The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membraneembedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein-and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gramnegative pathogens.RND efflux transporters | multidrug resistance | efflux pump inhibitors | X-ray crystallography | molecular dynamics simulation

cMembers of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC of Escherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of the Enterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versus E. coli AB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 M) in combination with 0.016 g/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) of E. coli AB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 g/ml CIP alone. In contrast, phenyl-arginine-␤-naphthylamide (PA␤N), a known EPI, did not increase the bactericidal activity of 0.016 g/ml CIP at concentrations as high as 100 M. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active against Enterobacteriaceae species and Pseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens. Multidrug resistance (MDR) in Gram-negative pathogens, including Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp., and Stenotrophomonas maltophilia, poses a significant threat to the effective treatment of infections caused by these organisms (1-4). The MDR threat has been exacerbated by the recent decrease in commercial efforts to discover and develop new antibacterial agents. In addition, antibacterial agents that have been introduced recently into the clinic or are in development, such as daptomycin, gemifloxacin, telithromycin, and telavancin, are not active against Gram-negative pathogens. Recently FDAapproved agents with activity against Gram-negative bacteria include tigecycline and doripenem. While tigecycline is active against bacteria producing a tetracycline-specific pump in vitro, it is pumped out rapidly by the ubiquitous multidrug pumps, and its pharmacokinetic properties limit its use for treating urinary tract infections (UTIs) and bloodstream infections (5), as will the evolution of resistance during therapy (6). Clearly, novel strategies for effectively treating infections caused by MDR Gram-negative pathogens are urgently needed.The MDR phenotype has been attributed to both acquired and intrinsic mechanisms of resistance. However, the resistance-nodulation-division (RND) efflux pumps of Gram-negative bacteria play a major role in MDR. Because of their broad substrate specificity, overexpression of these efflux pumps results in decreased susceptibility to a diverse array of antibacterial agents and biocides (7). The major ef...

An in vitro assay is presented for culturing staphylococcal biofilms and biofilms of nonmotile Gram‐positive bacteria under static conditions in microtiter assay plates, and for the quantification of biofilm growth, using a simple staining procedure that measures amounts of bacterial cells and extracellular matrix. This basic assay can be adapted readily to study several aspects of biofilm formation, for high‐throughput screening to identify small molecule inhibitors of biofilm formation or biofilm‐defective mutants, and for quantifying the anti‐biofilm activity of biofilm inhibitors. Curr. Protoc. Pharmacol. 50:13A.8.1‐13A.8.23. © 2010 by John Wiley & Sons, Inc.

Recently we described a novel pyranopyridine inhibitor (MBX2319) of RND-type efflux pumps of the Enterobacteriaceae. MBX2319 (3,3-dimethyl-5-cyano-8-morpholino-6-(phenethylthio)-3,4-dihydro-1H-pyrano[3,4-c]pyridine) is structurally distinct from other known Gram-negative efflux pump inhibitors (EPIs), such as 1-(1-naphthylmethyl)-piperazine (NMP), phenylalanylarginine-β-naphthylamide (PAβN), D13-9001, and the pyridopyrimidine derivatives. Here, we report the synthesis and biological evaluation of 60 new analogs of MBX2319 that were designed to probe the structure activity relationships (SARs) of the pyranopyridine scaffold. The results of these studies produced a molecular activity map of the scaffold, which identifies regions that are critical to efflux inhibitory activities and those that can be modified to improve potency, metabolic stability and solubility. Several compounds, such as 22d–f, 22i and 22k, are significantly more effective than MBX2319 at potentiating the antibacterial activity of levofloxacin and piperacillin against Escherichia coli.

Staphylococcus epidermidis and Staphylococcus aureus are the leading causative agents of indwelling medical device infections because of their ability to form biofilms on artificial surfaces. Here we describe the antibiofilm activity of a class of small molecules, the aryl rhodanines, which specifically inhibit biofilm formation of S. aureus, S. epidermidis, Enterococcus faecalis, E. faecium, and E. gallinarum but not the gram-negative species Pseudomonas aeruginosa or Escherichia coli. The aryl rhodanines do not exhibit antibacterial activity against any of the bacterial strains tested and are not cytotoxic against HeLa cells. Preliminary mechanism-of-action studies revealed that the aryl rhodanines specifically inhibit the early stages of biofilm development by preventing attachment of the bacteria to surfaces.Indwelling medical devices have become integral components of modern health care. The surfaces of these devices can be colonized by bacterial pathogens that are capable of forming biofilms. Resulting biofilms frequently compromise the function of the device or cause serious systemic infections. These device-related infections are often very difficult to eradicate with conventional antibiotics, as bacteria and fungi growing in the biofilm mode of growth are extremely resistant to antibiotics, biocides, and attack from the immune system (9). Consequently, device-related infections result in increased patient morbidity, mortality, and health care costs (7,46).Because device colonization precedes device-related infections, several approaches have been used to prevent biofilm colonization of indwelling devices, such as central venous catheters. These include the implementation of stringent infection control measures by health care workers during the insertion and maintenance of vascular access devices. Such measures have been shown to decrease the incidence of catheter-related bloodstream infections when there is strict compliance with established protocols (38). Alternatively, medical devices coated with antimicrobial compounds have been tested for prevention of bacterial colonization. Central venous catheters impregnated with a combination of antibacterial agents (rifampin [rifampicin] and minocycline) or coated with a combination of antiseptics (chlorhexidine and silver sulfadiazine) have been shown to be effective in reducing catheter colonization and catheter-related bloodstream infections in clinical studies (reviewed in reference 45). However, there are lingering concerns related to the use of antiseptics and antibiotics as coatings for medical devices. For example, it is possible that the use of antibiotic-impregnated catheters could lead to increases in the occurrence of strains resistant to antimicrobial agents. While increased resistance to antibiotics used as catheter coatings has not been detected in several in vitro and in vivo studies (14,27,32,45), evidence suggests that rifampinminocycline-impregnated catheters are more susceptible to colonization when challenged with a rifampin-resistant str...