The University of Chicago Magazine October 1995
Building a Better
ASPIRIN
CONTINUED
Biochemist Michael Garavito
Garavito was originally drawn to X-ray crystallography, he says, because he likes to visualize (his hobby is photography). He learned to think in three dimensions while doing electron microscopy at the Scripps Institute of Oceanography, where he did research while in college at the University of California-San Diego. In 1978-after earning his Ph.D. at Purdue University, studying under Michael Rossmann, an X-ray crystallographer who determined the structure of the cold virus-he headed to Basel.
"The person who had the office next to mine was a theoretical chemist who was working on the interaction of aspirin-like molecules on their target," recalls Garavito, now an associate professor of biochemistry and molecular biology at Chicago. What scientists already knew was that aspirin's target in the body is an enzyme called prostaglandin H2 synthase, or PGHS. The PGHS enzyme produces prostaglandins-hormone-like messenger molecules that trigger many body processes, including inflammation. Aspirin and other non-steroidal anti-inflammatory drugs, or NSAIDs, like ibuprofen and indomethacin, work by inhibiting PGHS.
It was also known that the PGHS enzyme is tightly linked to the cell membrane-much like the protein Garavito had just succeeded in crystallizing. Yet little was known about the structure of PGHS or about how aspirin and the other NSAIDs inhibit it, so the motivation to crystallize the protein was clear enough: "It's always nice to have a physical or mental concept or image of the system you're working on," Garavito explains. And X-ray crystallography "is the only way to get an atomic-level depiction of enzyme structure.
"You can make inferences from other techniques," says Garavito, "but you're still working with a black box. It's like inferring the mechanism of a car engine without lifting the hood."
The question was whether PGHS could be crystallized-and whether anyone would bet his or her career on trying. "The prevailing consensus was that it was either impossible or extremely difficult," Garavito says. "In those days, only the classical proteins had been crystallized." His laboratory neighbor who was studying PGHS-another American, by the name of Ernie Mehler-offered a challenge: "Since you're so good now, why don't you try this one?" is how Garavito recalls it.
Though Garavito is reluctant to call Mehler's suggestion a dare, he concedes it took some courage to accept it. "This was just before I left Switzerland to return to the States, and I was looking for a project I could make my own," he explains. "And when I looked over what was known about the biochemistry, it was actually a project that looked feasible-though not necessarily easy."
While still in Basel, Garavito began initial experiments, including some encouraging attempts to purify the protein and screen for possible crystallization conditions. He and his co-workers even got the first small crystals to grow. His major collaborator was an affable French-Swiss student named Daniel Picot, who was earning both an M.D. and Ph.D.-a rare combination in Europe-and who took summers off to raise sheep and goats in the south of France to make cheeses.
In 1986, the research duo left Switzerland for Chicago, where Garavito joined the faculty and Picot became a postdoc. They were joined by another research associate, Patrick Loll, a former chemical engineer and high school teacher from Baltimore who had taken up X-ray crystallography. With grants from the American Cancer Society and the Block Fund, the team seriously set about refining conditions for large-scale preparation of PGHS. Their search turned out to be a laborious exercise in trial and error (see sidebar, page 28). Years went by-but in the meantime a stunning breakthrough emerged at two other laboratories, a finding that superheated the entire field and added new importance to determining the precise structure of PGHS.
PGHS, it turned out, had an evil twin.
In 1991 and 1992, groups at UCLA and the University of Rochester found that PGHS is not one enzyme, but two: an ever-ready PGHS-1, present in nearly all cells and necessary for basic housekeeping duties; and its problem sibling, PGHS-2, made only occasionally and only by those cells involved in inflammation and immune responses. All cells except the red blood cells need PGHS-1 just to go about their normal business. But PGHS-2 causes pain and swelling. In response to physical damage or infection, it pumps out prostaglandins from cells, increasing the leakiness of blood-vessel walls and attracting immune cells to the damaged tissues. And in chronic inflammatory diseases, such as rheumatoid arthritis, it runs amok.
IT WAS TO HELP HIS AILING ARTHRITIC FATHER that, in 1893, a German dye chemist named Felix Hoffmann had performed a simple chemical modification of the willow bark compound. Working at the Bayer division of the chemical giant I. G. Farber, Hoffmann created acetylsalicylic acid-aspirin, after the German. Aspirin was a marked improvement, and a century later Americans alone consume 16,000 tons of it each year. Substitute drugs have also come along. But unfortunately for pain sufferers-and especially for rheumatoid arthritis patients, who must take whopping doses daily-none of the current crop of 16 non-steroidal anti-inflammatory drugs can discriminate between the two forms of PGHS. Before these drugs can trickle into the bloodstream and alleviate inflammation by reining in PGHS-2, they land with a thud in the stomach, where they knock out PGHS-1, causing excess acid secretion and stomach upset or ulcers.
The discovery of the dual PGHS enzymes meant that it was at least theoretically possible to design drugs that block pain and inflammation without causing side effects. "If you had only a single target and that molecule did everything," notes Garavito, "then no matter how you attacked inflammation you would knock out prostaglandin synthesis everywhere." From a practical standpoint, the discovery meant that the detailed structural information Garavito and his coworkers were seeking-all of a sudden they were no longer studying PGHS, but PGHS-1-would be vital to the rational design of drugs that would block one form but not the other.
In early 1994 Garavito's team published the X-ray crystal structure of PGHS-1. The dare had been met, but his research continued.
Besides electrifying the pharmaceutical industry, which saw its utility for developing new drugs to expand the $5-billion global NSAID market, the PGHS-1 structure was fascinating in itself. Running through the middle of the enzyme, the researchers found a deep tunnel. The raw material that PGHS acts on-a long, fat-like molecule called arachidonic acid-must pass through this tunnel to reach the enzyme's core, where it is converted into prostaglandin. More surprising was the protein's location on the cell membrane.
While most membrane proteins are threadlike molecules that lace back and forth across the two layers of membrane, PGHS-1-balled up against one surface-turned out to be the first-charted "monotopic" membrane protein. The existence of such proteins had long been supposed but never proven. "It floats on the membrane like a boat," explains Garavito. Two loops of the protein act as pontoons, forming the main contact points with the membrane, and the mouth of the tunnel opens into the membrane's middle-exactly where one would expect to find an oily substance like arachidonic acid.
The researchers had crystallized PGHS-1 by letting it latch onto a NSAID called flurbiprofen. But now they really wanted to see how aspirin worked. Again crystallizing the enzyme-this time in the presence of an aspirin molecule that contained a bromine atom to aid the X-ray analysis-they got another surprise.
The structure they found, published in the August 1995 issue of Nature/Structural Biology, showed that aspirin splits into two parts, and one part-the part that Hoffmann had added in 1893-becomes permanently attached to the enzyme, altering its chemical structure. Aspirin is the only NSAID known to work in this manner: It transfers an acetyl group to the enzyme, where it acts as a gate to block the tunnel and prevent arachidonic acid from reaching the enzyme's core. The researchers also showed that this gate can be in two positions, either fully or partially closed, and that the gate's position may differ between the two forms of the enzyme found in the body. Identifying such differences between the two forms is the key to developing improved NSAIDs, Garavito says.
"We know that PGHS-2 is only partly blocked by aspirin, while PGHS-1 is completely knocked out. This shows why this might be so," Garavito says. "The bottom line is that although the two forms of the enzyme seem very similar, their active sites are subtly different, and this could be a basis for rational drug design."
While Loll and Picot have gone on to start their own academic careers-Loll at Penn and Picot at the Institut de Biologie Physico-Chimique in Paris-Garavito, with support from the National Institutes of Health, is now trying to crystallize PGHS-2, working in collaboration with researchers at Syntex and G. D. Searle.
Drug developers are most interested in targeting PGHS-2, the culprit behind inflammation. Much of its structure can be inferred from Garavito's PGHS-1 determination, since the two are cousins-two-thirds identical in their chemical makeup, with the differences all known. PGHS-1 is an attractive subject itself, because it not only underlies aspirin's bad side effects but also its side benefits. Aspirin's new-found ability to prevent vascular disease and heart attacks is thought to be a PGHS-1 phenomenon, so improved anti-clotting drugs may soon be in the offing.
Other benefits of the ancient drug are still emerging: It now seems that long-term, regular aspirin use lowers one's risk for colon and rectal cancer, the second leading cause of cancer death after lung cancer. And antiinflammatory drugs tend to reduce the symptoms of Alzheimer's disease, probably by acting on PGHS-2, which is known to be the enzyme's predominant form in the brain.
All of these emerging benefits add new meaning to Michael Garavito's decade of work. Then there's the close-to-home angle: Garavito confesses he has at least a minor personal stake in his current research. "Yes, I get headaches," he says with a shy grin, "and arthritis runs in my family, so I like to think I'm helping Mom and Dad."
Click here for a sidebar article, "Take a pound of frozen sheep seminal vesicles, grind well..."