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

Abstract

BackgroundSeveral 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 FindingsHere 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/SignificanceThese 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.

Pore-forming activity of clostridial binary toxins

“…The small cationic blockers including tetraalkylammonium, chloroquine, quinacrine and related compounds (see [119]) as mentioned above efficiently inhibit PA and C2-II but poorly Ib. Similarly, positively charged heterocyclic azazolopyridinium salts are efficient inhibitors of anthrax toxins and C2 toxin in cell model [129,130]. The rationale to use cyclodextrin derivatives is to develop low molecular cyclic compound having a shape able to block the toxin pores and to prevent the translocation of the enzymatic components [119].…”

Inhibitors of pore-forming toxins

“…By showing that blocker activity is determined by the sophisticated interplay between the different hydrophobic and electrostatic effects [115], researchers have opened ample avenues for rational discovery of the universal pore-inhibiting agents of the future. Thus, several synthesized azolopyridinium salts [97,98] and commercially available cationic PAMAM dendrimers [118] were recently shown to be universally effective against both anthrax and C2 bacterial toxins. When optimized, these or other positively charged molecules might be considered for further pharmaceutical development.…”

“…These findings show that the substances carrying both the positively charged and the bulky hydrophobic aromatic groups could represent lead compounds suitable for further rational modifications as binary toxin inhibitors. A group of heterocyclic azolopyridinium salts was recently examined to probe their inhibitory activity against PA 63 and C2IIa components in the model bilayers and their protective effect against anthrax and C2 toxins in cell assays [97,98]. Several of these compounds, while being active in the low-micromolar concentrations in vitro, were fully protective against toxin action in cell assays and had only negligible cytotoxic effects.…”

“…In contrast, the off-rate showed significantly higher variations, increasing when both the size of the ligands and the number of aromatic groups decreased. The highest binding affinity was observed for salts that had a nitroxyl group bound to an aromatic ring [97].…”

Exploiting endocytic pathways to prevent bacterial toxin infection

“…The pore formed by anthrax toxin can be blocked by heterocyclic azolopyridinium salts, resulting in an almost complete inhibition of toxicity in J774.A1 cells [112]. Indeed, targeting the intracellular trafficking of toxins has proved to be beneficial against a number of bacterial toxins.…”