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      Identification of Natural Compound Inhibitors for Multidrug Efflux Pumps of Escherichia coli and Pseudomonas aeruginosa Using In Silico High-Throughput Virtual Screening and In Vitro Validation

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          Abstract

          Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH) generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug resistant strains of P. aeruginosa and E. coli.

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          Most cited references24

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          Pseudomonas aeruginosa: all roads lead to resistance.

          Pseudomonas aeruginosa is often resistant to multiple antibiotics and consequently has joined the ranks of 'superbugs' due to its enormous capacity to engender resistance. It demonstrates decreased susceptibility to most antibiotics due to low outer membrane permeability coupled to adaptive mechanisms and can readily achieve clinical resistance. Newer research, using mutant library screens, microarray technologies and mutation frequency analysis, has identified very large collections of genes (the resistome) that when mutated lead to resistance as well as new forms of adaptive resistance that can be triggered by antibiotics themselves, in in vivo growth conditions or complex adaptations such as biofilm growth or swarming motility. Copyright © 2011 Elsevier Ltd. All rights reserved.
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            Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria.

            Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed.
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              Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader.

              Oxidative stress (OS) has been implicated in various degenerative diseases in aging. In an attempt to quantify OS in a cell model, we examined OS induced by incubating for 30 min with various free radical generators in PC12 cells by using the dichlorofluorescein (DCF) assay, modified for use by a fluorescent microplate reader. The nonfluorescent fluorescin derivatives (dichlorofluorescin, DCFH), after being oxidized by various oxidants, will become DCF and emit fluorescence. By quantifying the fluorescence, we were able to quantify the OS. Our results indicated that the fluorescence varied linearly with increasing concentrations (between 0.1 and 1 mM) of H2O2 and 2,2'-azobios(2-amidinopropane) dihydrochloride (AAPH; a peroxyl radical generator). By contrast, the fluorescence varied as a nonlinear response to increasing concentrations of 3-morpholinosydnonimine hydrochloride (SIN-1; a peroxynitrite generator), sodium nitroprusside (SNP; a nitric oxide generator), and dopamine. Dopamine had a biphasic effect; it decreased the DCF fluorescence, thus acting as an antioxidant, at concentrations <500 microM in cells, but acted as a pro-oxidant by increasing the fluorescence at 1 mM. While SNP was not a strong pro-oxidant, SIN-1 was the most potent pro-oxidant among those tested, inducing a 70 times increase of fluorescence at a concentration of 100 microM compared with control. Collectively, due to its indiscriminate nature to various free radicals, DCF can be very useful in quantifying overall OS in cells, especially when used in conjunction with a fluorescent microplate reader. This method is reliable and efficient for evaluating the potency of pro-oxidants and can be used to evaluate the efficacy of antioxidants against OS in cells.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                15 July 2014
                : 9
                : 7
                : e101840
                Affiliations
                [1 ]Department of Bioinformatics, School of Bioengineering, Faculty of Engineering & Technology, SRM University, Kattankulathur, Tamilnadu, India
                [2 ]Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, Tamilnadu, India
                University of Cambridge, United Kingdom
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: VA KD DV WH. Performed the experiments: VA KD NM. Analyzed the data: VA KD WH. Contributed to the writing of the manuscript: VA KD WH.

                [¤]

                Current address: Department of Biology, McGill University, Montreal, QC, Canada.

                Article
                PONE-D-14-14077
                10.1371/journal.pone.0101840
                4099075
                25025665
                6ac03c15-3c98-472e-beec-d1e9b69c443b
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 29 March 2014
                : 12 June 2014
                Page count
                Pages: 13
                Funding
                This study was supported by the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, for funding the research and SRM University, India for support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Computational Biology
                Microbiology
                Medicine and Health Sciences
                Infectious Diseases
                Research and Analysis Methods
                Biological Cultures
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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