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 ciprofloxacin 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.

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.

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.

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).

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.