Symmetry Requirements for Effective Blocking of Pore-Forming Toxins: Comparative Study with α-, β-, and γ-Cyclodextrin Derivatives

Abstract

We compared the abilities of structurally related cationic cyclodextrins to inhibit Bacillus anthracis lethal toxin and Staphylococcus aureus ␣-hemolysin. We found that both ␤-and ␥-cyclodextrin derivatives effectively inhibited anthrax toxin action by blocking the transmembrane oligomeric pores formed by the protective antigen (PA) subunit of the toxin, whereas ␣-cyclodextrins were ineffective. In contrast, ␣-hemolysin was selectively blocked only by ␤-cyclodextrin derivatives, demonstrating that both symmetry and size of the inhibitor and the pore are important.Previously, we proposed a novel approach for the discovery of inhibitors of pore-forming toxins that involves the blockage of the pores using molecules with comparable dimensions and the same symmetry as the target pores. It allows for the identification of lead compounds faster and significantly more cheaply in comparison with the existing industry standards. First, this approach was successfully tested on anthrax lethal toxin (LeTx), which plays a key role in anthrax infection. The toxin was disabled by the blockage of the pore formed by protective antigen (PA 63 ), an essential component of anthrax toxin, by rationally designed compounds. Based on the 7-fold symmetry of the PA 63 pore, we synthesized and tested cyclic molecules that had 7-fold symmetry using ␤-cyclodextrin (␤-CD) as a starting molecule (Fig. 1). The discovered inhibitors of anthrax toxin were successfully tested in vitro and in vivo (1-5, 7, 10). The broader applicability of this approach was demonstrated using as targets two other toxins that form transmembrane pores with 7-fold symmetry: ␣-hemolysin (␣-HL) of Staphylococcus aureus (2, 11) and ε-toxin produced by Clostridium perfringens (unpublished data).To investigate how the structural features of the pore blockers affect their activities, we evaluated the ability of structurally related derivatives of ␣-, ␤-, and ␥-cyclodextrins to inhibit the cytotoxic activities of anthrax lethal toxin (LeTx) and staphylococcal ␣-HL as well as to block the ion current through the channels formed by PA 63 and ␣-HL in planar lipid membranes.

Role of Pore-Forming Toxins in Bacterial Infectious Diseases

“…Inhibiting the effects of PFTs on host epithelia also increases survival of the host during S. pneumoniae infection (436). Further examples are cyclodextrin derivatives, which can disable B. anthracis PA pores and S. aureus alpha-toxin pores in vitro and protect mice from killing by anthrax in vivo (477,478), and pore-dead PFTs or other compounds used as competitive inhibitors of live PFTs (479,480). Other, hypothetical mechanisms include the administration of decoy membranes (330) and methods to inhibit expression or release of PFTs.…”

Inhibitors of pore-forming toxins

“…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.…”

“…Activity of the guanidine-substituted cyclodextrin compounds was decreased only slightly compared to the aminoalkyl analogs. To identify an optimum length of the linkers [104,105,107] which connect the positively charged ligands with the 7+β-CD scaffold, the blocker effect was examined using a group of hepta-6-thioaminoalkyl derivatives with alkyl spacers of various lengths both against PA 63 channels in planar lipid bilayer membranes and in cell models.…”

Cyclodextrins: A Weapon in the Fight Against Antimicrobial Resistance

“…69 However, they also showed -hemolysin produced from S. aureus which was selectively blocked only by -CD derivatives, suggesting the pore inhibition mode depends on both symmetry and size of the CD inhibitor.…”