The Francisella tularensis FabI enoyl-acyl carrier protein reductase gene is essential to bacterial viability and is expressed during infection.

Francisella tularensis is classified as a category A priority pathogen and causes fatal disseminated disease in humans upon inhalation of less than 50 bacteria. Although drugs are available for treatment, they are not ideal because of toxicity and route of delivery, and in some cases patients relapse upon withdrawal. We have an ongoing program to develop novel FAS-II FabI enoyl-ACP reductase enzyme inhibitors for Francisella and other select agents. To establish F. tularensis FabI (FtFabI) as a clinically relevant drug target, we demonstrated that fatty acid biosynthesis and FabI activity are essential for growth even in the presence of exogenous long-chain lipids and that FtfabI is not transcriptionally altered in the presence of exogenous long-chain lipids. Inhibition of FtFabI or fatty acid synthesis results in loss of viability that is not rescued by exogenous long-chain lipid supplementation. Importantly, whole-genome transcriptional profiling of F. tularensis with DNA microarrays from infected tissues revealed that FtfabI and de novo fatty acid biosynthetic genes are transcriptionally active during infection. This is the first demonstration that the FabI enoyl-ACP-reductase enzyme encoded by F. tularensis is essential and not bypassed by exogenous fatty acids and that de novo fatty acid biosynthetic components encoded in F. tularensis are transcriptionally active during infection in the mouse model of tularemia.

Synthesis and antitubercular activity of heterocycle substituted diphenyl ether derivatives.

Despite being an ancient disease, tuberculosis (TB) remains the leading single-agent infectious disease killer in the world. The emerging serious problem of TB control and clinical management prompted us to synthesize a novel series of heterocyclic substituted diphenyl ether derivatives and determine their activity against the H37Rv strain of Mycobacterium. All ten compounds inhibited the growth of the H37Rv strain of Mycobacterium at concentrations of 1 microg/mL. This activity was found to be comparable to the reference drugs rifampicin and isoniazid at the same concentration. While the antimicrobial activity of other diphenyl ether analogues, such as triclosan, is associated with the inhibition of enoyl-ACP reductase (ENR), the synthesised substituted diphenyl ether derivatives did not affect this enzyme activity in spite of their structural similarity with triclosan. Therefore, these compounds appear to have a novel mechanism of action against M. tuberculosis, and their structural features should be studied further for their potential as new antitubercular drugs.

Functional expression of Francisella tularensis FabH and FabI, potential antibacterial targets.

Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40 microM with IC(50) value of 2 microM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5 microM with IC(50) value of 6 microM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia.