multidrug efflux
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Author(s):  
Simon Patching

The aim of this work was to test polyamines as potential natural substrates of the Acinetobacter baumannii chlorhexidine efflux protein AceI using near-UV synchrotron radiation circular dichroism (SRCD) spectroscopy. The Gram-negative bacterium A. Baumannii is a leading cause of hospital-acquired infections and an important foodborne pathogen. A. Baumannii strains are becoming increasingly resistant to antimicrobial agents, including the synthetic antiseptic chlorhexidine. AceI was the founding member of the recently recognised PACE family of bacterial multidrug efflux proteins. Using the plasmid construct pTTQ18-aceI(His6) containing the A. Baumannii aceI gene directly upstream from a His6-tag coding sequence, expression of AceI(His6) was amplified in E. coli BL21(DE3) cells. Near-UV (250-340 nm) SRCD measurements were performed on detergent-solubilised and purified AceI(His6) at 20 °C. Sample and SRCD experimental conditions were identified that detected binding of the triamine spermidine to AceI(His6). In a titration with spermidine (0-10 mM) this binding was saturable and fitting of the curve for the change in signal intensity produced an apparent binding affinity (KD) of 3.97 +/- 0.45 mM. These SRCD results were the first experimental evidence obtained for polyamines as natural substrates of PACE proteins.


2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Coline Plé ◽  
Heng-Keat Tam ◽  
Anais Vieira Da Cruz ◽  
Nina Compagne ◽  
Juan-Carlos Jiménez-Castellanos ◽  
...  

AbstractEfflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump.


2021 ◽  
Vol 12 ◽  
Author(s):  
Kunihiko Nishino ◽  
Seiji Yamasaki ◽  
Ryosuke Nakashima ◽  
Martijn Zwama ◽  
Mitsuko Hayashi-Nishino

Multidrug efflux pumps are inner membrane transporters that export multiple antibiotics from the inside to the outside of bacterial cells, contributing to bacterial multidrug resistance (MDR). Postgenomic analysis has demonstrated that numerous multidrug efflux pumps exist in bacteria. Also, the co-crystal structural analysis of multidrug efflux pumps revealed the drug recognition and export mechanisms, and the inhibitory mechanisms of the pumps. A single multidrug efflux pump can export multiple antibiotics; hence, developing efflux pump inhibitors is crucial in overcoming infectious diseases caused by multidrug-resistant bacteria. This review article describes the role of multidrug efflux pumps in MDR, and their physiological functions and inhibitory mechanisms.


Author(s):  
Christian Bonifer ◽  
Clemens Glaubitz

ATP-binding cassette (ABC) transporters play an important role in various cellular processes. They display a similar architecture and share a mechanism which couples ATP hydrolysis to substrate transport. However, in the light of current data and recent experimental progress, this protein superfamily appears as multifaceted as their broad substrate range. Among the prokaryotic ABC transporters, MsbA can serve as a paradigm for research in this field. It is located in the inner membrane of Gram-negative bacteria and functions as a floppase for the lipopolysaccharide (LPS) precursor core-LPS, which is involved in the biogenesis of the bacterial outer membrane. While MsbA shows high similarity to eukaryotic ABC transporters, its expression in Gram-negative bacteria makes it conveniently accessible for many experimental approaches from spectroscopy to 3D structure determination. As an essential protein for bacterial membrane integrity, MsbA has also become an attractive target for the development of novel antibiotics. Furthermore, it serves as a model for multidrug efflux pumps. Here we provide an overview of recent findings and their relevance to the field, highlight the potential of methods such as solid-state NMR and EPR spectroscopy and provide a perspective for future work.


Author(s):  
Andrew H. Moeller

Bacteria in the human gut contend with numerous fluctuating environmental variables, including bouts of extreme selective agents like antibiotics. Theory predicts that oscillations in the adaptive landscape can impose balancing selection on bacterial populations, leaving characteristic signatures in the sequence variation of functionally significant genomic loci. Despite their potential importance for gut bacterial adaptation, the metagenomic targets of balancing selection have not been identified. Here, I present population genetic evidence that balancing selection maintains allelic diversity in multidrug efflux pumps of multiple predominant bacterial species in the human gut metagenome. Metagenome wide scans of 566,958 core open reading frames (CORFs) from 287 bacterial species represented by 118,617 metagenome assembled genomes (MAGs) indicated that most CORFs have been conserved by purifying selection. However, dozens of CORFs displayed positive Tajima’s D values that deviated significantly from their species’ genomic backgrounds, indicating the action of balancing selection. The AcrB subunit of a multidrug efflux pump (MEP) in Bacteroides dorei displayed the highest Tajima’s D of any CORF, and AcrB and other MEPs from a diversity of bacterial species were significantly enriched among the CORFs with the highest Tajima’s D values. Crystal structures indicated that the regions under balancing selection bind tetracycline and macrolide antibiotics. Other proteins identified as targets of balancing selection included synthases, hydrolases, and ion transporters. Intriguingly, bacterial species experiencing balancing selection were the most abundant in the human gut based on metagenomic data, further suggesting fitness benefits of the allelic variation identified.


2021 ◽  
pp. 105286
Author(s):  
Thiago S. Freitas ◽  
Jayze C. Xavier ◽  
Raimundo L.S. Pereira ◽  
Janaína E. Rocha ◽  
Fábia F. Campina ◽  
...  

2021 ◽  
Vol 14 (11) ◽  
pp. 1087
Author(s):  
Yaogeng Wang ◽  
Rolf W. Sparidans ◽  
Sander Potters ◽  
Rahime Şentürk ◽  
Maria C. Lebre ◽  
...  

Selpercatinib is a targeted, FDA-approved, oral, small-molecule inhibitor for the treatment of rearranged during transfection (RET) proto-oncogene mutation-positive cancer. Using genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporters, and the drug-metabolizing CYP3A complex in selpercatinib pharmacokinetics. Selpercatinib was efficiently transported by hABCB1 and mAbcg2, but not hABCG2, and was not a substrate of human OATP1A2, -1B1 or -1B3 in vitro. In vivo, brain and testis penetration were increased by 3.0- and 2.7-fold in Abcb1a/1b-/- mice and by 6.2- and 6.4-fold in Abcb1a/1b;Abcg2-/- mice, respectively. Oatp1a/1b deficiency did not alter selpercatinib pharmacokinetics. The ABCB1/ABCG2 inhibitor elacridar boosted selpercatinib brain penetration in wild-type mice to the levels seen in Abcb1a/1b;Abcg2-/- mice. Cyp3a-/- mice showed a 1.4-fold higher plasma AUC0–4h than wild-type mice, which was then 1.6-fold decreased upon transgenic overexpression of human CYP3A4 in liver and intestine. In summary, ABCG2, and especially ABCB1, limit brain and testis penetration of selpercatinib. Elacridar coadministration could mostly reverse these effects, without causing acute toxicity. CYP3A-mediated metabolism can limit selpercatinib oral exposure and hence its tissue concentrations. These insights may be useful in the further clinical development of selpercatinib.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1761
Author(s):  
Wenlong Li ◽  
Rolf W. Sparidans ◽  
Maria C. Lebre ◽  
Jos H. Beijnen ◽  
Alfred H. Schinkel

Repotrectinib shows high activity against ROS1/TRK/ALK fusion-positive cancers in preclinical studies. We explored the roles of multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporter(s), and the CYP3A complex in pharmacokinetics and tissue distribution of repotrectinib in genetically modified mouse models. In vitro, human ABCB1 and ABCG2, and mouse Abcg2 efficiently transported repotrectinib with efflux transport ratios of 13.5, 5.6, and 40, respectively. Oral repotrectinib (10 mg/kg) showed higher plasma exposures in Abcg2-deficient mouse strains. Brain-to-plasma ratios were increased in Abcb1a/1b−/− (4.1-fold) and Abcb1a/1b;Abcg2−/− (14.2-fold) compared to wild-type mice, but not in single Abcg2−/− mice. Small intestinal content recovery of repotrectinib was decreased 4.9-fold in Abcb1a/1b−/− and 13.6-fold in Abcb1a/1b;Abcg2−/− mice. Intriguingly, Abcb1a/1b;Abcg2−/− mice displayed transient, mild, likely CNS-localized toxicity. Oatp1a/1b deficiency caused a 2.3-fold increased oral availability and corresponding decrease in liver distribution of repotrectinib. In Cyp3a−/− mice, repotrectinib plasma AUC0–h was 2.3-fold increased, and subsequently reduced 2.0-fold in humanized CYP3A4 transgenic mice. Collectively, Abcb1 and Abcg2 restrict repotrectinib brain accumulation and possibly toxicity, and control its intestinal disposition. Abcg2 also limits repotrectinib oral availability. Oatp1a/1b mediates repotrectinib liver uptake, thus reducing its systemic exposure. Systemic exposure of repotrectinib is also substantially limited by CYP3A activity. These insights may be useful to optimize the therapeutic application of repotrectinib.


Biochemistry ◽  
2021 ◽  
Author(s):  
Lauren Ammerman ◽  
Sarah B. Mertz ◽  
Chanyang Park ◽  
John G. Wise

Author(s):  
Samiksha Kukal ◽  
Debleena Guin ◽  
Chitra Rawat ◽  
Shivangi Bora ◽  
Manish Kumar Mishra ◽  
...  

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