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A liposomal method for evaluation of inhibitors of H(+)-coupled multidrug transporters
- Source :
- Journal of pharmacological and toxicological methods. 77
- Publication Year :
- 2015
-
Abstract
- Introduction This paper describes a novel technique, Fluorosomes, applied to investigating the interaction of antimicrobials with proton driven microbial efflux transporters. These transporters remove toxic compounds from the cytoplasm, including antibiotics and are involved in antibiotic resistance. Methods To assess transporter activity we developed a methodology to generate a proton gradient across Fluorosome membranes into which selected purified fully active efflux transporters were reconstituted. The interior of the Fluorosome particle (a unilamellar liposome) contains a fluorescent drug sensing probe whose fluorescence is quantitatively quenched by transporter substrates. Using an injecting fluorescence plate reader to initiate a proton gradient and to monitor subsequent fluorescence change, real time transport kinetics can be followed and transport inhibition characterized. Results Fluorosomes containing the Escherichia coli EmrE efflux pump demonstrated transport of two known EmrE substrates, ethidium and methyl viologen upon creation of a proton gradient. For Fluorosomes containing the inactive EmrE mutant, E14Q, no transport was observed. When the gradient was fully collapsed by the addition of nigericin, full inhibition of substrate transport was observed. The IC50 for nigericin inhibition of ethidium was shown to be 0.71 μM. Discussion We have for the first time prepared and validated a single bacterial efflux pump assay, Fluorosome-trans-EmrE, that faithfully mimics properties of the transporter in vivo. It is faster than whole cell screens, simple to use, amenable to robotics, and reports on very specific targets. We have demonstrated proof of principle with EmrE and have created the first of an intended series of proton driven Fluorosomes.
- Subjects :
- 0301 basic medicine
Pharmacology
Liposome
Nigericin
030106 microbiology
Membrane Transport Proteins
Transporter
Biological Transport
Toxicology
03 medical and health sciences
chemistry.chemical_compound
Membrane
chemistry
Biochemistry
Anti-Infective Agents
Ethidium
Liposomes
Escherichia coli
Unilamellar liposome
Efflux
Multidrug Resistance-Associated Proteins
Protons
Electrochemical gradient
Plate reader
Fluorescent Dyes
Subjects
Details
- ISSN :
- 1873488X
- Volume :
- 77
- Database :
- OpenAIRE
- Journal :
- Journal of pharmacological and toxicological methods
- Accession number :
- edsair.doi.dedup.....0d99fe39ba87db41ca3d2fa44c4cfa87