"This novel approach to the design of anthrax antitoxin is an important advance, not only for the value it may have in anthrax treatment, but also because this technique could be used to design better therapies for cholera and other diseases," says NIH Director Elias A. Zerhouni, M.D.
The research appears in the April 23 online edition of the journal Nature Biotechnology.
Led by NIAID grantees Ravi S. Kane, Ph.D., of Rensselaer Polytechnic Institute, in Troy, NY, and Jeremy Mogridge, Ph.D., of the University of Toronto, the investigators built a fatty bubble studded with small proteins that can cling tightly to the cell membrane receptor-binding protein used by anthrax toxin to gain entry into a host cell.
The protein-spiked fatty bubble, or "functionalized liposome," hampers a critical early step in the assembly process that anthrax toxin must undergo to become fully active. In test-tube experiments, the inhibitor, which is covered with multiple short proteins (peptides), was 10,000 times more potent than unattached peptides.
"If the effectiveness of anthrax inhibitors designed in this fashion is confirmed by additional testing, they could one day be important adjuncts to standard antibiotic therapy for the treatment of inhalation anthrax," says NIAID Director Anthony S. Fauci, M.D.
The spore-forming bacterium Bacillus anthracis produces a toxin that causes anthrax symptoms. Antibiotics are used to treat anthrax, but even with such therapy, inhalation anthrax, the most severe form of the disease, has a fatality rate of 75 percent.
"There would be real value to having an additional form of therapy available to physicians confronting a case of inhalation anthrax," notes Phillip J. Baker, Ph.D., anthrax program officer at NIAID.
Anthrax toxin has three parts: protective antigen (PA), a protein that binds to a receptor on the target cell surface; and two enzymes that must be transported into the cell to cause damage. The enzymatic portions of the toxin enter the cell through a pore created for them by PA after it binds to the cell's outer surface. PA can be seen as a bundle of seven cigar-shaped parts, a molecular arrangement referred to as "polyvalent," meaning it displays multiple binding sites.
The inhibitor designed by Dr. Kane and his colleagues is also polyvalent. Just as a glove matches the shape of a hand more closely than a mitten, and so fits more snugly, the polyvalent inhibitor binds the toxin at multiple sites and is orders of magnitude more potent than an inhibitor that binds at a single site. The multiple peptides on the functionalized liposome are arranged with the same average spacing as the binding sites of the PA molecule, which permits a firmer bond between the two, explains Dr. Kane. When the inhibitor is bound tightly to PA, the subsequent steps of enzyme entry cannot occur and the toxin is effectively neutralized.
The investigators tested the anthrax inhibitor in rats. When given in relatively small doses, injection of the inhibitor at the same time as anthrax toxin prevented five out of nine rats from becoming ill. Slightly higher doses of the inhibitor prevented eight out of nine rats from being sickened by anthrax toxin. Nine additional rats were injected with anthrax toxin only. Of these, eight became gravely ill. This experiment was the first to show the efficacy of a liposome-based polyvalent inhibitor in animals, says Dr. Kane.
Dr. Kane says the recent experiments demonstrate a proof of principle and suggest that polyvalent inhibitors could be used along with antibiotics in a clinical setting. Aside from its inherent toxicity, anthrax toxin also accelerates the disease process. Thus, combining antibiotics with a toxin inhibitor might act synergistically to lessen or halt anthrax symptoms, notes Dr. Kane.
Using the same technique of placing multiple peptides on a liposome, the researchers also created a polyvalent inhibitor of cholera toxin that functioned well in test-tube experiments.
In the next phase of their research, Drs. Kane and Mogridge and their colleagues plan to test the action of their inhibitor in animals after infecting them with B. anthracis and allowing the disease process to begin. They also will evaluate the inhibitor with and without adjunct antibiotic therapy.