CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application 61/482,108 filed on May 3, 2011, the entire content of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the development of symmetry-based small molecule blockers of pore-forming virulence factors and their use as anti-infectives.

BACKGROUND OF THE INVENTION

We have previously proposed an approach for the discovery of inhibitors of pore-forming toxins that involves selection of molecules with comparable dimensions and the same symmetry as the target pores and capable of blocking the pores. We suggested that this approach would allow highly efficient identification of leads using orders of magnitude fewer initial screenings of candidate compounds compared to the standard High-Throughput Screening approach employed in pharmaceutical drug discovery. The identification of small molecule drug candidates can be performed faster and significantly cheaper in comparison with the existing industry standards. We have successfully applied this approach to anthrax toxin which plays a key role in anthrax infection. The toxin was disabled using high-affinity blockage of the pore formed by protective antigen (PA) subunit of anthrax lethal toxin (LeTx) by rationally designed low-molecular weight compounds. Guided by the seven-fold symmetry of the PA pore, cyclic molecules of seven-fold symmetry using β-cyclodextrin as a starting molecule were synthesized and tested (Fig. 1). The hydroxyls at positions 2 and 3 form hydrogen bonds and are required to keep the molecule rigid, making the 6-OH group a favorable site for modifications. We successfully tested the discovered inhibitors of anthrax toxin in vitro and in vivo. ^1-6

SUMMARY OF THE INVENTION

We identified the structural features of the pore blocking cyclodextrin (CD) compounds that affect their ability to inhibit the cytotoxic activities of anthrax lethal toxin, α-hemolysin (α-HL) toxin of Staphylococcus aureus, and ε-toxin produced by Clostridium perfringens as well as to block the ion current through the channels formed by PA and α-HL in artificial membranes. These toxin targets form trans-membrane pores with sevenfold symmetry ^5,7

The inventor discovered that not only β-CD derivatives, but also certain α- and γ-CDs can be utilized as blockers of pore-forming virulence factors and as anti- infectives. Furthermore, it was also established that cyclodextrins carrying substitute groups at positions 2 and 3, rather than at position 6, can be utilized as blockers of pore-forming virulence factors and as anti-infectives.

Accordingly, one aspect of the present invention is directed to a compound for use in treating a disease condition caused by a pore-forming toxin, said compound having the general formula:

, wherein n = 6, 7, or 8, depending on the geometry of the pore opening; R ₂ = R ₃ = H, OH, ethylamine, or butylamine; and R ₁ = any of the substituents shown in tables 1 and 2.

In one preferred embodiment, the pore-forming toxin is α-HL or ε-toxin, and the compound is one in which:

R ₂ = R ₃ = H, OH, (CH ₂ ) ₂ NH ₂ or (CH ₂ ) ₃ NH ₂ and

or

N(CH ₂ ) ₄ CH ₃ . wherein n = 7, R ₂ = R ₃ = H, OH, (CH ₂ ) ₂ NH ₂ or (CH ₂ ) ₃ NH ₂ and

or

In another preferred embodiment, the pore-forming toxin is anthrax toxin and the compound is one in which:

orN(CH ₂ )4CH ₃ .

In yet another preferred embodiment, the pore-forming toxin is anthrax toxin, and the compound is one in which:

n = 7, R.2 = R3 = ethylamine or butylamine, and Rj = OH, NH ₂ , N(CH ₂ ) ₄ CH ₃ .

In another preferred embodiment, a compound having a formula of any of the compounds presented in tables 1 and 2 is used for treating a disease condition caused by a pore-forming toxin.

DESCRIPTION OF THE FIGURES

Figure 1. Schematic illustration of β-cyclodextrin molecule in comparison with anthrax PA (left) and staphylococcal α-HL (right) pores.

Figure 2. Protection of RAW 264.7 cells from LeTx action by compound 3.

Figure 3. Channel blocking activity of compounds 1-3.

Figure 4. Protection of rabbit erythrocytes from α-HL action by compound 12. Figure 5. Multichannel α-HL conductance upon cis-addition of CD derivatives. Figure 6. Activation of ε-prototoxin with trypsin. 10% SDS-polyacrylamide gel stained by Coomassie brilliant blue.

Figure 7. Protection of MDCK cells from ETX-induced cell death by monoclonal antibodies against ETX. Cells were incubated with different concentrations of anti- ETX mAb with or without ε-toxin. Each experimental condition was performed in duplicates. Cell viability was determined by MTS colorimetric assay. Error bars represent standard deviations.

Figure 8. Protection of MDCK cells from ε-toxin-induced cytotoxicity by compound 5105. MDCK cells were incubated with different concentrations of compound 5105 with or without ε-toxin. Each experimental condition was performed in duplicates. Error bars represent standard deviations.

DETAILED DESCRIPTION OF THE INVENTION

Compounds 1-9 and 13 (see Table 1) were tested for their ability to protect mouse macrophage cells from LeTx -induced cell death (Fig. 2). For some of these compounds, their ability to block the ion current though the pores formed by PA in planar lipid membranes (Fig. 3) was also evaluated. First, the activities of per-6- amino derivatives carrying unmodified hydroxyls at positions 2 and 3 (compounds 1-3) were compared with the 2,3-methylated ones (compounds 4-6). None of the methylated derivatives displayed anti-toxin activities while the unmodified compounds 2 and 3 showed activity at low micromolar concentrations. This data demonstrates the importance of the rigidness of the cyclodextrin core provided by free OH groups. In contrast, compound 13 carrying ethylamino groups at positions 2 and 3 displayed anti - LeTx activity at low micromolar concentrations.

The activities of various α-, β-, and γ-cyclodextrin derivatives carrying the same modifications were also investigated. One α-cyclodextrin derivative (compound 7) showed a detectible level of LeTx inhibiting activity, while another α- cyclodextrin derivative (compound 1) demonstrated no such activity. Although not wanting to be bound by a theory, these results could be explained by smaller size or by structural features of α-CDs, which could provide a less favorable spatial orientation of the substituting groups.

All the β- and γ-cyclodextrin derivatives with free OH groups at positions 2,3 displayed anti-LeTx activity in the micromolar range. When structurally similar derivatives of β- and γ-cyclodextrins were compared, it appeared that the γ-CDs displayed comparable activities with the β-CD derivatives (compounds 2 and 3; 8 and 9). Although not wanting to be bound by a theory, these results could be explained by a recent discovery that, in addition to the heptameric pores, PA can form octameric pores having the same eight-fold symmetry as γ-CD. ⁸

Channel blocking activity was tested for some of the compounds. The results demonstrate that there is a general correlation between the cytotoxicity inhibition and channel blocking activity. Similar to the results of the cytotoxicity experiments, cc-CD derivative (compound 1) exhibited a much lower blocking activity than β- and y-CD derivatives (compounds 2 and 3) (Pig. 3).

The activities of two α- andy-CDs (compounds 10 and 12) structurally related to a β-CD derivative (compound 11) which inhibits α-HL as shown previously, were also evaluated. ³ The compounds were tested using a rabbit erythrocyte assay (Pig. 4). Both α- and γ-CDs did not display any activity. Similar results were obtained when the compounds' ability to block ion current though the pores formed by α-HL in planar lipid membranes (Fig. 5) was compared. Although not wanting to be bound by a theory, this difference in the activities of anthrax and staphylococcal toxins could be explained by the fact that in contrast to α-HL, PA can form octameric pores having the same eight-fold symmetry as γ-CD. This data demonstrates that size and conformation as well as the match of the symmetry of a blocking molecule and a pore play important roles in the activity of the inhibitors of pore-forming toxins.

A cell-based assay was developed for the screening of chemical libraries. The procedure includes incubation of target cells with ε-toxin in the presence or absence of tested compounds followed by the colorimetric detection of the cytotoxic effect. First, ε-toxin was prepared using the optimized protocol to obtain the product with highest yield and activity. The mature ε-toxin was prepared using the optimized conditions and its quality was assessed by 10% SDS-PAGE (Fig. 6).

The activated ETX was assessed for its cytotoxicity. Only one cell line with a reasonable sensitivity to ε-toxin has been identified - the Madin-Darby canine kidney (MDCK) epithelial cell line, and the concentration of the toxin reducing cell culture viability by 50% (LCw) was 2μg/ml according to the literature. This cell line was utilized for ETX cytotoxicity testing and for the development of a screening assay in a 96-well format. Cytotoxicity was monitored with the use of the MTS bioreduction cell viability assay kit (Promega).

The assay was adapted to a 96-well format. Using anti-ETX monoclonal antibody (mAb) as a toxin inhibitor, it was demonstrated that anti-ETX mAb dose- dependently protected MDCK cells from ε-toxin-mediated cytotoxicity (Fig. 7).

The final protocol developed was successfully utilized for the screening of the library of β-CD derivatives. Over one hundred β-cyclodextrin derivatives at a concentration of 50 uM were screened using the cell-based assay.

Compounds that showed at least 50% inhibition of ε-toxin cytotoxicity at a 50 μΜ concentration were selected. They were serially diluted and tested to determine the IC50 values. Four compounds displayed dose-dependent inhibition of ε-toxin cytotoxicity (Fig. 8, Table 2).

The obtained results demonstrate that the pore blocking cyclodextrin (CD) compounds with the above-described structural features inhibit the cytotoxic activities of anthrax lethal toxin, α-hemolysin (α-HL) toxin of Staphylococcus aureus, and ε-toxin produced by Chstridium perfringens. Thus, this invention demonstrates that such CD compounds can be used for treating, preventing, or delaying a disease condition caused by Bacillus anthracis, Staphylococcus aureus, and Clostridium perfringens. Compound having a formula of any of the compounds presented in Table 1 and 2 of this application can be used for treating, preventing, or delaying a disease condition caused by a pore-forming toxin. In particular, compounds having a formula of any of the compounds presented in Table 2 of this application are particularly useful for testing, preventing, or delaying a disease condition caused by ε-toxin of Clostridium perfringens. The structures of the compounds identified by the inventor is being utilized for the further design, synthesis and testing of a biased library of β-cyclodextrin derivatives.

REFERENCES

The following references are cited herein. The entire disclosure of each reference is relied upon and incorporated by reference herein.

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