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Herndon, VA, United States

Bezrukov S.M.,U.S. National Institutes of Health | Liu X.,Catholic University of America | Karginov V.A.,Innovative Biologics | Wein A.N.,National Institute of Allergy and Infectious Diseases | And 4 more authors.
Biophysical Journal | Year: 2012

Cationic β-cyclodextrin derivatives were recently introduced as highly effective, potentially universal blockers of three binary bacterial toxins: anthrax toxin of Bacillus anthracis, C2 toxin of Clostridium botulinum, and iota toxin of Clostridium perfringens. The binary toxins are made of two separate components: the enzymatic A component, which acts on certain intracellular targets, and the binding/translocation B component, which forms oligomeric channels in the target cell membrane. Here we studied the voltage and salt dependence of the rate constants of binding and dissociation reactions of two structurally different β-cyclodextrins (AmPrβCD and AMBnTβCD) in the PA63, C2IIa, and Ib channels (B components of anthrax, C2, and iota toxins, respectively). With all three channels, the blocker carrying extra hydrophobic aromatic groups on the thio-alkyl linkers of positively charged amino groups, AMBnTβCD, demonstrated significantly stronger binding compared with AmPrβCD. This effect is seen as an increased residence time of the blocker in the channels, whereas the time between blockages characterizing the binding reaction on-rate stays practically unchanged. Surprisingly, the voltage sensitivity, expressed as a slope of the logarithm of the blocker residence time as a function of voltage, turned out to be practically the same for all six cases studied, suggesting structural similarities among the three channels. Also, the more-effective AMBnTβCD blocker shows weaker salt dependence of the binding and dissociation rate constants compared with AmPrβCD. By estimating the relative contributions of the applied transmembrane field, long-range Coulomb, and salt-concentration- independent, short-range forces, we found that the latter represent the leading interaction, which accounts for the high efficiency of blockage. In a search for the putative groups in the channel lumen that are responsible for the short-range forces, we performed measurements with the F427A mutant of PA 63, which lacks the functionally important phenylalanine clamp. We found that the on-rates of the blockage were virtually conserved, but the residence times and, correspondingly, the binding constants dropped by more than an order of magnitude, which also reduced the difference between the efficiencies of the two blockers. © 2012 Biophysical Society. Source


Nestorovich E.M.,Catholic University of America | Nestorovich E.M.,U.S. National Institutes of Health | Karginov V.A.,Innovative Biologics | Popoff M.R.,Institute Pasteur Paris | And 2 more authors.
PLoS ONE | Year: 2011

Background: Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxin consists of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components. Methodology/Principal Findings: Here we demonstrate that a 7-fold symmetrical positively charged ß-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We discovered that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound strongly blocked transmembrane channels formed by the B-components of C2 and iota toxin in planar lipid bilayers in vitro. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component. Conclusions/Significance: The described ß-cyclodextrin derivative was previously identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria. Source


Ragle B.E.,University of Chicago | Karginov V.A.,Innovative Biologics | Wardenburg J.B.,University of Chicago
Antimicrobial Agents and Chemotherapy | Year: 2010

Staphylococcus aureus pneumonia is a common, potentially life-threatening infection caused by this human pathogen. The only therapies available to treat S. aureus pneumonia are antibiotics, a modality that is jeopardized by the organism's remarkable ability to acquire antimicrobial resistance. S. aureus alpha-hemolysin is a pore-forming cytotoxin that is essential for the pathogenesis of pneumonia. Strains lacking this cytotoxin are avirulent in a murine model of pneumonia; likewise, vaccine-based strategies that antagonize the toxin afford protection against lethal disease. Disruption of the function of this toxin therefore provides a potent mechanism to prevent and/or treat S. aureus pneumonia. β-Cyclodextrin derivatives are small molecules with a sevenfold symmetry that mirrors the heptameric alpha-hemolysin. These compounds block the assembled alpha-hemolysin pore, compromising toxin function. We report that a modified β-cyclodextrin compound, IB201, prevents alpha-hemolysin-induced lysis of human alveolar epithelial cells. This protective effect does not result from the ability of the β-cyclodextrin to impair formation of the oligomeric alpha-hemolysin on the cell surface, supporting a role for this molecule in blockade of the lytic pore. An examination of IB201 in murine S. aureus pneumonia demonstrated that administration of this compound prevents and treats disease, protecting against mortality. Consistent with the vital importance of alpha-hemolysin in pneumonia caused by methicillin-sensitive and highly virulent methicillin-resistant S. aureus strains, IB201 protects against lethal challenge with both types of isolates. These observations, coupled with a favorable safety profile of β-cyclodextrin compounds, provide a novel strategy that may be developed to combat S. aureus pneumonia. Copyright © 2010, American Society for Microbiology. All Rights Reserved. Source


The invention provides methods for treating, delaying, and preventing pathological conditions caused by pore-forming toxins such as anthrax toxin, -hemolysin toxin, and -toxin using a class of low molecular weight compounds that block the pore formed by these toxins. Specific compounds useful for treating, preventing, or delaying a disease condition caused by


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 300.00K | Year: 2009

DESCRIPTION (provided by applicant): We have recently demonstrated that alpha-hemolysin produced by S. aureus plays a key role in the pathogenesis of staphylococcal pneumonia, and identified beta-cyclodextrin derivatives that potently inhibit the cytotoxic effects of this secreted bacterial toxin. The overall goal of this feasibility study is to examine the ability of these inhibitors to provide in vivo protection in a mouse model of S. aureus pneumonia. The specific aims of this Phase I study are: (1) Establish and validate analytical methods for the detection of cyclodextrins in biological samples. (2) Evaluate toxicity and preliminary pharmacokinetic properties of the compounds and determine the optimal dosing schedules for the efficacy studies. (3) Quantify alpha-hemolysin inhibition by beta-cyclodextrin derivatives in a tissue culture model of human alveolar cell injury. (4) Evaluate the efficacy of beta-cyclodextrin derivatives in the prevention and treatment of S. aureus pneumonia in mice. The data derived from this feasibility study will lay the foundation for future prophylactic and therapeutic strategies in S. aureus pneumonia, including infection caused by methicillin resistant strains. In the long-term, subsequent pre-clinical and clinical studies of these compounds will lead to the development of novel drugs against S. aureus infection. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus is one of the most significant causes of serious hospital- and community-acquired bacterial infections, and a sharp increase in the incidence of severe S. aureus pneumonia has been noted for nearly a decade. Increasing antimicrobial resistance among S. aureus strains and the appearance of particularly virulent isolates of this pathogen within the community have rendered conventional antimicrobial therapies obsolete, resulting in increased mortality and cost of care. Novel therapeutic approaches to both prevent and treat S. aureus-mediated invasive pulmonary infection, especially methicillin resistant S. aureus (MRSA), are necessary.

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