Send Orders for Reprints to reprints@benthamscience.net Current Topics in Medicinal Chemistry, 2013, 13, 000-000 1 1568-0266/13 $58.00+.00 © 2013 Bentham Science Publishers RND Efflux Pumps: Structural Information Translated into Function and Inhibition Mechanisms Paolo Ruggerone 1 , Satoshi Murakami 2 , Klaas M. Pos 3 and Attilio V. Vargiu 1,* 1 Department of Physics, University of Cagliari, S.P. 8, km 0.700, 09042 Monserrato (CA), Italy; 2 Life Science Depart- ment, School and Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, J2-17, 4259 Na- gatsuta-cho, Midori-ku, Yokohama 226-8503, Japan; 3 Institute of Biochemistry, Goethe Universität Frankfurt, Max-von- Laue-Straße 9, D-60438 Frankfurt, Germany Abstract: Efflux pumps of the Resistance Nodulation Division (RND) superfamily play a major role in the intrinsic and acquired resistance of Gram-negative pathogens to antibiotics. Moreover, they are largely responsible for multi-drug resis- tance (MDR) phenomena in these bacteria. The last decade has seen a sharp increase in the number of experimental and computational studies aimed at understanding their functional mechanisms. Most of these studies focused on the RND drug/proton antiporter AcrB, part of the AcrAB-TolC efflux pump actively recognizing and expelling noxious agents from the interior of bacteria. These studies have been focused on the dynamical interactions between AcrB and its substrates and inhibitors, on the details of the proton translocation mechanisms, and on the way AcrB assembles with protein part- ners to build up a functional pump. In this review we summarize these advances focusing on the role of AcrB. Keywords: Multi-drug resistance, membrane barrier, efflux pumps, RND transporters, proton motive force, AcrAB-TolC, anti- biotics, EPIs. ANTIBIOTIC RESISTANCE AND THE MEMBRANE BARRIER The re-emergence of bacterial resistance to known and new antimicrobials in the last decades is one of the major threats to public health all over the world [1-9]. New and re- emerging diseases are thought to be responsible for more than 13 million deaths worldwide each year [10]. Moreover, lethal bacterial strains, strictly confined to nosocomial set- tings until the recent past, are found these days in the com- munity with a severe frequency [11, 12]. This is a conse- quence of several factors. First, the intense (ab)use of antibiotics, biocides and her- bicides begun in the last century has prompted the evolution of defense strategies and the selection of resistant strains in a wide variety of microorganisms [13-16]. Second, despite the recognized need for new antimicro- bial agents [17, 18], pharmaceutical companies have cut their investments in antibiotic development [17, 19-21], and only in the last few years a new effort is ongoing in the field of antibacterial research [22]. As a result, only a few new classes of antibiotics have been brought to market in the last 30 years, and many companies have left the field [6, 23, 24]. Third, in addition to non-scientific concerns, the discovery and the development of novel antibacterial agents against multi-drug resistance has shown to be one of the most diffi- cult challenges for the scientific community [25-28]. Tradi- *Address correspondence to this author at the Department of Physics, Uni- versity of Cagliari, S.P. 8, km 0.700, 09042 Monserrato (CA), Italy; Tel: +390706754847; Fax: +390706753191; Email: vargiu@dsf.unica.it tional screening protocols having largely failed to address the complexity of bacterial resistance, which requires instead new and more powerful methods [11, 26, 29-31]. In view of these considerations, it is not surprising that some pathogenic bacterial strains have acquired today resis- tance to almost all known antibiotics [32-34]. These phe- nomena, known as multi, extensive or total (or pan) drug resistance (MDR, XDR and TDR (or PDR) respectively, depending on how many classes of antibiotics are effective in the treatment of the disease [35]), are related to the occur- rence of specific resistance mechanisms such as target and drug modification, and of more general ones which reduce the flux of antibiotics to the bacterial cytoplasm, where their targets reside [26, 36-38]. The reduction of intracellular drug concentration occurs by changes in membrane permeability (alterations and/or repression of porin expression), which slows down the influx of most drugs [21, 26, 29, 31, 36, 38- 41]. However, this mechanism is not sufficient to explain the high levels of resistance found in pathogenic bacteria, and the additional contribution of active exporters, the so-called efflux pumps, is necessary in order to achieve the character- istic levels of intrinsic resistance [36, 37, 40, 42-54]. The interplay between influx and efflux endows bacteria with a general mechanism of resistance that effectively keeps the concentration of noxious agents within bacteria at suble- thal levels. Furthermore, it gives bacteria the opportunity to reinforce more specific mechanisms such as enzymatic inac- tivation and modification of the drug target(s). In view of the mechanism behind MDR, it is not surprising that this phe- nomenon is particularly effective in Gram-negative bacteria, where two membranes (inner and outer respectively) enve- lope the periplasm [55-58].