Many pathogens utilize the formation of trans-membrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors, which makes them attractive targets for the discovery of new therapeutic agents. Our research is based on the idea that compounds designed to block the pores can inhibit the action of virulence factors, and that the chances to find high affinity blocking agents increases if they have the same symmetry as the target pore. Recently, we demonstrated that derivatives of β-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the trans-membrane pore formed by the protective antigen (PA) subunit of the toxin. To test the broader applicability of this approach, we sought β-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcas aureus α-hemolysin (α-HL), which is regarded as a major virulence factor playing an important role in staphylococcal infection. We identified several amino acid derivatives of β-cyclodextrin that inhibited the activity of α-HL and LeTx in cell-based assays at low micromolar concentrations. One of the compounds was tested for the ability to block ion conductance through the pores formed by α-HL and PA in artificial lipid membranes. We anticipate that this approach can serve as the basis for a structure-directed drug discovery program to find new and effective therapeutics against various pathogens that utilize pore-forming proteins as virulence factors.

Recently, using structure-inspired drug design, we demonstrated that aminoalkyl derivatives of ␤-cyclodextrin inhibited anthrax lethal toxin action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. In the present study, we evaluate a series of new ␤-cyclodextrin derivatives with the goal of identifying potent inhibitors of anthrax toxins. Newly synthesized hepta-6-thioaminoalkyl and hepta-6-thioguanidinoalkyl derivatives of ␤-cyclodextrin with alkyl spacers of various lengths were tested for the ability to inhibit cytotoxicity of lethal toxin in cells as well as to block ion conductance through PA channels reconstituted in planar bilayer lipid membranes. Most of the tested derivatives were protective against anthrax lethal toxin action at low or submicromolar concentrations. They also blocked ion conductance through PA channels at concentrations as low as 0.1 nM. The activities of the derivatives in both cell protection and channel blocking were found to depend on the length and chemical nature of the substituent groups. One of the compounds was also shown to block the edema toxin activity. It is hoped that these results will help to identify a new class of drugs for anthrax treatment, i.e., drugs that block the pathway for toxin translocation into the cytosol, the PA channel.

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.

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We evaluated the in vivo efficacy of three ␤-cyclodextrin derivatives that block the anthrax protective antigen pore. These compounds were at least 15-fold more potent than previously described ␤-cyclodextrins in protecting against anthrax lethal toxin in a rat model. One of the drugs was shown to protect mice from bacterial infection.