Target Practice

In the “The Three Little Pigs,” the wisest pig protects himself by building the house with the strongest walls. In the world of bacteria, the microbe that causes tuberculosis employs a similar strategy. Mycobacterium tuberculosis packs extra layers of sugars and lipids into its cell wall to make a structure almost impenetrable by human–immune system defenses and by many antibiotics.

“As a general rule, they are one of the least permeable bacteria on the planet,” says Clifton E. Barry III, who studies tuberculosis at the National Institute of Allergy and Infectious Diseases in Rockville, Md.

Like the wolf in the story, scientists are working to breach the microbe’s tough defenses. It’s a critical fight: The TB bacterium infects one-third of the world’s population, and it kills nearly 2 million people every year. “More people die of TB today than [of] any other single infectious bacterium,” says John S. Blanchard, a biochemist at the Albert Einstein College of Medicine in New York.

A cough or sneeze from a person with TB symptoms sends out droplets containing the bacteria. If someone nearby inhales the droplets, that person, too, can become infected. Researchers working with the microbe have to wear masks and protective clothing, and their laboratories have to have special air-filtration systems and safety cabinets that can contain infectious agents.

The need for new TB therapies is especially pressing because the current treatment, four drugs taken in various combinations with medical supervision over 6 months or longer, is a difficult enterprise in many parts of the world. What’s more, some TB microbes remain undaunted by even that regimen.

“The incidence of multidrug-resistant tuberculosis is growing all the time and will continue to grow,” says Blanchard. The World Health Organization estimates that 450,000 new multidrug-resistant TB cases occur every year. There are few back-up antibiotics to treat strains that evade the standard defense.

In the search for biochemical processes for new therapies to target, some researchers have focused on the super reinforced cell walls of the TB bacteria. They are continuing to describe not only how the microbe synthesizes that reinforced cell wall, but also other ways in which the bacterium fends off drugs. Some teams are targeting processes essential to the microbe’s survival, such as the pathways for iron uptake. Aided by the complete sequence of the M. tuberculosis genome, reported in 1998, and funding boosts from public and private sources, researchers are optimistic that these efforts and others like them will soon result in new therapies for TB.

Cell wall secrets

The microbe’s unusually well-fortified cell wall gives it several advantages. When M. tuberculosis enters a person’s body, immune system cells called macrophages take it up. Whereas these cells typically destroy microbial invaders, the TB microbe manages to survive within them. Because of this hardiness, the bacterium can persist in a person’s body for years.

M. tuberculosis doesn’t immediately cause illness in most people. In 9 out of 10 people infected, the bacteria lie dormant in the lungs and don’t produce the fever, weight loss, fatigue, and other symptoms characteristic of an active TB infection. However, AIDS patients and others with weakened immune systems are at high risk of becoming ill.

Scientists attribute the microbe’s persistence—as well as its resistance to drugs—in large part to the cell wall. Like other bacteria, M. tuberculosis‘ inner cell membrane attaches to a wall composed of peptidoglycan, a polymer of amino acids and sugars. The TB microbe adds to that another layer of sugars, called galactans and arabinans. That’s topped off by a water-repelling layer of lipids, including very long chains of fatty acids called mycolic acids. The attraction between these lipids prevents most substances from diffusing across the boundary, Barry explains.

Researchers have spent decades searching for the many enzymes that build M. tuberculosis‘ cell wall. Some of these enzymes may be good targets for drugs against TB. Indeed, isoniazid and ethambutol, two of the primary antibiotics now used to treat tuberculosis, interfere with cell wall biosynthesis.

Recent work has provided more details about the cell wall’s machinery. Todd L. Lowary of the University of Alberta in Edmonton and his coworkers are looking for inhibitors of the enzymes that assemble galactans for the middle layer of the cell wall when the microbe replicates. Toward that goal, they’ve coaxed the bacterium Escherichia coli to produce one of those enzymes, called glfT, in the lab and have begun to characterize the enzyme.

With the E. coli–made glfT in hand, the team plans to work out the three-dimensional structure of the enzyme and use it to design new compounds likely to bind to and thereby block glfT. The researchers will also search existing lists of known structures for compounds that fit the bill, Lowary says.

Sheryl Tsai of the University of California, Irvine and her colleagues have determined the three-dimensional structure of a different M. tuberculosis enzyme, one that supplies building blocks for some of the lipids in the microbe’s cell wall. In the Feb. 28 Proceedings of the National Academy of Sciences (PNAS), the researchers reported that this enzyme, called AccD5, provides the starting material for the synthesis of a type of branched fatty acid that the bacterium inserts among the mycolic acids in its lipid layer.

To look for potential drugs, the team used a computer simulation to screen compounds that might inhibit the enzyme. One candidate binds to AccD5 20 times as tightly as the enzyme’s substrates do, so it should prevent AccD5 from carrying out its normal function. The researchers plan to test this compound in biochemical and animal studies.

Defensive maneuvers

M. tuberculosis‘ protective strategy doesn’t rest solely with its cell wall. Charles J. Thompson of the University of British Columbia in Vancouver reports that a recent “fishing expedition” revealed a gene that coordinates the microbe’s internal resistance against many different classes of antibiotics. The gene acts as a second line of defense when a drug permeates the fortified cell wall, he says.

Thompson and his colleagues began with bacteria from the genus Streptomyces, soil bacteria that share an ancestor with Mycobacterium and are champions of producing antibiotics that fend off other microbes. The scientists isolated a mutant strain of Streptomyces lividans that is sensitive to a variety of structurally unrelated antibiotics that kill some other bacteria but don’t deter M. tuberculosis.

The researchers identified a gene, called whiB7, that was mutated in this drug-sensitive strain. A similar gene turned up in a search of M. tuberculosis‘ genome and received the same name.

When the scientists removed whiB7 from a strain of M. tuberculosis, the mutant became sensitive to several antibiotics, such as erythromycin, that it normally resists. Reinserting a copy of the gene into the microbe rendered it again resistant to those drugs.

The team then turned to a related microbe, derived from a strain that causes TB in cows. When the scientists silenced whiB7 in that microbe within human cells growing in the lab, the mutant strain became 10 times as sensitive to spectinomycin as the normal strain was, the researchers reported in the Aug. 23, 2005 PNAS.

The researchers found evidence that low concentrations of antibiotics activate whiB7. Thompson speculates that this gene regulates a group of other genes, located at various sites throughout the microbe’s genome. These genes, which normally have different functions, “came under the control of this master regulator,” says Thompson, where they add to the bacterium’s resistance to many drugs.

Thompson plans where they search for compounds that inhibit whiB7. “If we could inactivate the function of whiB7 chemically, then we would make M. tuberculosis susceptible to many, many drugs that aren’t effective now,” he says.

Drug developments

Aside from targeting cell machinery that M. tuberculosis uses for defense, researchers are homing in on ways to shut down metabolic pathways that keep the microbe alive day to day. Courtney C. Aldrich of the University of Minnesota in Minneapolis and his colleagues have designed drugs to disrupt the microbe’s acquisition of iron.

“Almost every form of life needs iron,” says Aldrich. Once inside its host, M. tuberculosis sends out small molecules called mycobactins to rip iron out of the human proteins that carry the metal, he says. Other groups of researchers had identified about a dozen enzymes that sequentially assemble mycobactins. If you knock out even one of these enzymes, Aldrich explains, “you will hinder all subsequent steps.”

Aldrich and his colleagues have focused on one enzyme, called MbtA, that begins the reaction sequence that produces mycobactins. To find an inhibitor for MbtA, the scientists designed a mimic of a compound to which the enzyme binds tightly during its normal activity. The team then substituted different chemical groups at various spots in the structure. In tests of M. tuberculosis growing in the laboratory, one of the seven resulting structures halted microbial growth nearly as well as does isoniazid, one of the first-line tuberculosis drugs. The team’s findings appeared in the Jan. 12 Journal of Medicinal Chemistry.

The researchers have since made additional modifications to their structures and plan to test their optimal compound in animal studies, “I hope by the end of the year,” says Aldrich.

Another anti-tuberculosis drug, a nitroimidazole called PA-824, has already reached clinical trials. Unlike that of Aldrich’s compound, however, PA-824’s biochemical target is unknown.

The PA-824 story began in the 1970s, when researchers selected molecules being considered as anticancer agents, and screened them against M. tuberculosis. In 2000, Barry’s team demonstrated that PA-824 shows activity against the microbe both when it is replicating in oxygen-rich conditions and when it is in oxygen-poor areas and not dividing.

Other research has suggested that the microbe maintains its latent, non-replicating state in oxygen-poor microenvironments in the body, says Barry.

Some drugs attack only an actively dividing microbe. The nitroimidazole’s potential effectiveness against latent M. tuberculosis is “part of the excitement with PA-824,” Barry says.

Barry and his colleagues are working out how PA-824 does the job. In the Jan. 10 PNAS, they reported on the discovery of a microbial enzyme within M. tuberculosis that activates PA-824, making the compound chemically reactive and thus harmful to the bacterium. The researchers plan to determine this enzyme’s three-dimensional structure, which will be useful for designing additional drug candidates similar to PA-824.

Turning tide

The newest tuberculosis drug developed specifically for the disease appeared more than 30 years ago. Lack of innovation since then is “probably typical of diseases that don’t affect the developed world right now,” says Barry. “That’s a consequence of the fact that we have poor chemotherapy that works adequately under very stringent conditions but doesn’t work so well in the developing world.”

Besides the biochemical challenges that the microbe presents, technical difficulties in working with it have also hindered progress. Scientists need special facilities to avoid the spread of M. tuberculosis. “The construction of these labs is very expensive,” Blanchard says.

Also, the experiments take a long time. “You can grow 10⁹E. coli organisms overnight,” Blanchard says. “It takes 3 to 4 months to grow that many of tuberculosis.”

Nevertheless, scientists expect progress toward a better treatment, partly because funding for TB research is increasing. The Bill and Melinda Gates Foundation has brought hundreds of millions of dollars and international attention to the issue. For example, that foundation supports organizations such as the Global Alliance for TB Drug Development, which is conducting the clinical trials of PA-824. And the National Institutes of Health plans to invest $158 million in TB research in 2007.

“There’s been a sea change in the amount of tuberculosis research going on,” Barry reports.

Still, adds Blanchard, “There’s a lot to do…. I would encourage everybody to think about how to deal with TB.”