title: 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.
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.
authors.affiliation: Department of Bioinformatics, School of Bioengineering, Faculty of Engineering & Technology, SRM University, Kattankulathur, Tamilnadu, India.
authors.forename: Vasudevan
authors.initials: V
authors.lastname: Aparna
authors.affiliation: Department of Bioinformatics, School of Bioengineering, Faculty of Engineering & Technology, SRM University, Kattankulathur, Tamilnadu, India.
authors.forename: Kesavan
authors.initials: K
authors.lastname: Dineshkumar
authors.affiliation: Department of Bioinformatics, School of Bioengineering, Faculty of Engineering & Technology, SRM University, Kattankulathur, Tamilnadu, India.
authors.forename: Narasumani
authors.initials: N
authors.lastname: Mohanalakshmi
authors.affiliation: Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, Tamilnadu, India.
authors.forename: Devadasan
authors.initials: D
authors.lastname: Velmurugan
authors.affiliation: Department of Bioinformatics, School of Bioengineering, Faculty of Engineering & Technology, SRM University, Kattankulathur, Tamilnadu, India.
authors.forename: Waheeta
authors.initials: W
authors.lastname: Hopper
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D001688:Biological Products / Q000737:chemistry / Q000494:pharmacology*; D003198:Computer Simulation; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays; D066298:In Vitro Techniques; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D064209:Phytochemicals / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000378:metabolism*; D015203:Reproducibility of Results; D016415:Sequence Alignment
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001688:Biological Products; D029968:Escherichia coli Proteins; D004996:Ethidium; D008024:Ligands; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D064209:Phytochemicals; C083496:AcrB protein, E coli; C118597:MexB protein, Pseudomonas aeruginosa