Efficient synthetic inhibitors of anthrax lethal factor

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   Fig. 1.

   Inhibition of anthrax LF. (a) ¹⁹F NMR spectra of the peptide substrate in presence of LF. (b) Effect of GM6001 (20 μM). (c) Effect of BI MFM3 (20 μM). (d) Effect of BI-11B3 (0.8 μM). (e)IC₅₀ evaluation for compound BI-MFM3;(f) Lineweaver–Burk K _m and K _m(app) evaluation for LF, BI-MFM3, and BI-11B3, respectively. Each measurement was performed in triplicate. (g) Synthetic scheme adopted for the synthesis of compounds listed in Table 2.

   
   
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   Fig. 2.

   In vitro and cell-based evaluation. (a) BI-11B2 efficiently protects the purified MAPKK-1 against LF cleavage in vitro. BI-11B2 and GM6001 (as control) were each coincubated with LF and MAPKK1. The digest samples were analyzed by SDS/PAGE to determine the specific conversion of MAPKK1 into the 45-kDa cleavage product. (b) Inhibitors BI-11B2 and BI-11B3 are effective in protecting MAPKK1 and murine macrophage RAW264.7 cells against LF. Cells were coincubated with anthrax PA (500 ng/ml) and LF (40 ng/ml). The indicated concentrations of the inhibitors were added to the cells. In 4 h, the residual viable cells were measured by adding the tetrazolium salt 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The data show that inhibitors BI-11B2 (open circles) and BI-11B3 (filled circles) protect cells from the cytotoxic effect by LF and PA.

   
   
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   Fig. 3.

   Crystal structure of the LF-BI-MFM3–zinc complex. (a) Detailed view of the electron density trace and overall model fit of BI-MFM3.(b) Detail of the binding site of LF for MFM3 (both shown in stick representation). These data are at a resolution limit of 2.67 Å. The small molecule appears to be interacting with the zinc atom in the LF active site via an S atom. Additional interactions are mainly of hydrophobic nature involving the aromatic rings of the inhibitor and hydrophobic side chains of LF. Prepared by using spock (http://quorum.tamu.edu/spock) and sybyl (Tripos Associates, St. Louis).

   
   
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   Fig. 4.

   Comparison of survival rates between different treatments regimes. DBA2 mice were infected with B. anthracis Sterne spores at a dosage of 2 × 10⁷ per mouse in 200 μl of PBS on day 0 through i.p. injection. Animals were treated with ciprofloxacin alone or in combination with lethal toxin blocking substance B1-11B3. Similar data were obtained with compound BI-11B1 (not shown). Treatment was started 24 h postexposure and continued for 10 days. Nontreated mice were used as a control. Animals were monitored for 14 days after infection.