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MICROBIOLOGY
Drugs Thwart Anthrax Toxin By Protecting Key PathwayAnthrax has cast a terrifying spell over humans since biblical times--the plague of boils unleashed upon Pharoah's livestock is thought to have been due to the cutaneous form of the disease. Millenia later, anthrax still inspires fear, and for good reason. Once the anthrax-causing microbe invades the cells of the immune system--and it is more likely to do so if inhaled or ingested rather than rubbed on the skin--it releases a deadly poison into the bloodstream and tissues. Researchers still have not come up with an antidote to the bacterium's lethal toxin. Five out of 11 people infected with inhalation anthrax in the 2001 bioterrorist attacks died of the disease, and many who survived remain symptomatic.
A team of HMS scientists has made a discovery that could give humans the upper hand in their renewed struggle with the ancient nemesis. Anthrax lethal toxin works its destructive effects by dismantling a key survival pathway inside cells. Benjamin Turk, Lewis Cantley, and their colleagues are investigating how exactly the toxin gains its stranglehold on the life-giving MAP kinase pathway. What is more, they have devised small molecules that are capable of keeping the toxin from disrupting this molecular cascade. While a key thrust of other anti-anthrax approaches is to stop lethal toxin from entering cells (see Focus May 4, 2001, and Sept. 12, 2003), the current findings, which appear in the Jan. 8 Nature Structural & Molecular Biology, represent a new strategy, namely stopping the toxin from attacking its intracellular target. Fooling a FoeResearchers have known for some time that anthrax lethal factor, a subunit of the toxin, attacks the MAP kinase pathway by binding and cleaving a particular pathway protein, MAP kinase kinase. Using a novel method devised by Turk, the HMS team figured out how exactly the protease recognizes and grabs onto its quarry--that is, by what optimal arrangement of amino acid residues. With this template as a guide, they then built small replicas in the hope that the molecules would act as decoys. "That would basically clog the active site of the enzyme and block access to the MAP kinase kinase substrate," said Turk, HMS research fellow in medicine at Beth Israel Deaconess Medical Center.
Cantley and his colleagues have shared their blueprint with a multi-institutional team headed by researchers at the U.S. Army Medical Research Institute of Infectious Diseases, which has used it to pluck out potential lethal factor inhibitors from tens of thousands of druglike molecules. The Army team reports in the same issue of Nature Structural & Molecular Biology that three of the identified compounds showed promise in inhibiting anthrax toxin in cultured cells. Not Ready for Prime TimeNeither the HMS nor Army researchers see their inhibitors as a final product. "We do not envision these as being the ultimate drugs," said Turk. Both teams are working to create inhibitors with higher affinity for lethal factor, better ability to penetrate the cell, and minimal side effects--an effort Cantley and his colleagues hope will be aided by industry."We felt at this stage that by making this information publicly available to pharmaceutical companies, they could then start from here and address those toxicity and bioavailability issues, and use this information to find something that can be used in humans without side effects," he said.
Benjamin Turk (right) and Lewis Cantley have devised small molecules that may become the basis of a new approach against anthrax infection. (Photo by Graham Ramsay) The findings seem tailor-made for a post 9/11 world and yet Turk and his colleagues began work on the anthrax protease inhibitor well before the World Trade Center disaster. Building on the work of a graduate student in Cantley's lab, Turk had developed a method for identifying how proteases recognize their substrates. "What we get is a collection of amino acid residues on each side surrounding the cleavage site for a protease. So we know which ones are preferred by that enzyme," he said. Turk was looking for new proteases to experiment with when he heard about the MAP kinase kinase-splitting activities of anthrax lethal toxin. Protease-inhibitor BandwagonIt was a good match. "We were already interested in the MAP kinases. But we also saw the potential medical relevance," said Cantley. "The HIV protease success in AIDS suggested that this might be a good way to attack bacteria." Turk performed a library screen to see what, exactly the anthrax lethal toxin was looking for in a partner. Using the information, he generated a substrate that bound with very high affinity to the active site--all this by the time the twin towers were hit.Army researchers got word of the peptide and used it to screen for anthrax lethal toxin inhibitors. Meanwhile, the HMS team was designing and testing in cultured cells even more efficient inhibitors. In fall 2001, with their template and best inhibitors in hand, they worked to generate a set of images with Robert Liddington, of the Burnham Institute, and John Collier, the Maude and Lillian Presley professor of microbiology and molecular genetics at HMS, who had collaborated from the beginning of the study and had come up with the first lethal toxin crystal structure. "The biggest bottleneck for us was getting the crystal structure. Lethal toxin is a very large protein and crystals are difficult to grow in a way where you can get high resolution structures," said Turk. The clarity of the images (see image) could speed the researchers' effort to devise more effective lethal toxin inhibitors. In addition to that effort, they are embarking on what may be an even more difficult project--understanding how exactly the protease subunit wreaks such havoc. "Even if knowing that pathway does not really suggest a new method of treating an infection, it is likely to tease out something new about cell signaling or cell survival that we do not yet understand," Cantley said. "So it would likely be of interest to a wide variety of biologists if we could figure it out." --Misia Landau |
Anthrax lethal toxin (tan) forms a deep hydrophobic pocket into which an optimal MAP kinase kinase-like substrate fits. Inhibitors modeled after this substrate stopped the toxin from killing cells. (Image courtesy of Benjamin Turk)