Identification of novel host-targeted compounds that protect from anthrax lethal toxin-induced cell death

Abstract

Studying how pathogens subvert the host to cause disease has contributed to the understanding of fundamental cell biology. Bacillus anthracis, the causative agent of anthrax, produces the virulence factor lethal toxin to disarm host immunity and cause pathology. We conducted a phenotypic small molecule screen to identify inhibitors of lethal toxin-induced macrophage cell death and used an ordered series of secondary assays to characterize the hits and determine their effects on cellular function. We identified a structurally diverse set of small molecules that act at various points along the lethal toxin pathway, including inhibitors of endocytosis, natural product inhibitors of organelle acidification (e.g., the botulinum neurotoxin inhibitor, toosendanin), and a novel proteasome inhibitor, 4MNB (4-methoxy-2-[2-(5-methoxy-2-nitrosophenyl)ethyl]-1-nitrosobenzene). Many of the compounds, including three drugs approved for use in humans, also protected against the related Clostridium difficile toxin TcdB, further demonstrating their value as novel tools for perturbation and study of toxin biology and host cellular processes and highlighting potential new strategies for intervening on toxin-mediated diseases.

Figure 1

Cytoplasmic delivery of anthrax toxin 1. PA binds to one of two anthrax toxin receptors on the cell surface; 2. PA is processed by a host furin protease; 3. PA₆₃ remains bound to the receptor and oligomerizes into a heptamer or octamer; 4. binding of EF and/or LF; 5. clathrin-mediated endocytosis of the toxin-receptor complexes and trafficking to the endosome; 6. upon endosome acidification, PA undergoes a conformational change and forms a pore in the endosomal membrane; 7. translocation of EF and LF through the PA pore into the cytosol; 8. LF cleaves NLRP1b; 9. caspase-1 activation is dependent on proteasome activity; 10. caspase-1-dependent cell death.

Figure 2

Small molecule screen: primary data from screening day 2. The plot shows the z score for replicates A and B for each compound screened. Compounds that were cherrypicked and retested are shown in red.

Figure 3

Structures of selected hits from the small molecule screen. Structures of compounds from Table 2.

Figure 4

Internalization inhibitors of LT that inhibit endosome acidification Inhibition of lysotracker staining requires a higher concentration of compound than protection from LT.

Figure 5

4MNB protects against LT–induced cytotoxicity by inhibiting the proteasome a) Structure of 4MNB. b) J774 cells were treated with compound, followed by LT, and cell survival was measured. ConA is the internalization inhibitor concanamycin A and Mg132 is proteasome inhibitor that prevents LF-induced cytotoxicity. c) J774 cells were treated with compound (Boc-D-CMK 10, 5, 2.5 μM and 4MNB 80, 40, 20 μM), washed, exposed to LT, and cell survival measured. d) J774 cells were treated with compound (0.5 μM ConA and 40 μM 4MNB), exposed to ET, and cAMP levels were measured by ELISA. e) J774 cells were treated with compound (4MNB 40 μM and ConA 0.5 μM), exposed to LT, and lysates were probed for MEK3 cleavage. f) Lysates from J774 cells were treated with compound (Epoxo 10 μM and 4MNB 40 μM), incubated with substrates of the three proteasome proteases, and activity was determined by measuring fluorescence of cleaved substrates. g) J774 cells were treated with 4MNB and incubated with substrates of the proteasome or T. Proteasome activity was determined by measuring luminescence of leaved substrate and cell survival was measured. h) J774 cells were treated with compound (4MNB 80 μM and Mg132 20 μM), exposed to LPS (100 ng ml⁻¹), and lysate was collected and probed for evidence of IκBβ degradation.