Tailored Cyclodextrin Pore Blocker Protects Mammalian Cells from Clostridium difficile Binary Toxin CDT

Abstract

Some Clostridium difficile strains produce, in addition to toxins A and B, the binary toxin Clostridium difficile transferase (CDT), which ADP-ribosylates actin and may contribute to the hypervirulence of these strains. The separate binding and translocation component CDTb mediates transport of the enzyme component CDTa into mammalian target cells. CDTb binds to its receptor on the cell surface, CDTa assembles and CDTb/CDTa complexes are internalised. In acidic endosomes, CDTb mediates the delivery of CDTa into the cytosol, most likely by forming a translocation pore in endosomal membranes. We demonstrate that a seven-fold symmetrical positively charged β-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-β-cyclodextrin, which was developed earlier as a potent inhibitor of the translocation pores of related binary toxins of Bacillus anthracis, Clostridium botulinum and Clostridium perfringens, protects cells from intoxication with CDT. The pore blocker did not interfere with the CDTa-catalyzed ADP-ribosylation of actin or toxin binding to Vero cells but inhibited the pH-dependent membrane translocation of CDTa into the cytosol. In conclusion, the cationic β-cyclodextrin could serve as the lead compound in a development of novel pharmacological strategies against the CDT-producing strains of C. difficile.

Chloroquine Analog Interaction with C2- and Iota-Toxin in Vitro and in Living Cells

“…Possible candidates are tailored azolopyridinium salts and tailored cyclic dextrines [33,34,35,36]. In previous studies, we have demonstrated that low concentrations of chloroquine were able to inhibit intoxication of target cells by C2-toxin in cell-based assays and pore-formation by C2IIa in lipid bilayer membranes [37,38].…”

Inhibitors of pore-forming toxins

“…This chapter has provided a vivid example of one of the first successful designs of such wide-spectrum antitoxins. Indeed, the custom-synthesized, positively charged polyvalent cyclodextrin-based AMBnTβCD compound was shown to be protective against the cytotoxicity induced by four different toxins: anthrax toxins of B. anthracis [104], clostridial C2, iota [106], and CDT toxins [84]. The universality idea is easy to perceive and appreciate because a potent, wide-spectrum antidote would be expected to be active against more than one virulent agent.…”

“…Multiple compounds, which are charged positively at mildly acidic pH values, were shown to interact with PA 63 , C2IIa, and Ib channels incorporated into planar bilayers [38,67,72,74,75,83,84] (Figure 38.2). The ability of tetraalkylammonium ions to translocate through PA 63 , reversibly blocking the K + current [76,85,86], was used to estimate the minimum physical size of the PA 63 pore lumen to be about 12 Å.…”

“…This led researchers to a design of synthetic tailor-made cationic 7-positively charged blockers based on a sevenfold symmetrical β-cyclodextrin (7+βCD) core (reviewed in [103]) (Table 38.2). The designed compounds have shown in vitro, in cell cultures, and in vivo (in the case of anthrax toxin) activity against four different binary bacterial toxins (i.e., anthrax, C2, iota, and CDT), thus representing the potential for the development of universal blockers against these toxins [77,84,89,[104][105][106][107]. The cyclodextrins, which are cyclic oligomers of glucose, have a long and successful history of being employed in the pharmaceutical, agrochemical, environmental, cosmetic, and food industries [108] because of their ability to form water-soluble inclusion complexes with different small molecules and portions of large molecules.…”

“…The most active of them so far identified is a β-CD-based inhibitor of PA 63 , AMBnTβCD. It fully protects cultured cells from intoxication with three clostridial binary toxins (C2, iota [106] and CDT [84]) by inhibiting the membrane translocation of the A components (C2I, Ia, CDTa) and blocking the ion current through heptameric pores formed by C2IIa and Ib in planar lipid bilayers in vitro [106]. Because PA 63 insertion is always directional, trans-blocker addition was also examined [77].…”