Aspirin target deciphered

January 20, 1994

Roadmap to designing better pain relievers

X-ray crystal structure of unusual membrane protein may spell relief for millions who take non-steroidal anti-inflammatory drugs.

Millions of arthritis sufferers and others who regularly take aspirin to reduce pain and inflammation may be able to look forward to improved drugs with fewer side effects now that researchers at the University of Chicago Medical Center have determined in atomic detail the three-dimensional structure of the target molecule of these drugs. The finding, reported in the January 20 issue of the journal Nature, shows that the target belongs to a long-sought and elusive class of biomolecules and could also aid cancer research.

Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) like ibuprofen and indomethacin work by inhibiting an enzyme that produces prostaglandins--hormone-like messenger molecules that trigger many processes in the body, including inflammation. Less than three years ago several groups found that the enzyme, prostaglandin H2 synthase, exists in two forms: an ever-ready PGHS-1, present in nearly all cells for basic housekeeping duties, and PGHS-2, made only as needed and just by those cells involved in inflammation and immune responses.

Unfortunately for pain sufferers--and especially for rheumatoid arthritis patients, who must take huge doses daily--none of the current crop of 16 NSAIDs discriminates between the two enzyme forms. Before it can trickle into the bloodstream and alleviate inflammation by reining in PGHS-2, the drug lands with a thud in the stomach, where it knocks out PGHS-1, causing excess acid secretion and stomach upset or ulcers.

"Just three years ago the consensus in the pharmaceutical community was you couldn't build a better aspirin," says X-ray crystallographer Michael Garavito, assistant professor of biochemistry and molecular biology. He and research associates Daniel Picot and Patrick Loll grew crystals of PGHS-1 and determined its molecular shape by analyzing the diffraction pattern of X-rays passed through a crystal. Because the gene and protein sequences of both enzymes are known, "now we can take the whole repertoire of drugs and see how they bind to PGHS-1, predict how they might bind to PGHS-2, and try to fashion whole new families of drugs that would be highly specific for one form or the other," Garavito said.

PGHS-1 is tightly bound to an intracellular membrane. Such proteins are notoriously difficult to study because the detergents needed to separate the protein from the greasy membrane make crystallization difficult. The researchers spent six years devising conditions for growing and handling the crystals. Using PGHS-1 isolated from sheep seminal vesicles, they bound the molecule to the NSAID flurbiprofen and slowly changed the composition of the solution and the surrounding vapor over a period of weeks to grow brown, rod shaped crystals less than one sixteenth of an inch long that they then were able to X-ray.

The structure held some surprises. Most membrane proteins are threadlike molecules that lace back and forth across the two layers of the membrane. PGHS-1 turns out to be the first "monotopic" membrane protein, balled up against just one surface. The existence of such proteins was long supposed but never proven. "It floats on the membrane like a boat," said Garavito. Two loops of the protein act as pontoons, forming the main Contact points with the membrane.

Drug developers are most interested in targeting PGHS-2, whose structure can now be inferred and will be the next object of study in Garavito's laboratory. But aspirin's beneficial effects in preventing vascular disease and heart attacks are thought to be a PGHS-1 phenomenon, and improved anti-platelet drugs may derive from today's study, which was funded by the National Institutes of Health.

PGHS-1 has also been implicated in the activation of carcinogens in the body, and recent studies have shown daily aspirin may decrease the incidence of colon cancer. Garavito is now doing American Cancer Society-sponsored research to study how PGHS-1 activates carcinogens through a free radical mechanism. Knowing how the enzyme grabs compounds and converts them into carcinogens might allow chemists to engineer safer substances, Garavito said. "It may be possible to modify industrially promising chemicals to biologically inert forms that can't be activated."