Dec. 30, 2003 BOSTON -- A research team led by scientists at Beth Israel Deaconess Medical Center (BIDMC) and Harvard Medical School (HMS) has identified a group of small molecules that inhibit a deadly toxin associated with inhalational anthrax. Described in the January 2004 issue of Nature Structural & Molecular Biology, these findings could eventually lead to the development of a protease inhibitor drug, which in combination with antibiotics could be used to treat anthrax cases later in the disease, at a point when antibiotics alone are no longer effective.
"Unlike most types of bacteria, Bacillus anthracis has the ability to produce large amounts of a toxin that can kill the patient even after antibiotics have destroyed the bacteria," explains the study's senior author Lewis Cantley, Ph.D., Chief of the Division of Signal Transduction at BIDMC and Professor of Systems Biology at HMS. "This toxin is released within days of the initial infection, and is impervious to antibiotics." Because the initial symptoms of the disease – fever, cough and chest pain – mimic colds and flu, early diagnosis is extremely difficult; as a result, some 90 percent of all cases of inhalational anthrax prove fatal.
"Toxins act in two ways," adds BIDMC scientist and first author Benjamin Turk, Ph.D. "First, they cripple the cells that fight bacterial infection, thereby enabling the spread of bacteria early in the disease. Later in the process," he adds, "they contribute to the death of macrophage cells, leading to the shutdown of the body's immune system." In fact, autopsies of patients who have died from inhalational anthrax reveal that the high doses of antibiotics have killed the bacteria, indicating that the patients have died from the toxins rather than a persistent infection.
Using a "mixture-based peptide library" technique developed by Turk, the researchers analyzed trillions of peptides to determine an optimal peptide substrate for lethal factor, the active agent in the anthrax toxin. Based on the structure of the optimal substrate, small molecule inhibitors were identified. Finally, crystal structures of lethal factor protease bound to its optimal substrate and to small molecule inhibitors revealed new approaches to enable the design of better inhibitors that might prove effective for clinical use.
The mechanism by which anthrax lethal factor kills human cells is not yet clear. The protease activity of this toxin is known to attack a family of protein kinases called map kinase kinases (MEKKs), which mediate many cellular responses, including cytokine release and cell survival. The availability of drug inhibitors may facilitate the understanding of the effect of lethal factor on these pathways. Protease inhibitor drugs have gained popularity in recent years, notably in the treatment of HIV infections. They work by disabling native protease enzymes and like a key fitting perfectly into a lock, "lock up" the enzyme, rendering it ineffectual
"There could be a number of advantages to taking this approach in attacking inhalational anthrax," notes Cantley. "Unlike an anti-serum, which would require that whole populations be vaccinated -- regardless of whether or not an anthrax outbreak developed -- a therapeutic combination of antibiotics and protease inhibitor drugs wouldn't have to be used except in the incidence of actual disease. This approach would not only reduce the risk of side effects, but could also prove cost effective."
Study coauthors include Robert Liddington, Ph.D., Thiang Yian Wong, Ph.D., and Robert Schwarzenbacher, Ph.D., of The Burnham Institute, La Jolla, California; R. John Collier, Ph.D., of the Department of Microbiology and Molecular Genetics, Harvard Medical School; and Emily Jarrell of BIDMC.
Funding for this study came from the National Institutes of Health, the U.S. National Science Foundation and the U.S. Army.
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