What with well-concealed weapons of mass destruction, killer viruses and the threat of planet-knocking asteroids, Homo sapiens looks more and more like an endangered mammalian species.
Pending such catastrophes waiting to happen, the No. 1 threat to humankind's survival — at least statistically — is tuberculosis. Of our planet's 6 billion-plus population, TB infects nearly one-third — 1.9 billion.
In the next 10 years, by the World Health Organization's estimate, Mycobacterium tuberculosis will infect another 300 million people, of whom 30 million will die of the disease.
Things weren't always this gloomy. Half a century ago, in 1952, a sure-fire anti-tuberculosis synthetic compound called isoniazid came into clinical use. Unlike other antibiotics and antibacterials, isoniazid was a one-pathogen drug, useless against any and all other infections.
Why this was so, nobody really knew. In fact, nobody knew how isoniazid worked to do in M. tuberculosis.
Now, after 56 years of worldwide clinical use, researchers have elucidated the antibiotic's mechanism, as reported in the current issue of Science, dated June 5, 1998. The paper's title: "Inhibition of a Mycobacterium tuberculosis beta ketoacyl ACP [acyl carrier protein] synthase by isoniazid."
Its co-senior authors are biochemist Clifton Barry, at the Rocky Mountain Laboratories of the National Institute of Allergy and Infectious Diseases (NIAID), in Hamilton, Mont., and pathologist James Musser, of the Baylor College of Medicine, in Houston.
"Isoniazid is still a frontline drug," Barry told BioWorld Today. "If you come down with symptoms of TB, the Centers for Disease Control in Atlanta recommends that patients be put right away on a three-drug antibiotic regimen that includes isoniazid, rifampin and pyrazinamide."
As clinicians learned to their cost as early as 1954, isoniazid given by itself quickly causes strains of M. tuberculosis to develop drug resistance.
"It's still used mistakenly as a monotherapy in much of the world," Barry pointed out, "so resistance rates to isoniazid in many Third World countries now are astronomical."
Not to mention certain First World countries.
"New York City is a pretty bad place for resistance to isoniazid," he continued. "Its resistance rate is probably in the 20- to 30- percent range. Not quite as bad as Asia's 60 to 70 percent."
Until two years ago, Barry — like other TB scientists — was looking at how the bacterium becomes resistant to the drug, and what physiologic changes go along with that phenomenon. (See BioWorld Today, June 18, 1996, p. 1.)
Revised Research Agenda Blew Bacterium's Cover
"The direction we've taken since is quite different," he said. "We really started asking: What's the molecular target? What does isoniazid do to the bacterium to kill it? Based on that knowledge, can we think of a better way to intervene?"
The Science co-authors' answers to these questions, Barry explained, center on "a very specific molecular target, the enzyme ketoacyl synthase (KasA). It's involved in making the cell wall of M. tuberculosis.
"Isoniazid is covalently incorporated into the KasA enzyme," he explained, "which destroys the ability of KasA to carry out its catalytic function. That then prevents the bacterial strain from synthesizing the mycolic acid that goes into its cell wall, and therefore kills the cell.
"Once we had figured out biochemically what the target was," Barry recounted, "Jim Musser at Baylor took that into his collection of recent clinical isolates of M. tuberculosis and started looking at the genetics of how these strains become resistant.
"Specifically, they altered four different residues that are all placed very near to the active site of the enzyme. The bacterial strain fights back," Barry went on, "by changing the KasA amino-acid sequence. And that change, we presume, affects the rate at which the bacterium inactivates the isoniazid."
He continued, "Musser's method in determining whether a gene is involved in drug resistance — particularly in TB — was very straightforward. The level of amino-acid changes in a protein that isn't selected for by virtue of drug resistance is very small. So when you see such changes, they're generally correlated with drug resistance."
Seeking Super-Isoniazid By High-Speed Screening
Barry made the point that "studying patterns of gene regulation and protein expression in response to known chemotherapies, we can identify proteins that are upregulated to use as reporters for development of more improved therapies, based on the same target.
"In the case of isoniazid," Barry recalled, "what we've done is identify a protein whose expression is regulated based on inhibition of its function by the drug.
"Then we turned that around by putting the promoter region responsive to the inhibition by isoniazid behind a luciferase gene. That gave us a powerful reporter that now tells us when that protein is inhibited in vivo.
"Now we're turning that around, together with Kirk McMillan, a principal scientist at Pharmacopaeia Inc., [in] Princeton, N.J."
Enzymologist McMillan is collaborating with Barry under a Cooperative Research and Development Agreement, sponsored by NIAID.
"Barry has an assay," McMillan told BioWorld Today, "that in a few hours can identify inhibitors of cell-wall biosynthesis. This is a well-validated area therapeutically, considering that isoniazid is the primary agent.
"Beginning in September," McMillan continued, "we intend to screen one-million-plus compounds, looking for novel anti-tuberculars. "This assay will search for agents that inhibit mycolic acid biosynthesis, and mycobacterial cell-wall assembly."
Barry concluded: "Coming up with a new drug is always a risky game. But I do want to point out that using this sort of technology, we can run through a library of several million compounds, such as Pharmacopeia has, inside of a few weeks, whereas if we were using growth-based inhibition, that would take us a few years." *