Small Molecule Inhibitors of Bacillus anthracis Protective Antigen Proteolytic Activation and Oligomerization

Abstract

Protective antigen (PA), lethal factor, and edema factor, the protein toxins of Bacillus anthracis, are among its most important virulence factors and play a key role in infection. We performed a virtual ligand screen of a library of 10,000 members to identify compounds predicted to bind to PA and prevent its oligomerization. Four of these compounds slowed PA association in a FRET-based oligomerization assay, and two of those protected cells from intoxication at concentrations of 1–10 μM. Exploration of the protective mechanism by Western blot showed decreased SDS-resistant PA oligomer on cells, and surprisingly, decreased amounts of activated PA. In vitro assays showed that one of the inhibitors blocked furin-mediated cleavage of PA, apparently through its binding to the PA substrate. Thus, we have identified inhibitors that can independently block both PA’s cleavage by furin and its subsequent oligomerization. Lead optimization on these two backbones may yield compounds with high activity and specificity for the anthrax toxins.

Monitoring the Kinetics of the pH-Driven Transition of the Anthrax Toxin Prepore to the Pore by Biolayer Interferometry and Surface Plasmon Resonance

“…Using BLI or SPR label free approaches, one could then directly test if synthesized protein stabilizer candidates inhibit the PA pore transitions in endosomal-like solution environments. A similar in silico approach was implemented by Wein et al, where they used initially used virtual screening methodologies to identify potential inhibitors to PA prepore oligomerization (32) . They then tested a number of positive candidates directly using cell based systems and found that in addition to oligomer inhibition, some of these compounds also inhibited the furin based proteolytic processing of the PA protective antigen (32) .…”

Anthrax Pathogenesis

“…Additional therapeutics targeting PA and its action include receptor decoys (225,231,247), polyvalent competitive peptides (14), and small molecules that inhibit receptor-toxin interaction (50). Inhibitors that act on the PA translocation function include dominant negative mutant PA proteins (28, 229), small-molecule oligomerization inhibitors (195,268), β-cyclodextrin-based blockers of the PA oligomer channel (114), and other pore-blocking agents (for a review see 180). A different approach has been to interfere with EF/LF binding to PA (178).…”

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

“…Combination of this agent with chloroquine had additive effects in the macrophage lysis assay. Studies described below found that compounds targeting PA monomer oligomerization could also inhibit furin cleavage of PA [71]. …”

“…Cisplatin protected BALB/cJ mice and Fisher rats when co-administered with an LT challenge, but not when administered 2 h before or after LT challenge. Another group employed ICMPocketFinder to identify pockets in the PA monomer interface necessary for oligomerization and then screened the Chem-bridge library for compounds predicted to bind with these sites [71]. Four compounds [5180717(17), 5181401(01), 5181385(85) and 5117235(35)] were identified that targeted a pocket close to the furin-binding site, protected against LT in the macrophage lysis assay and inhibited PA oligomerization in vitro .…”