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

Identification of Natural Compound Inhibitors for Multidrug Efflux Pumps of Escherichia coli and Pseudomonas aeruginosa Using In Silico HighThroughput Virtual Screening and In Vitro Validation Vasudevan Aparna1, Kesavan Dineshkumar1, Narasumani Mohanalakshmi1¤, Devadasan Velmurugan2, Waheeta Hopper1* 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 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. Citation: Aparna V, Dineshkumar K, Mohanalakshmi N, Velmurugan D, Hopper W (2014) 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. PLoS ONE 9(7): e101840. doi:10. 1371/journal.pone.0101840 Editor: Hendrik W. van Veen, University of Cambridge, United Kingdom Received March 29, 2014; Accepted June 12, 2014; Published July 15, 2014 Copyright: ß 2014 Aparna et al. 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. Data Availability: 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. 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. Competing Interests: The authors have declared that no competing interests exist. * Email: ¤ Current address: Department of Biology, McGill University, Montreal, QC, Canada. reduced permeability is owed to two reasons, efflux pumps and low porin protein expression [2]. The major mechanism of resistance in these organisms is the efflux pumps, which have their substrate specificity based on their polarity [3]. Multidrug resistance in Escherichia coli is also a major difficulty in the treatment of the infectious diseases caused by them, with efflux pumps as one of the mechanisms of resistance. The multidrug efflux pumps are membrane proteins that are involved in the pumping out of antibiotics and are classified into the resistant nodulation division (RND) family, the major facilitator super family (MFS), the staphylococcal multi-resistance (SMR) and the multidrug and toxic Introduction Multidrug resistant bacteria resist a broad range of antimicrobials thereby reducing the treatment options and hence increasing the mortality. There is an increase in incidence of infectious diseases in developing countries where the use of antibiotics is high. This problem of antimicrobial resistance is of great concern. The World Health Organization has urged on to ‘‘evaluate the strategies to overcome and control the spread of antimicrobial resistant micro-organisms’’ [1]. Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium, resistant to multiple drugs, mainly due to low permeability of its cell membrane. This PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e101840 Phytochemical Inhibitors of RND Efflux Pumps compound extrusion (MATE) family [4]. P. aeruginosa and E. coli have efflux pumps that belong to the RND family. AcrAB-TolC and MexAB-OprM are RND pumps that form a tripartite assembly in the bacterial membrane, contributing to the intrinsic and acquired antibiotic resistance in E. coli and P. aeruginosa respectively. They confer resistance to a large array of drugs which include quinolones, macrolides, tetracycline, chloramphenicol, novobiocin, and b-lactam [5]. Deletion of MexAB-OprM in wildtype strain of P. aeruginosa had made the strain hypersusceptibile to many drugs [4], thus giving the scope for the development of agents that could possibly block the activity of these pumps thereby making the organisms susceptible to the drugs. It is reported that combating the resistance could be done by targeting the mechanism responsible for it, in this case by developing specific inhibitors against the efflux pumps [6]. Compounds that could interact with specific efflux pump proteins could restore the organism’s susceptibility to drugs. This approach could counteract pathogens that harbour efflux pumps and compounds, the efflux pump inhibitors (EPIs) can be used as chemotherapeutics, along with the antibiotics. As efflux pumps provide both innate and higher-level resistance to antibiotics in bacteria, EPIs should ideally increase the activity of an antibiotic in multidrug-resistant cells [7] and this indicates the significance for deve (...truncated)