Novel chemotherapeutics for treating multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) are required to combat the spread of tuberculosis, a disease that kills more than 2 million people annually. Using structure-based drug design, we have developed a series of alkyl diphenyl ethers that are uncompetitive inhibitors of InhA, the enoyl reductase enzyme in the MTB fatty acid biosynthesis pathway. The most potent compound has a Ki' value of 1 nM for InhA and MIC99 values of 2-3 microg mL(-1) (6-10 microM) for both drug-sensitive and drug-resistant strains of MTB. Overexpression of InhA in MTB results in a 9-12-fold increase in MIC99, consistent with the belief that these compounds target InhA within the cell. In addition, transcriptional response studies reveal that the alkyl diphenyl ethers fail to upregulate a putative efflux pump and aromatic dioxygenase, detoxification mechanisms that are triggered by the lead compound triclosan. These diphenyl ether-based InhA inhibitors do not require activation by the mycobacterial KatG enzyme, thereby circumventing the normal mechanism of resistance to the front line drug isoniazid (INH) and thus accounting for their activity against INH-resistant strains of MTB.

Acyl carrier proteins play a central role in metabolism by transporting substrates in a wide variety of pathways including the biosynthesis of fatty acids and polyketides. However, despite their importance, there is a paucity of direct structural information concerning the interaction of ACPs with enzymes in these pathways. Here we report the structure of an acyl-ACP substrate bound to the Escherichia coli fatty acid biosynthesis enoyl reductase enzyme (FabI), based on a combination of x-ray crystallography and molecular dynamics simulation. The structural data are in agreement with kinetic studies on wild-type and mutant FabIs, and reveal that the complex is primarily stabilized by interactions between acidic residues in the ACP helix ␣2 and a patch of basic residues adjacent to the FabI substrate-binding loop. Unexpectedly, the acyl-pantetheine thioester carbonyl is not hydrogen-bonded to Tyr 156 , a conserved component of the short chain alcohol dehydrogenase/reductase superfamily active site triad. FabI is a proven target for drug discovery and the present structure provides insight into the molecular determinants that regulate the interaction of ACPs with target proteins. Acyl carrier proteins (ACPs)7 play an essential role in a diverse array of metabolic pathways including the biosynthesis of fatty acids (1, 2), polyketides (3), membrane-derived oligosaccharides (4), lipopolysaccharides (5, 6), and phospholipids (7). In each case the growing substrate is attached via a thioester to the ACP phosphopantetheine group. ACPs must therefore be able to recognize and interact, in an acyl group-dependent manner, with a wide variety of enzymes. In eukaryotic type I fatty acid synthesis (FASI) and in polyketide biosynthesis, the ACP occurs as part of a larger polypeptide that is also associated with other catalytic activities. In contrast, in bacterial type II fatty acid biosynthesis (FASII), each of the enzyme activities as well as the ACP are encoded by separate polypeptide chains (2). ACPs that function in FASII-mediated biosynthesis are small, highly soluble, acidic proteins that vary in molecular mass from 7.5 kDa (Escherichia coli) to 13 kDa (Mycobacterium tuberculosis) (1, 8 -11).Despite the central role that ACPs play in metabolism, structural details of their interaction with target proteins are sparse. Whereas the structures of ACPs from a variety of different species have been determined by x-ray crystallography (12) and NMR spectroscopy (see for example, Refs. 13 and 14), only one structure has been determined of ACP in complex with another protein, the holo-ACP synthase (AcpS) (15), and no structural information is available for the interaction between ACP and enzymes of the fatty acid biosynthesis pathway. AcpS attaches the phosphopantetheine to the ACP serine and thus, although valuable, the complex of AcpS and ACP differs fundamentally from other ACP-protein complexes and does not provide insight into the delivery of substrate by ACP.The NMR studies reveal that ACPs are highly flexible, a structural f...

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High-throughput sequencing was applied to compare the intestinal microbiota in largemouth bronze gudgeon either healthy or affected by furunculosis. Proteobacteria, Actinobacteria, Tenericutes, Firmicutes and Bacteroidetes were detected as the predominant bacterial phyla in the gut of both diseased and healthy fish. The abundance of Proteobacteria differed significantly between the two groups of fish, mainly due to the overwhelming prevalence of Aeromonas in the diseased fish (81% ± 17%), while the genus was unevenly spread among the apparently healthy fish (33% ± 33%). The bacterial diversity in the intestine of diseased fish was markedly lower than in healthy fish. Analysis revealed the significant dissimilarity between the gut microbiota of diseased and healthy fish. The bacterial profiles in the gut were further characterized with the 28 phylotypes that were shared by the two groups. In diseased fish, two shared OTUs (OTU0001 and OTU0013) were closely related to Aeromonas salmonicida, their total proportion exceeding 70% of the sequences in diseased fish, while averaging 5.2% ± 4.6% in the healthy fish. This result suggested the presence of healthy carriers of pathogenic A. salmonicida among the farmed fish, and the gut appeared as a probable infection source for furunculosis in largemouth bronze gudgeon.

Chemicals targeting the liver stage (LS) of the malaria parasite are useful for causal prophylaxis of malaria. In this study, four lichen metabolites, evernic acid (1), vulpic acid (2), psoromic acid (3) and (+)-usnic acid (4) were evaluated against LS parasites of Plasmodium berghei. Inhibition of P. falciparum blood stage (BS) parasites was also assessed to determine stage specificity. Compound 4 displayed the highest LS activity and stage specificity (LS IC50 value 2.3 μM, BS IC50 value 47.3 μM). The compounds 1-3 inhibited one or more enzymes (PfFabI, PfFabG and PfFabZ) from the plasmodial fatty acid biosynthesis (FAS-II) pathway, a potential drug target for LS activity. To determine species specificity and to clarify the mechanism of reported antibacterial effects, 1-4 were also evaluated against FabI homologues and whole cells of various pathogens (S. aureus, E. coli, M. tuberculosis). Molecular modelling studies suggest that lichen acids act indirectly via binding to allosteric sites on the protein surface of the FAS-II enzymes. Potential toxicity of compounds was assessed in human hepatocyte and cancer cells (in vitro) as well as in a zebrafish model (in vivo). This study indicates the therapeutic and prophylactic potential of lichen metabolites as antibacterial and antiplasmodial agents.