Med Chem Res DOI 10.1007/s00044-016-1761-1 MEDICINAL CHEMISTR Y RESEARCH ORIGINAL RESEARCH Inhibitors of multidrug efflux pumps of Pseudomonas aeruginosa from natural sources: An in silico high-throughput virtual screening and in vitro validation Gianmarco Mangiaterra 1 ● Emiliano Laudadio 1 ● Marta Cometti 1 ● Giovanna Mobbili 1 ● Cristina Minnelli 1 ● Luca Massaccesi 1 ● Barbara Citterio 2 ● Francesca Biavasco 1 ● Roberta Galeazzi 1 Received: 6 July 2016 / Accepted: 2 December 2016 © Springer Science+Business Media New York 2016 Abstract Pseudomonas aeruginosa is resistant to a wide range of antibiotics, thus making troublesome the infection treatment. Efflux systems are the main mechanisms involved; among these, MexAB-OprM is a tripartite efflux pump responsible for resistance to ciprofloxacin, aztreonam, gen- tamicin, tetracycline and tobramycin. In an attempt to con- trast antibiotic efflux, databases of natural compounds were tested for their ability to bind MexB, the inner membrane channel, using a high-throughput virtual screening approach. The comparison of their common pharmacophoric features was the basis for inhibitor identification and selection pro- cess. In silico screening against the MexB protein was per- formed by Autodock/Vina and further refined using a minimization/focused docking protocol on the obtained complexes. The compounds showing the best docking and resulting potentially active at nanomolar concentration have been selected and used in combination with antibiotics usually exported by MexAB-OprM in antimicrobial in vitro synergy tests (checkerboard and time kill assays) against multidrug-resistant P. aeruginosa clinical isolates. The combinations morelloflavone and pregnan-20-one-derivative/ ciprofloxacin showed a four-fold MIC decrease and 100-fold increase of the bacterial killing compared to the antibiotic alone. The two chosen hits were validated by ethidium bro- mide accumulation assay for their efflux inhibition potency. These compounds showed the ability to increase the accumulation of ethidium bromide inside the bacterial cells as evidenced by the increase of its fluorescence in the pre- sence of the each of them. Finally, their toxicity has been preliminary tested through hemolysis assay. The observed good correlation between in silico docking and in vitro synergy tests, indicates these two compounds as promising drugs to be used in combination therapy against MDR and MexAB-OprM overexpressing P. aeruginosa. Keywords High-throughput virtual screening ● Molecular docking ● In vitro synergy testing ● MDR P. aeruginosa ● Efflux pumps inhibitors Introduction Antibiotic-resistant bacteria have increased in recent years and such resistance can compromise the efficacy of anti- microbial therapy (Taubes 2008). Multidrug-resistant (MDR) bacteria have been associated with infections char- acterized by a higher mortality than those caused by antibiotic-susceptible strains and nowadays MDR strains are cause of great concern. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is frequently associated with life-threatening nosocomial infections, particularly in immune-compromised and cystic fibrosis (CF) patients. Intrinsically resistant to many drugs and readily developing high-level resistance during anti- microbial therapy, P. aeruginosa infections are often diffi- cult to counteract. Efflux systems are among the major contributors to the emergence of MDR strains (Breidenstein et al. 2011; Ball et al. 2006; Ho-Fung Lau et al. 2014). The physiological function of multidrug efflux pumps is to push solutes out of a cell, allowing the microorganisms to * Roberta Galeazzi r.galeazzi@univpm.it 1 Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, via Brecce Bianche, Ancona 60128, Italy 2 Dipartimento di Scienze Biomolecolari, sez. di Biotecnologie, Università degli Studi di Urbino “Carlo Bo”, Urbino 61029, Italy