By David N. Leff

It¿s not the anthrax bacterium that kills, nor even its ever-lurking spores. What makes Bacillus anthracis so deadly to man and beast is the toxin it releases.

¿Nature probably endowed the bacillus with this poison,¿ explained biochemist R. John Collier at Harvard University, ¿so it could disable our immune system cells, particularly those that engulf and destroy the anthrax bacterium. These toxins are little missiles that the organism produces to disable or destroy those cells in our body that attack them.

¿This anthrax soil microorganism mainly affects herbivores ¿ cattle, sheep, goats, etc. ¿ probably because of a step in its transmission cycle. The spores are the infectious forms. The organism converts to spores, which repopulate the soil, only after the animal dies and decays. Anthrax is not effectively transmitted from one infected animal to another while the first animal is living. That¿s probably why it wants to kill people.

¿As for the mode of killing,¿ Collier continued, ¿we don¿t know for sure. The lethal toxin destroys macrophages, but also probably forces these immune system cells to produce certain cytokines that are likely what kills its victim. But this is not clear.¿

Collier pursues his anthrax research as professor of microbiology and molecular genetics at Harvard School of Medicine in Boston. He is senior author of a paper in the October issue of Nature Biotechnology, titled: ¿Designing a polyvalent inhibitor of anthrax toxin.¿

¿This is the first example of a peptide inhibiting any sort of anthrax toxin,¿ Collier told BioWorld Today. ¿And to my understanding, it¿s the first polyvalent inhibitor [PVI] that works well in vivo. So it¿s a possible route to therapy of anthrax. You¿d probably give it together with antibiotics,¿ he observed, ¿which eradicate the bacteria from their hosts.

¿Whether or not you¿d give the inhibitor to everyone who¿s contacted anthrax spores, I don¿t know; that would be a question. This is something that one hopes will be effective in the later stages of systemic, inhalational anthrax ¿ such as the fellow in Florida died from. Possibly PVI would have been able to rescue him, and obviously we don¿t know that.¿

Antibiotics + Inhibitor = Rescue

¿Here we¿re talking about something that most likely would be stockpiled for the event of major anthrax biowar attacks,¿ Collier conjectured. ¿In such case, it might take a day or so before one knew what hit the population, by which time people are already symptomatic. At that point they¿re not responsive to antibiotics. You can¿t rescue them from inhalation anthrax with antibiotics. But,¿ he suggested, ¿this toxin inhibitor might be something that one could administer together with antibiotics ¿ and save a much higher percentage of the exposed population.

¿What we report is that we can create a very effective inhibitor of anthrax toxin by using phage display to identify peptides that bind to the heptomeric [seven-molecule] form of the delivery protein, PA ¿ thus blocking its interaction with EF and LF ¿ then coupling those peptides to a flexible backbone, giving a polyvalent inhibitor, effective in vitro, in cell culture, and in vivo.

¿We injected a lethal dose of the anthrax lethal toxin ¿ not the organism itself but the toxin ¿ into the penile vein of male rats,¿ Collier recounted. ¿The animals died in about 90 minutes. Symptoms of anthrax intoxication in rats,¿ he noted, ¿include extremely labored breathing, beginning 15 minutes or so before death, and then prostration. When we mixed our inhibitor with the toxin before we injected it, the animals exhibited no symptoms whatsoever and survived indefinitely.¿

Building on this experimental success, his team is now ¿waiting to go into infected animal models,¿ Collier said, ¿so we can test PVI against the actual infection first in mouse, then rabbit, and if those work we go into nonhuman primates.¿ The university, he allowed, has applied to patent the new inhibitor, which it will license to PharmAthene Inc. in Cambridge, Mass. ¿ co-founded early this year by Collier and others.

In fact, he has not one but two candidate anthrax toxin inhibitors on his drawing board ¿ one, the polyvalent peptide inhibitor he reported in Nature Biotechnology; the other, a dominant negative inhibitor he announced in Science dated April 27, 2001. (See BioWorld Today, May 3, 2001, p. 1.)

The anthrax toxin complex is like a microscopic Lego erector set, composed of several discrete subunit peptides, loitering on the surface of a target cell, and ready to pounce inside. Its three basic components are a single receptor-binding moiety termed protective antigen [PA] ¿ the basis for existing anthrax vaccines ¿ and two enzymatic moieties, edema factor [EF] and the 776-amino-acid lethal factor [LF]. Once self-assembled, these three proteins bind to a heptameric complex of PA, so the PA itself heptamerizes, forms the seven-member ring, and then EF and LF bind to that structure, which then penetrates the cell and infects it.

Two Candidates Better Than One

¿Our polyvalent inhibitor,¿ Collier related, ¿acts by blocking the binding to the enzymatic moieties of the toxin ¿ the delivery component, the last step in toxin assembly. What we¿ve got is a polymeric inhibitor that binds in a polyvalent manner to the heptomeric PA delivery protein ¿ binds very tightly because of its multiple contact points. The efficacy of PVI in blocking the action of anthrax toxin in vivo suggests that it, or another inhibitor developed by a similar approach, could be a useful therapeutic ally against clinical anthrax

¿Our second candidate, the dominant negative inhibitor,¿ Collier went on, ¿consists of a mutant form of PA, with only one or two amino acid changes we engineered. If we add them to normal wild-type PA, they will bind together with it on the cell surfaces, and interact to form these heptameric rings. It appears that we need only one molecule of the mutant form of the protein to be incorporated into a ring to inactivate the whole toxin complex, thus preventing poisoning. So that blocks toxin action by a different mode; it inhibits the translocation step across the cell membrane.

¿Both those candidate inhibitors are promising,¿ Collier concluded. ¿They may lead to a double-pronged pharmaceutical that is equally efficacious as a vaccine and as a faster-acting therapy than currently available.¿

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