Small-Molecule Inhibitors of Lethal Factor Protease Activity Protect against Anthrax Infection

Abstract

bBacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.

Anthrax lethal and edema toxins in anthrax pathogenesis

“…One potential advantage of CMG2-Fc is that deliberately engineered B. anthracis strains having variants of PA that evade individual neutralizing anti-PA monoclonal antibodies would remain subject to inhibition by CMG2-Fc. Small molecule inhibitors of LF are also under active development [92]. Adefovir dipivoxyl, an ATP analogue used for hepatitis B treatment in humans, was found to be an effective inhibitor of EF in vitro and in some in vivo models [72, 93, 94].…”

Efficacy of Single and Combined Antibiotic Treatments of Anthrax in Rabbits

“…Postexposure treatment with penicillin (10)(11)(12), doxycycline (10), ciprofloxacin (10,13), and levofloxacin (13) efficiently protected rhesus monkeys following inhalation of lethal doses of virulent B. anthracis spores. In guinea pigs, treatment with penicillin (14), doxycycline (15), tetracycline (16), ciprofloxacin (15,16), and erythromycin (16) protected the animals, but upon treatment termination, the animals died due to anthrax relapse (12,15,16). To develop improved/alternative treatment protocols for systemic anthrax, we previously evaluated the efficacy of antibiotic treatment at different stages based on bacteremia levels (17).…”

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.…”