Inhibitions of the translocation pore of Clostridium botulinum C2 toxin by tailored azolopyridinium salts protects human cells from intoxication.

C2 toxin from Clostridium botulinum represents the prototype of clostridial binary actin ADP-ribosylating toxins which destroy the actin-cytoskeleton of mammalian cells and cause severe enteric diseases in humans and animals. After receptor-mediated endocytosis of the C2 toxin complex, the binding/translocation component C2IIa forms a heptameric transmembrane pore in membranes of acidified endosomal vesicles. The separate ADP-ribosyltransferase component C2I translocates through this C2IIa-pore from the endosomal lumen into the cytosol. Here we demonstrate that positively charged heterocyclic azolopyridinium salts which were developed as pore blockers for the anthrax toxins, efficiently protect cultured mammalian cells from intoxication with C2 toxin. The inhibitors had no effects on enzyme activity of C2I or receptor binding of C2 toxin but inhibited the pH-dependent membrane translocation of C2I in living cells, most likely by blocking the C2IIa-translocation pores. In vitro, the substances blocked C2IIa-pores in black lipid bilayer membranes when applied to the cis-side of the membrane which corresponds to the endosomal lumen of cells. Thus, heterocyclic azolopyridinium salts could represent lead compounds for development of novel therapeutics against binary clostridial toxins.

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 PA63 binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the PA63-channel in a dose dependent way. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the PA63-channel in the µM range, when both, inhibitor and PA63 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 PA63-channel function also efficiently block intoxication of the cells by the combination lethal factor and PA63 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 PA63-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax.