Enoyl-acyl carrier protein (ACP) reductases are critical for bacterial type II fatty acid biosynthesis and thus are attractive targets for developing novel antibiotics. We determined the crystal structure of enoyl-ACP reductase (FabK) from Streptococcus pneumoniae at 1.7 Å resolution. There was one dimer per asymmetric unit. Each subunit formed a triose phosphate isomerase (TIM) barrel structure, and flavin mononucleotide (FMN) was bound as a cofactor in the active site. The overall structure was similar to the enoyl-ACP reductase (ER) of fungal fatty acid synthase and to 2-nitropropane dioxygenase (2-ND) from Pseudomonas aeruginosa, although there were some differences among these structures. We determined the crystal structure of FabK in complex with a phenylimidazole derivative inhibitor to envision the binding site interactions. The crystal structure reveals that the inhibitor binds to a hydrophobic pocket in the active site of FabK, and this is accompanied by induced-fit movements of two loop regions. The thiazole ring and part of the ureido moiety of the inhibitor are involved in a face-to-face p-p stacking interaction with the isoalloxazine ring of FMN. The side-chain conformation of the proposed catalytic residue, His144, changes upon complex formation. Lineweaver-Burk plots indicate that the inhibitor binds competitively with respect to NADH, and uncompetitively with respect to crotonoyl coenzyme A. We propose that the primary basis of the inhibitory activity is competition with NADH for binding to FabK, which is the first step of the two-step ping-pong catalytic mechanism.Keywords: FabK; enoyl-acyl carrier protein reductase; fatty acid biosynthesis; antibiotics; inhibitor Streptococcus pneumoniae causes community-acquired infections such as pneumonia, otitis media, and meningitis. The increase of penicillin-and/or macrolide-resistant S. pneumoniae is of great concern worldwide, and, moreover, the emergence of quinolone-resistant S. pneumoniae has been reported recently (Cohen 1992;Bartlett et al. 1998;Johnson et al. 2005). A key strategy to overcoming antibiotic resistance is the discovery of antibacterial agents with novel mechanisms of action that have no crossresistance.The bacterial type II fatty acid synthase complex comprises discrete enzyme activities encoded by discrete genes, in contrast to the multifunctional type I fatty acid synthase in mammals (Rock and Cronan 1996). These bacterial enzymes are attractive targets for the development of novel selective antibacterial agents (Heath et al. 2001). Enoyl-acyl carrier protein (ACP) reductase is Reprint requests to: Jun Saito, Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan; e-mail: jun_saito@meiji.co.jp; fax: 81-45-545-3152.Abbreviations: ACP, acyl carrier protein; FAD, flavin adenine dinucleotide; FAS II, bacterial type II fatty acid biosynthesis; FMN, flavin mononucleotide; MAD, multiple-wavelength anomalous dispersion; NADH, nicotinamide adenine dinucleotide; ER, enoyl-ACP ...

Polytheonamide B is by far the largest non-ribosomal peptide known at present, and displays extraordinary cytotoxicity (EC(50) = 68 pg ml(-1), mouse leukaemia P388 cells). Its 48 amino-acid residues include a variety of non-proteinogenic d- and l-amino acids, and the absolute stereochemistry of these amino acids alternate in sequence. These structural features induce the formation of a stable β-strand-type structure, giving rise to an overall tubular structure over 30 Å in length. In a biological setting, this fold is believed to transport cations across the lipid bilayer through a pore, thereby acting as an ion channel. Here, we report the first chemical construction of polytheonamide B. Our synthesis relies on the combination of four key stages: syntheses of non-proteinogenic amino acids, a solid-phase assembly of four fragments of polytheonamide B, silver-mediated connection of the fragments and, finally, global deprotection. The synthetic material now available will allow studies of the relationships between its conformational properties, channel functions and cytotoxicity.

FabI and FabK are bacterial enoyl-acyl carrier protein (ACP) reductases that catalyze the final and rate-limiting step of bacterial fatty acid biosynthesis (FAS) and are potential targets of novel antibacterial agents. We have reported 4-pyridone derivative 3 as a FabI inhibitor and phenylimidazole derivative 5 as a FabK inhibitor. Here, we will report phenylimidazole derivatives of 4-pyridone as FabI and FabK dual inhibitors based on an iterative medicinal chemistry and crystallographic study of FabK from Streptococcus pneumoniae/compound 26. A representative compound 6 showed strong FabI inhibitory (IC50 = 0.38 microM) and FabK inhibitory (IC50 = 0.0045 microM) activities with potent antibacterial activity against S. pneumoniae (MIC = 0.5 microg/mL). Since elevated MIC value was observed against S. pneumoniae mutant possessing one amino acid substitution in FabK, the antibacterial activity of the compound was considered to be due to the inhibition of FabK. Moreover, this compound showed no significant cytotoxicity (IC50 > 69 microM). These results support compound 6 as a novel agent for the treatment of bacterial infections.

We examined the combined effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa in biofilms by using an in vitro experimental system with a modified Robbins device. Sessile cells in a mature or immature biofilm, developed on a silicon disk, were used, and an ATP bioluminescence assay was employed to assess antibacterial effects. A synergistic effect of fosfomycin and ofloxacin was clearly detected when concentrations at which each drug independently produced no detectable decrease in the bioactivity of sessile cells were used. Exposure of the cells in a mature biofilm to fosfomycin at concentrations of one-eighth of the MIC to 10 times the MIC (6.25 to 500 g/ml) and ofloxacin at three or 10 times the MIC (18.75 or 62.5 g/ml) resulted in reduction of the bioactivity to 1.5 to 4.5% after 72 h. Young sessile cells in an immature biofilm were more susceptible to this combination therapy. With a combination of fosfomycin at three times the MIC and ofloxacin at three times the MIC, complete eradication was confirmed by both ATP assay and scanning electron microscopy.Pseudomonas aeruginosa is a problematic organism in acute and chronic human infections, including urinary tract infections (14). In chronic infections, the cells are protected by an extracellular polymeric substance (glycocalyx) and exhibit resistance to most antibiotics at high doses (4,11,17). In our in vitro studies with a modified Robbins device, only fluoroquinolones showed a considerable killing effect on P. aeruginosa in biofilms (15). However, the effect of quinolones is limited and there is no available single drug which is sufficiently active against the cells in a biofilm to be of clinical significance.In the present study, we examined the combination of fosfomycin with ofloxacin against P. aeruginosa in biofilms. It is believed that the barrier to drug penetration formed by the exopolysaccharide and the low growth rate of bacteria in biofilms confer relative drug resistance (11,15). Fluoroquinolones are drugs which penetrate exopolysaccharide and attack slowly growing cells relatively well (11,15,17). Fosfomycin [(2R-cis)-(3-methyloxiranyl)-phosphonic acid] is a unique antibiotic which is chemically unrelated to any other known antimicrobial agent. Fosfomycin is actively taken into bacterial cells through their two nutrient transport systems (12) and inhibits the initial step in cell wall synthesis by inhibiting phosphoenolpyruvate synthesis (10,12). This characteristic entry and action of fosfomycin were not necessarily affected by the growth rate (9), and the penetration of fosfomycin was not inhibited by Pseudomonas exopolysaccharides in the penetration assay using a sandwich cup method (17). Therefore, we selected fosfomycin for use in combination with a fluoroquinolone for elimination of P. aeruginosa in biofilms. MATERIALS AND METHODSBacteria and culture conditions. P. aeruginosa 24) isolated from a patient with a catheter-associated urinary tract infection was used throughout this study. The culture conditions used to produce ...