Researchers Elucidate Machinery of Major Anti-Cancer Drug Target
DURHAM, N.C. -- Duke University Medical Center biochemists have reported the detailed function of an enzyme that is a key component of a signaling pathway important for about 30 percent of cancers. Their surprising findings about how the enzyme, called farnesyl transferase (FTase), works could help improve the FTase-inhibiting drugs that pharmaceutical companies are now testing to fight a broad spectrum of cancers.
In an article in the October 10, 2002, Nature, biochemists Lorena Beese, Ph.D., Patrick Casey, Ph.D., and Stephen Long, Ph.D., reported detailed X-ray crystallographic studies of FTase "frozen" in configurations in which it is attached to the molecules -- called substrates -- on which it acts catalytically. Together, the structures of these configurations produce the equivalent of a stop-motion animation of the enzyme's reaction path. The scientists' work was supported by the National Institutes of Health.
FTase works as a cellular seamstress, connecting a fatty farnesyl molecule to newly built protein enzymes when they emerge from the cell's production machinery. This chemical stitchery adds a molecular "flag" to a new enzyme that signals the cell to send it off on its metabolic mission in the cell.
Ten years ago, Casey and other researchers discovered that among the enzymes that FTase flags to trigger into action is the notorious cancer-causing enzyme Ras. Although Ras usually plays a normal, essential role in the machinery of cell growth when it is under the precise control of the cell's regulatory systems, Ras can mutate to become constantly active and thereby help trigger uncontrolled cancerous cell growth. Mutant Ras enzymes are associated with nearly a third of human cancers, including up to 90 percent of pancreatic cancers, half of all colon cancers and a quarter of all lung cancers.
Thus, pharmaceutical companies including Schering-Plough, Bristol Myers-Squibb and a subsidiary of Johnson & Johnson are now testing drugs that jam FTase to block the function of mutant Ras proteins and other substrates of the enzyme that are important in uncontrolled growth.
Almost five years ago, Beese and Casey reported the first detailed structure of the FTase protein. In such structural studies, Beese uses X-ray crystallography, a technique that involves shining X-rays through a crystallized protein and deducing the molecule's structure from the resulting pattern of diffracted spots. The initial FTase structure identified the enzyme's "active site," where the catalytic joining of substrate molecules takes place. However, those initial structure determinations still did not explain the details of how the enzyme works, said Beese.
"Even our initial structures of FTase complexed with substrates bound to the active site raised a lot of questions," she said. "For one thing, they showed a significant gap between the molecules that had to come together to form a bond."
Now, however the new series of FTase structures published in Nature have revealed some startling new details of the enzyme's action, said Beese. For one thing, the scientists realized that the enzyme doesn't release its product -- the Ras protein attached to the farnesyl molecule -- until another substrate molecule arrives to bind to FTase. Such a mechanism is highly unusual for an enzyme that is not 'processive,' said Beese. Such processive enzymes, she explained, act on a succession of molecules; an example is the cell's protein-making machinery.
"That was an absolute shock when we saw that," she said. "At first I was afraid it was an artifact, and we asked Steve Long to repeat the experiment a number of times before I actually believed it." Among the implications of the finding regarding substrate binding, said Beese, is that FTase might play a role beyond that of molecular seamstress.
"The enzyme may be involved in transporting their substrates to the next part of the reaction pathway," said Beese. "And so it doesn't release the substrate until it gets where it needs to be." Such insights could aid both basic understanding of FTase and the development of drugs that thwart the enzyme pathway in a new way, said Beese.
Also helpful will be the researchers' discovery that FTase remains in the same basic shape, or conformation, throughout its catalytic process.
"Many enzymes undergo conformational changes of their protein structure as part of the reaction cycle," said Beese. "However, with FTase, we saw that during each step, the protein 'scaffold' remains basically unchanged, and it is the substrates that undergo conformational changes." Such a finding is good news for drug companies, said Beese.
"This means that the drug structures that companies are now testing are going to be very good models for the enzyme with the drug bound," said Beese. "If the FTase protein changed its conformation every time it bound a drug, the computational algorithms for positioning drugs into active sites wouldn't work."
Just as they deduced the reaction path of FTase, Beese and her colleagues have now tackled the detailed mechanism of FTase's close cousin, "geranylgeranyl transferase." The difference between the two enzymes is that FTase stitches the 15-carbon farnesyl molecule onto proteins, while geranylgeranyl transferase adds a longer 20-carbon geranylgeranyl molecule. Like FTase, geranylgeranyl transferase is thought to play a role in many cancers.
Beese said the latest findings may apply not only to cancer but also to development of drugs for such diseases as malaria and sleeping sickness. The structural information on human FTase could reveal important differences between the human enzyme and corresponding enzymes found in the Plasmodium microbe that causes malaria and the Trypanosome that causes sleeping sickness, said Beese.
"Thus, it might be possible to make very specific inhibitors of the FTases in these organisms, without affecting the human enzyme. And there is some excellent work going on at other institutions to develop FTase-inhibiting drugs for these major diseases," she said.
Beese emphasized that the ability to elucidate the reaction path of FTase and understand the enzyme's function depended on both the X-ray crystallography done in her laboratory and biochemistry experiments done in Casey's laboratory.
"Pat has made seminal contributions to the understanding of FTase, and our structural studies depended on the kinetic pathway worked out in his laboratory to trap the protein in the different stages of the reaction," she said.
First-author Long is a postdoctoral fellow in biochemistry; Beese is an associate professor of biochemistry, and Casey is a James B. Duke Professor of Cancer Biology and director of the Center for Chemical Biology in the department of pharmacology and cancer biology.