When an asthma attack strikes, the airways within the lungs become inflamed and swollen. The tubes narrow because of smooth muscle constriction and might be further obstructed by sticky mucus. The patient develops wheezing that can progress to labored breathing accompanied by a sense of panic.
For people who suffer from asthma, a daily dose of cysteinyl leukotriene inhibitors often makes a significant improvement. Since these drugs can be taken orally, they have advantages over inhalers and are especially effective for children and for adults facing exercise-induced attacks. Up to half the patients who have used the drugs have improved lung function and quality of life.
This widely successful therapy, approved by the Food and Drug Administration in 1998, is indelibly tied to a lifetime of research by K. Frank Austen of Harvard Medical School (HMS). The AstraZeneca professor of respiratory and inflammatory diseases in the Department of Medicine at Brigham and Women’s Hospital and HMS, Austen led basic science investigations that enabled the development of these drugs. They are the first medications aimed at inhibiting substances that actually contribute to smooth muscle inflammation and airway constriction in asthmatic patients as well as to nasal symptoms underlying several immune system and allergic diseases.
Austen’s discoveries have taken years of detailed investigation, funded by the National Institutes of Health (NIH), including the National Institute of Allergy and Infectious Diseases (NIAID), the National Heart, Lung, and Blood Institute (NHLBI), and the National Institute of General Medical Sciences (NIGMS). According to Roy Soberman, HMS associate professor of medicine at Massachusetts General Hospital, a former postdoc in the Austen lab, “The NIH funding, which was constant for many years, allowed and still allows us to address the basic problems related to human diseases.”
Asthma ImpactAs diseases go, asthma is one of the most well known in the United States. Now the most common chronic illness in children, asthma affects more than 6.5 million kids and 15.7 million U.S. adults, with symptoms ranging from shortness of breath and wheezing to full-blown asthmatic attacks in which breathing can become completely obstructed. In 2004, patients made over 2.8 million hospital visits due to asthma complications, and an average of 5,000 people die each year from the disease.
Asthma is a product the sufferer's own immune system. When environmental irritants such as pollen or house dust enter the airways, immune system cells, such as mast cells, lymphocytes, and eosinophils that reside in the inner lining of these tubes react in defense. They produce inflammation-causing substances that attract immune cells, elicit a sticky mucus, and initiate contraction of the smooth muscle of the bronchial tubes.
Conventional therapies for combating the disorder use inhaled corticosteroid drugs to reduce inflammation and beta agonists to relax the smooth muscles of the bronchial tubes. Their important actions are to relieve symptoms. If the severity of the asthma requires the use of oral corticosteroids, there can be major side effects ranging from weight gain to stomach ulcers, easy bruising, and thinning of the bones.
A Long ShotIn 1959, as a recent medical school grad and postdoc, Austen had developed an idea about one possible mediator involved in bronchial asthma. While on a fellowship at the National Institute for Medical Research in England, where he had gone to study mast cells and their role in immunologic reactions, Austen shifted his focus after meeting Walter Brocklehurst, a researcher at the institute who was working on a poorly understood group of compounds referred to as slow-reacting substance of anaphylaxis (SRS-A). Produced by lungs during immunologic and allergic reactions, the substance would eventually be known as cysteinyl leukotrienes.
SRS-A smooth-muscle constriction is not inhibited by antihistamine, which quells many symptoms of allergic reactions in the nose, eyes, or skin. Since antihistamines do not work on asthma either, Austen drew a connection between the two phenomena: apparently SRS-A might be involved in asthma. “Obviously, it was a long shot,” Austen said, “but there weren’t any other candidates. And all our drugs for the management of asthma were directed at relaxing smooth muscle; they were not directed at blocking some molecules that actually tightened the muscle.” Austen developed evidence that SRS-A could be generated by allergic activation of mast cells, known to provide the positive skin test when allergic individuals are injected intradermally with an allergen to which they are sensitive. Bolstered by grant support from the NIH, Austen began to investigate SRS-A in depth, hoping that it was, in fact, a molecule that actually constricts the bronchial airways.
A Pure MotiveAusten soon started collaborating with two postdocs, Robert Orange and Robert Murphy to further understand the substance on the chemical level—even though the researchers had not yet identified an individual molecule. What they had was a stew of fluids produced by allergic reactions in animal tissues. The usual scientific tools for identifying a compound’s composition, such as mass spectrometry, and UV spectroscopy, gave jumbled and unclear information—there were too many other biological compounds mixed in with SRS-A. The researchers would have to somehow isolate the compound. “To get a pure structure, you need a pure molecule,” said Murphy.
So Austen’s group devoted itself to developing a purification procedure that would eliminate the other ingredients and yield only SRS-A. “Purification had two goals: to study the physiology and pharmacology and to actually get the structure so one could look for the receptors and look for the biosynthetic enzyme,” Austen explained.
Murphy, who worked extensively on purifying and identifying SRS-A, explained why isolating and determining the structure is so important: “If you don’t know the structure, you can’t engage in its synthesis or find its enzyme. If you don’t know it’s a biosynthetic enzyme, how are you going to inhibit its effect in a nasty disease?” They used every purification tool they could think of—gel filtration, ion exchange chromatography, thin-layer chromatography, and high-performance liquid chromatography. Eventually, they purified SRS-A enough to develop a rough sketch of the molecule—it was probably a lipid, it had a low molecular weight, it was enriched with sulfur, and it had an unusual UV spectrum. While these were important clues, the SRS-A samples were just not pure enough to provide more information. Fortunately, on his sabbatical in 1978, Murphy collaborated with Bengt Samuelsson of the Karolinska Institute in Sweden. Together, they circumvented the purification problems by using transformed mouse mast cells that could produce pure SRS-A–like activity when stimulated with calcium.
Now, using UV spectroscopy, mass spectrometry, high-performance liquid chromatography, and other tools to determine molecular composition, the scientists identified the origin of this SRS-A–like activity: a lipid with three conjugated double bonds derived from arachidonic acid linked with a sulfur-containing amino acid. Based on this new information, the substance was renamed cysteinyl leukotrienes (or cysLTs). The stereochemistry of the lipid and the structure of the conjugated polypeptide were then obtained in a collaboration with E. J. Corey, a professor in the Harvard chemistry department, with whom Samuelsson had previously studied. Corey was known for his skills in total organic synthesis, a technique that turns simple, common starting material into complex biological compounds by a series of chemical reactions. Using this technique, Corey synthesized candidate leukotriene molecules in various formations, and they were compared in function to the SRS-A–like substance characterized in Sweden and the biologically derived SRS-A in the Austen laboratory. This work established that the SRS-A–like material was a conjugate of eicosatetraenoic acid with glutathione, termed leukotriene C4 (LTC4). Analysis of SRS-A revealed that it was composed not only of LTC4 but of its metabolic products LTD4 and LTE4. Each of these three cysLTs constricted smooth muscle. Corey would go on to win the Nobel Prize in chemistry for his total body of work in natural-product synthesis.
Model BehaviorOnce Austen had Corey’s engineered cysLTs, he was eager to make sure that the substance behaved like the natural molecule; he decided to test it, and not just on an animal model but on a human—himself. As it turned out, Austen and two of his bold colleagues injected a small amount of Corey’s synthetic molecule into their skin and observed the hoped-for wheal and flare (hive) response due to induction of a plasma leak in the microvasculature. To this day, Austen’s arm bears the scar where his colleague biopsied the bumps produced by the cysLTs. Austen has no regrets, however, since the biopsies showed that their synthesized molecule acted exactly as the mysterious SRS-A did years before in animals. “You can’t imagine how exciting it was for us to find out that these things really did what they were supposed to,” said Austen.
Jeffrey Drazen, (current editor of The New England Journal of Medicine), and a prior postdoc in Austen’s lab, took the experiments one step further and inhaled a small amount of the LTC4 as did other healthy volunteers. The substance reduced airflow by formal measurements and induced a wheeze. The concentration of LTC4 that was active revealed that the compound was a thousand times as potent as histamine. These successful experiments confirmed that there was more than just one molecule—and that three forms, LTC4, LTD4 and LTE4, were active on the airways of normal humans and subjects with bronchial asthma.
The researchers knew that the enzyme that could generate LTC4 needed to be characterized. With the help of Soberman, the researchers found that in lung tissue, the two parent compounds, LTA4 and glutathione, converted quickly into LTC4. They called the unidentified enzyme involved LTC4 synthase.
Profiling a ProteinTo better understand the enzyme, the researchers needed more of it—yet it was too biologically scarce to harvest from the cells that naturally produce it. Austen and his colleagues turned to expression cloning, whereby a small amount of DNA is taken from enzyme-producing cells and put into yeast or bacteria cells that then churn out more of the desired protein. Using a highly sensitive, high-throughput assay invented by Austen and his colleague Bing Lam, HMS assistant professor of medicine at Brigham and Women’s Hospital, they isolated the complementary DNA that encodes LTC4 synthase (LTC4S) and determined the amino acid sequence of the enzyme. As they suspected, it was unusual. It lived only in the outer nuclear membrane, which explained its relative scarcity in the cell; it was, as suspected, the only source of the potent LTC4, which in turn gets converted by other extracellular enzymes into its progeny molecules, LTD4 and LTE4.
This tantalizing information encouraged the Austen group to seek the crystal structure of the enzyme. Austen teamed up with Yoshihide Kanaoka, HMS assistant professor of medicine in the Rheumatology, Immunology, and Allergy Division of the Department of Medicine at Brigham and Women’s Hospital. The researchers had to generate and purify relatively large quantities of intact LTC4 synthase from cells extracted with detergents. Once they had gathered enough viable enzyme, they had to crystallize the substance by adding the right chemical compounds to the enzyme—a complicated process that took about a year of testing. Then they provided the expressed protein and initial crystallization conditions to a collaborating group in Japan that specialized in X-ray crystallographic structure analysis. In July of 2007, after five years of experimentation, Austen and Kanaoka and their Japanese colleagues published a report in Nature detailing the exact crystal structure of LTC4 synthase. It is composed of three monomers that bind a glutathione between each set of adjacent monomers. As LTA4 is added by an activated cell, the compound is structurally modified by one monomer and coupled to glutathione by the adjacent monomer to provide the LTC4. “The exciting thing is, the crystal structure really does explain how the enzyme works,” said Austen.
To MarketUnmasking the enzyme helped to reveal the full arc of the leukotriene pathway. LTC4 synthase is already primed with glutathione. LTA4 is released from immune cells’ inner membranes during an allergic reaction and then is quickly taken up by LTC4 synthase for conversion to LTC4. LTC4 is then exported from the cell for metabolism to LTD4 and LTE4. There are two receptors, CysLT1 and CysLT2, which then interact with the three cysLTs. These receptors are not only on smooth muscle in airways and blood vessels, for example, but also on bone marrow–derived cells such as leukocytes, which mediate inflammatory responses.
Throughout Austen and his collaborators’ pursuit in understanding leukotrienes, their gradual discoveries were picked up by pharmaceutical companies in the United States and abroad. Lead scientist Jilly Evans at Merck and her colleagues worked to develop a drug that blocked the leukotriene receptors in the smooth muscles of the lungs, a product that entered the market in 1998. “Frank’s work on cysteinyl leukotriene signaling set the scene for our research,” said Evans.
Soberman agreed, saying that it was Austen’s “persistence over decades, driven by his recognition of the importance of the question, that eventually fueled the interest and drive to identify the components of SRS-A as leukotrienes … and eventually led to the development of drugs.”
While Austen’s work on leukotrienes has revealed the entire network of how and where these inflammatory molecules are made and where they go, many of their details are still tangled with uncertainty. The widespread presence and variability of the leukotriene receptors make them a difficult target for drugs to thwart. As effective as the current receptor blockers are, they only act at one of the two known receptors. Austen believes that developing drugs to block the source of all leukotrienes—LTC4 synthase—would prove most effective. And blocking the enzyme means preventing not only asthma but possibly other inflammatory diseases caused, in part, by cysLTs, such as atherosclerosis and pulmonary fibrosis. This idea has already proved promising in a strain of LTC4 synthase–knockout mice developed by the group, which lack the genes to make the enzyme. When the researchers induced pulmonary fibrosis in the enzyme-free mice, the animals were significantly protected against the disease.
Austen and his colleagues will continue to pursue the intricate biological workings of this pathway. Yet, as Soberman pointed out, ongoing NIH funding is critical: “It is important to understand that the solution of important questions doesn’t just ‘happen’ and that the process can be incremental and can take decades…. If this support is interrupted for short-sighted reasons, the impact will have long-term repercussions in delaying results.”
The Research Path
When immunologist K. Frank Austen began his investigation of bronchial asthma in the late 1950s, there were no medications that aimed at knocking out the actual cause of asthma attacks.
Austen focused his attention on a poorly understood group of compounds that went by the name of slow-reacting substance of anaphylaxis, or SRS-A, produced by the lungs during immunologic and allergic reactions. Though there was reason to believe that SRS-A might be involved in the muscle constriction of asthma,“it was a long shot,” Austen said. Nevertheless, with funding from the National Institutes of Health, he began an exhaustive study of the substance.
The primary scientific challenge was to uncover which molecules make up SRS-A. So Austen and colleagues set out to get a pure sample of the substance for analysis.
This is no simple task. The work required multiple purification and analytical technologies, teams of collaborating scientists, novel problem-solving approaches, and years of effort, not to mention funding. Ultimately, the researchers shed light on the composition and detailed chemistry of the substance, which was renamed cysteinyl leukotrienes (cysLTs), reflecting its molecular makeup.
The scientists also identified the key enzyme involved in producing the cysLTs that they had pinpointed. Their work in illuminating the structure of this enzyme clarified its molecular pathway, and further investigations uncovered the critical receptor proteins that interact with the cysLTs. Along the way, pharmaceutical companies developed drugs to block the receptors, inhibiting the muscle constriction that the leukotrienes induce. The result: the 1998 entrance into the market of the first asthma medication that inhibits muscle constriction.
