Try tasting this topical paradox:

A well-tried antibiotic called ciprofloxacin was much favored by physicians as therapy for UTI - urinary tract infection. Then seven months ago, "CIPRO" soared to the top of the charts as a highly specific and effective treatment for fatal inhalation anthrax.

Now comes the kicker. "What is clear from what we saw happen last fall," observed enzymologist Jeffrey Hermes, "is that current therapy is inadequate. Of the 11 cases of inhaled anthrax, five of the people died. So it's not a very good batting average. In anthrax infections," he continued, "much of the damage to the individual is caused by the toxin. So even though antibiotics such as CIPRO clear the infection and kill the bacteria, they don't neutralize the deadly effects of the toxins."

Hermes is dedicated to correcting this Catch-22 hang-up. A drug discovery specialist at Merck Research Laboratories in Rahway, N.J., he is lead author of a report in the current Proceedings of the National Academy of Sciences (PNAS), released online May 7, 2002, but dated May 14. Its title: "A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease." The paper's senior author is Edward Scolnik, president of the Merck Laboratories.

"We've developed an assay," Hermes told BioWorld Today, "for screening compound collections for leads to inhibit this metalloproteinase enzyme called anthrax lethal factor [LF]. It's the deadliest of the toxins produced by Bacillus anthracis, the anthrax pathogen. So our assay screen itself fills a void because to date there's been no suitable agent to detect the protease activity of anthrax lethal factor." (See BioWorld Today, Jan.24, 2002, p. 1.)

"The assay is quick," Hermes continued. "It takes 15 minutes; it's very sensitive, so requires small amounts of recombinant lethal factor. The assays that have been described to date," he went on, "have been gel-based or very cumbersome, whereas this is a simple sensitive test that allows quickly getting through thousands of compounds. There was nothing like that before."

Lethal Factor Chops Up Protective MAP Kinase

"It makes use of the synthetic 19-mer peptide substrate," Hermes went on, "so it doesn't use the natural substrates in the cells, which are believed to be MAP kinase kinases [MAP KKs] - a protein. When anthrax lethal factor is transported into cells - in particular immune-system macrophages - it causes cleavage and inactivation of MAP KKs, which results in their lysis. The inactivation of these MAP KKs is believed to cause the massive inflammatory response by the lysis of macrophages, which release cytokines such as tumor necrosis factor-alpha and interleukin-beta. The MAP KK is supposedly protective of that release. It's believed that when they are cleaved inside of cells, it results in the lysis of macrophages, and the resulting massive inflammatory reaction that eventually leads to death.

"The fatalities incurred by the residual anthrax toxin," Hermes pointed out, "would have to be inhalation. Releasing all of these inflammatory cytokines leads to shock and pulmonary complications that result in death. And this can happen anywhere from one to seven days after exposure. Inhalation of the bacillus itself is also lethal. You would have your normal bacteremia, but CIPRO will take care of that. Those antibiotics are pretty effective at killing B. anthracis. The problem is the residual secreted toxins inside blood cells - like macrophages - leading to this inflammatory response, which actually resembles shock. And that's the effect that we really want to inhibit. So we're trying to develop something that would be administered along with antibiotics, to take care of the secreted toxins - the root cause of the anthrax pathology.

"Merck's idea was to get to a pharmaceutical solution as quickly as possible. But that solution could come from anywhere - from academic or biotech labs that have been working in the matrix metalloprotease area for a period of 10 years. Those are also likely sources of lead compounds. So in the interest of protecting the U.S. population in time of crisis, we thought we'd just put the assay out there in the public domain so the whole project could move as quickly as possible.

"Anthrax lethal factor metalloproteases," Hermes explained, "use zinc as their active site. Like most proteases, they're involved in a cleavage reaction. They cleave proteins; that's their role in life. And for anthrax protease, it appears that some of the targets may be these MAP KKs. Our strategy is similar to the one people used early on with HIV protease. An HIV virus that has a protease that's not active does not cause AIDS. So it's the best way to validate a target for anthrax drug discovery - go after the LF proteases."

Rats, Mice Next; People Much Later

"We have found, from our own company collection, compounds that inhibit this enzyme. And we have an active medicinal chemistry program that would optimize the other properties needed in order to take this therapy into people. That would require the stability of the compound in blood. Then, of course, safety considerations. We're running a whole set of counter screens to make sure that LF protease is the only protease target. We're just getting to the point now where we're starting to test the effects in vivo, in rodents - mice and rats. It would take us a certain fixed period of time just to prove safety in those animals. Beyond that, we know we're going to have to do Phase I safety trials in people.

"But the tricky part here," Hermes observed, "is how are we going to do the efficacy patient studies, because it's not as if anthrax is out there to test like high blood pressure or lowering cholesterol. So we're talking to the FDA about how we're going to do that.

"As for how the final compound will be administered to patients," he said, "we imagine that with these anthrax-infected people who are very sick - they're in the hospital - and they're on intravenous drip anyway, we're looking at the drug as being injected. That speeds up development of the compound, without having to worry about oral activity. It would be given along with conventional antibiotics, such as CIPRO."

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