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.

Polyphenolic Extract from Maple Syrup Potentiates Antibiotic Susceptibility and Reduces Biofilm Formation of Pathogenic Bacteria

“…imization of further development of resistance (56), and (iv) reduced biofilm formation (57). In our study, the antimicrobial synergy of PRMSE with both ciprofloxacin (a fluoroquinolone) and carbenicillin (a ␤-lactam) suggests that it interacts with multiple membrane transporters.…”

Multidrug efflux pumps and their role in antibiotic and antiseptic resistance: a pharmacodynamic perspective

“…But the development of inhibitors targeting RND component of efflux pump is now the main approach that is described and tested in gram-negative bacteria. Several series of compounds from both natural [68,69] and synthetic [70][71][72][73][74] sources displaying an EPI activity have been identified (Table 1). Structure-activity relationship (SAR) studies give relevant information about the molecular and the physicochemical properties of EPIs useful in pharmacophore models to describe features that bind the target [75,76].…”

Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay

“…To combat MDR, efflux pump inhibitors (EPIs) are an attractive option, and several EPIs that act against the AcrAB-TolC efflux pump have already been described in the literature (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), among which arylpiperazine and arylmorpholine derivatives constitute some of the largest systematically examined compound classes.…”