Pharmacophore Selection and Redesign of Non-nucleotide Inhibitors of Anthrax Edema Factor

Abstract

Antibiotic treatment may fail to protect individuals, if not started early enough, after infection with Bacillus anthracis, due to the continuing activity of toxins that the bacterium produces. Stable and easily stored inhibitors of the edema factor toxin (EF), an adenylyl cyclase, could save lives in the event of an outbreak, due to natural causes or a bioweapon attack. The toxin’s basic activity is to convert ATP to cAMP, and it is thus in principle a simple phosphatase, which means that many mammalian enzymes, including intracellular adenylcyclases, may have a similar activity. While nucleotide based inhibitors, similar to its natural substrate, ATP, were identified early, these compounds had low activity and specificity for EF. We used a combined structural and computational approach to choose small organic molecules in large, web-based compound libraries that would, based on docking scores, bind to residues within the substrate binding pocket of EF. A family of fluorenone-based inhibitors was identified that inhibited the release of cAMP from cells treated with EF. The lead inhibitor was also shown to inhibit the diarrhea caused by enterotoxigenic E. coli (ETEC) in a murine model, perhaps by serving as a quorum sensor. These inhibitors are now being tested for their ability to inhibit Anthrax infection in animal models and may have use against other pathogens that produce toxins similar to EF, such as Bordetella pertussis or Vibrio cholera.

Efficacy of Single and Combined Antibiotic Treatments of Anthrax in Rabbits

“…In rabbits, treatment with ciprofloxacin (17), levofloxacin (18), or doxycycline (17) was efficient in preventing the death of animals with systemic anthrax. Furthermore, combination of anti-PA antibodies with either ciprofloxacin or levofloxacin therapy demonstrated improved efficacy compared to the single-antibiotic treatment (17,19).…”

Anthrax Pathogenesis

“…Thus, it is important for the next generation of therapeutics to target the EF and LF enzymatic activities in cells. Multiple approaches are being used to develop small-molecule inhibitors of these enzymes that can intervene at any stage of disease, and efficacy for some has been demonstrated in animal models (35, 109,110,121,168,222,228,235,237). For a complete and excellent recent review of all current anthrax therapeutics, see Reference 181.…”

An overview of investigational toxin-directed therapies for the adjunctive management ofBacillus anthracisinfection and sepsis

“…Adefovir inhibited EF-stimulated cAMP production and its cardiovascular effects in perfused heart and aortic ring models [22,25]. Other agents shown to have the potential to inhibit EF's enzymatic activity include 4-[4-(4-nitrophenyl)-thiazolylamino]-benzene-sulfonamide [140,141], 3-{(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid [142,143], and (M)Ant nucleotides [144-149] and allosteric inhibitors [150]. …”