation-shock" effects have been shown not to be negligible. The kinetic data have no simple biological significance per se, but they can serve to calculate cellular parameters of transport when combined with appropriate stereometric data. These considerations are relevant not only to our present problem, but to any type of compartmental analysis, whatever the plant sample and the absorbed substrate.     

Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ～1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ～1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ～500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.     

Multidrug transporters in prokaryotic and eukaryotic cells: physiological functions and transport mechanisms

Multidrug transporters mediate the extrusion of structurally unrelated drugs from prokaryotic and eukaryotic cells. As a result of this efflux activity, the cytoplasmic drug concentration in the cell is lowered to subtoxic levels and, hence, cells become multidrug resistant. The activity of multidrug transporters interferes with the drug-based control of tumours and infectious pathogenic microorganisms. There is an urgent need to understand the structure-function relationships in multidrug transporters that underlie their drug specificity and transport mechanism. Knowledge about the architecture of drug and modulator binding sites and the link between energy-generating and drug translocating functions of multidrug transporters may allow one to rationally design new drugs that can poison or circumvent the activity of these transport proteins. Furthermore, if one is to inhibit multidrug transporters in human cells, one should know more about their physiological substrates and functions. This review will summarize important new insights into the role that multidrug transporters in general, and P-glycoprotein and its bacterial homologue LmrA in particular, play in the physiology of the cell. In addition, the molecular basis of drug transport by these proteins will be discussed.     

Characterization of the Vibrio cholerae vexAB and vexCD efflux systems

Vibrio cholerae is an important human pathogen that causes the diarrheal disease cholera. Colonization of the human host is dependent upon coordinated expression of several virulence factors in response to as yet unknown environmental cues. Bile acids have been implicated in the in vitro regulation of several V. cholerae genes, including those involved in motility, chemotaxis, outer membrane protein production, and virulence factor production. Bile is toxic to bacteria and colonization of the intestinal tract is dependent upon bacterial resistance to bile acids. We have identified and characterized two bile-regulated RND-family efflux systems, named here vexAB and vexCD, that are involved in V. cholerae bile resistance. Mutational analysis revealed that the vexAB system is responsible for in vitro intrinsic resistance of V. cholerae to multiple antimicrobial compounds, including bile acids. In contrast, the vexCD efflux system was specific for certain bile acids and detergents and functioned in conjunction with the vexAB system to provide V. cholerae with high-level bile resistance. Mutants containing deletion of vexB, vexD, and vexB–vexD were able to efficiently colonize the infant mouse suggesting that these efflux systems were dispensable for V. cholerae growth in the small intestines of infant mice.     

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 6: exploration of aromatic substituents

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with aromatic substituents, were synthesized by the Suzuki cross-coupling method and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the anti-pseudomonas β-lactam aztreonam (AZT) in Pseudomonas aeruginosa. By incorporating hydrophilic substituents onto the aryl nucleus, we found a morpholine analogue that possessed improved solubility, retained activity in vitro, and displayed potentiation activity in vivo in a rat model of P. aeruginosa pneumonia.     

Species-specific differences in biomarker responses in two ecologically different earthworms exposed to the insecticide dimethoate

Earthworms ingest large amounts of soil and therefore are continuously exposed to contaminants through their alimentary surfaces. Additionally, several studies have shown that earthworm skin is a significant route of contaminant uptake as well. In order to determine effects of dimethoate, a broad-spectrum organophosphorous insecticide, two ecologically different earthworm species were used - Eisenia andrei and Octolasion lacteum. Although several studies used soil organisms to investigate the effects of dimethoate, none of these studies included investigations of dimethoate effects on biochemical biomarkers in earthworms. Earthworms were exposed to 0.001, 0.005, 0.01, 0.5 and 1 μg/cm(2) of dimethoate for 24 h, and the activities of acetylcholinesterase, carboxylesterase, catalase and efflux pump were measured. In both earthworm species dimethoate caused significant inhibition of acetylcholinesterase and carboxylesterase activities, however in E. andrei an hormetic effect was evident. Efflux pump activity was inhibited only in E. andrei, and catalase activity was significantly inhibited in both earthworm species. Additionally, responses of earthworm acetylcholinesterase, carboxylesterase and catalase activity to dimethoate were examined through in vitro experiments. Comparison of responses between E. andrei and O. lacteum has shown significant differences, and E. andrei has proved to be less susceptible to dimethoate exposure.     

Transporters in Absorption,Distribution, and Elimination

During the past decades, pharmacokinetics has been defined as the study of drug absorption, distribution, metabolism, and excretion (ADME), when the drug is introduced into a biological system, such as the human body. Pharmacokinetics is now challenged by the growing importance of transporters, a relatively new and potentially major factor in drug ADME. The recent intrusion of drug transporters means that there is no single mechanism by which drugs permeate through membranes. The presence of transporters in membranes modulates the traditional theory of ‘diffusional pharmacokinetics’ towards ‘vectorial pharmacokinetics’ in which ADME processes are governed more deterministically. Drug transporters are also clinically important. They can modulate the pharmacological activity of drugs by affecting their intracellular concentrations and causing toxicity in specific organs due to intracellular drug accumulation. Finally, they are key players in drug–drug interactions, where they are as important as the drug metabolizing enzymes.