Efficient synthetic inhibitors of anthrax lethal factor

Abstract

Inhalation anthrax is a deadly disease for which there is currently no effective treatment. Bacillus anthracis lethal factor (LF) metalloproteinase is an integral component of the tripartite anthrax lethal toxin that is essential for the onset and progression of anthrax. We report here on a fragment-based approach that allowed us to develop inhibitors of LF. The small-molecule inhibitors we have designed, synthesized, and tested are highly potent and selective against LF in both in vitro tests and cell-based assays. These inhibitors do not affect the prototype human metalloproteinases that are structurally similar to LF. Initial in vivo evaluation of postexposure efficacy of our inhibitors combined with antibiotic ciprofloxican against B. anthracis resulted in significant protection. Our data strongly indicate that the scaffold of inhibitors we have identified is the foundation for the development of novel, safe, and effective emergency therapy of postexposure inhalation anthrax.

Fluorine NMR‐Based Screening on Cell Membrane Extracts

“…n-FABS has been successfully applied to several pharmaceutically relevant targets resulting in the identification of novel inhibitors. [5][6][7][8][9][10][11] In a recent work, we reported its application to the screening of chemical fragments against a purified recombinant membrane enzyme. [12] Indeed, membrane proteins are challenging targets for biochemical assays.…”

Fluorine nuclear magnetic resonance-based assay in living mammalian cells

“…The samples were analyzed, as described before, by recording 19 F and 1 H NMR spectra. The integrals of the product 19 F signal were measured and plotted as a function of the inhibitor concentration in order to obtain the half-maximal inhibitory concentration (IC 50 ) value. The best data fit was performed using GraphPad Prism 5.…”

“…1, which shows the 19 F NMR spectra recorded as a function of incubation time. 19 F NMR signals labeled S and P correspond to the signal of the substrate, ARN1203, and the fluorinated product of the enzymatic reaction, 1-amino-3-fluoropropan-2-ol, respectively. As the incubation time increases, the area of the substrate signal decreases and the area of the product signal increases.…”

“…First, we show that it is possible to determine target inhibition in intact cells using 19 F NMR spectroscopy. Second, we report that coupling 1 H NMR to 19 F NMR spectroscopy allows obtaining a preliminary metabolic fingerprint of the cell system, which could reveal metabolic alterations and/or toxic effects of tested compounds.…”

“…Recently, 1 H NMR spectroscopy was used to screen small molecules in intact bacterial cells [10], showing that it is possible to use this technology in living cells for the identification of inhibitors against a specific target and for the evaluation of their potency. Other NMR approaches for screening are based on the use of 19 F NMR spectroscopy [11,12]. One of these is the functional assay n-FABS (n-fluorine atoms for biochemical screening) [13,14].…”