Designed Azolopyridinium Salts Block Protective Antigen Pores In Vitro and Protect Cells from Anthrax Toxin

BACKGROUND: Several intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels. This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Evidence has been presented that translocation of the enzymatic components of anthrax-toxin across the endosomal membrane of target cells and channel formation by the heptameric/octameric PA(63) binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the PA(63)-channel in a dose dependent way. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the PA(63)-channel in the µM range, when both, inhibitor and PA(63) are added to the same side of the membrane, the cis-side, which corresponds to the lumen of acidified endosomal vesicles of target cells. Noise-analysis allowed the study of the kinetics of the plug formation by the heterocycles. In vivo experiments using J774A.1 macrophages demonstrated that the inhibitors of PA(63)-channel function also efficiently block intoxication of the cells by the combination lethal factor and PA(63) in the same concentration range as they block the channels in vitro. CONCLUSIONS/SIGNIFICANCE: These results strongly argue in favor of a transport of lethal factor through the PA(63)-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax.