Cross-inhibition of pathogenic agents and the host proteins they exploit.

The traditional method of treating most human diseases is to direct a therapy against targets in the host patient, whereas conventional therapies against infectious diseases are directed against the pathogen. Unfortunately, the efficacy of pathogen-oriented therapies and their ability to combat emerging threats such as genetically engineered and non-traditional pathogens and toxins have been limited by the occurrence of mutations that render pathogen targets resistant to countermeasures. Thus, host proteins exploited by pathogens are potential targets for therapies1. Host proteins and pathways exploited by Bacillus anthracis toxins are well understood2. B. anthracis causes anthrax infections and leads to toxemia in humans and animals, rendering antibiotic therapies ineffective in the later stages of infection. The major virulence factors of the bacterium include an exotoxin protein complex consisting of protective antigen (PA) and lethal factor (LF), which act collectively to damage the host2. Proteases play important roles in anthrax toxin mediated host-cell killing. PA binds to host cellular receptors in the native form of 83 kDa (PA83)3,4, and once bound, host protease furin cleaves a 20 kDa fragment from the N-terminus of PA, thus activating the PA protein of 63 kDa (PA63)5. Following activation, PA forms a heptamer and binds LF6. The toxin undergoes clathrin-type endocytosis, mediated by another set of host proteases, calpains and cathepsin B7,8. A decrease in endosomal pH induces the formation of an endosomal membrane PA channel, by which LF translocates into the cytosol9. Once in the cytosol, LF itself acts as a protease that cleaves and inactivates host mitogen-activated protein kinase kinases (MAPKK) 1–4, 6, and 710. The MAPKK cleavage event prevents the passage of signals in the ERK1/2, p38, and c-Jun N-terminal kinase pathways10,11, which mediate responses to a variety of cellular stresses. In addition, rat NLRP1 and mouse NLRP1b proteins can also be directly cleaved by LF at sites near their N termini11,12. The cleavage of host proteins by LF results in the activation of the inflammasomes, resulting in rapid macrophage cell death mediated by additional host proteases, caspases-1 and -311,12. While the discovery of LF inhibitors has focused on new chemical compounds that either inhibit its protease activity or its cytoplasmic entry (reviewed in13), repurposing of existing drugs that simultaneously inhibit LF and the host proteases that assist LF, offers potential advantages. We used a fluorescence resonance energy transfer (FRET) assay, where LF cleaves a MAPKK2 peptide, to screen and identify approved drugs that affect the rate of the proteolytic reaction. We identified chemical and peptidic compounds that effectively inhibited cleavage of MAPKK2 peptide, as well as host furin, calpain, cathepsin B, and caspases. Two of those chemicals, ascorbic acid 6-palmitate and salmon sperm protamine, suppressed LF-induced cell death, as well as the cytotoxicity induced by cholera toxin and Pseudomonas aeruginosa exotoxin A. This study offers new solutions to treat these infectious diseases by using drugs that cross-inhibit pathogen and host targets.