DALLAS -- An enzyme critical to activating the protein called Ras, which triggers a wide range of cancers, is rapidly yielding its chemical secrets, researchers reported Monday.
What's more, the scientists say, drugs that inhibit the enzyme -- called farnesyl transferase (FTase) -- are proving in laboratory studies to effectively shut down cancer cells.
About one-fourth of all human cancers are caused by genetic malfunctions in the Ras biochemical pathway that result in the uncontrolled growth of cancers. These cancers include up to 90 percent of pancreatic cancers, half of all colon cancers and a quarter of all lung cancers.
The reports by university and pharmaceutical company researchers on progress in both understanding and inhibiting the enzyme were prepared for presentation at an American Chemical Society symposium on "Protein Prenylation."
FTase activates Ras by attaching a 15-carbon farnesyl molecule to the protein -- in the process called prenylation. The fatty farnesyl molecule, a member of a class of compounds called isoprenoids, tags the Ras protein for transport to the cell's outer membrane, where it transmits outside signals from hormones and growth factors that tell the cell to divide.
The symposium included:
- Pat Casey, of the Duke University Medical Center, who reported on research by a collaboration of three Duke laboratories into the detailed structure and biochemical mechanism of FTase. According to Casey, these structural and mechanistic insights are providing pharmaceutical companies with many new insights into how to design specific inhibitors.
- Dale Poulter, of the University of Utah, who reported on studies by him and his colleagues of how FTase binds the farnesyl molecule and the Ras protein, in preparation for enzymatically joining the two.
- Samuel Graham, of the Merck Research Laboratories, who reported on the biology of FTase inhibitors. The first such inhibitors were sulfur-containing thiol compounds, which mimicked the sulfur-containing amino acid cysteine on the Ras protein. However, those compounds were thought likely to trigger immune-related toxicity, so new inhibitors are being designed without thiols.
- Daniele Leonard, of Parke-David Pharmaceutical Research, who described mouse and cell culture studies revealing the efficacy of a series of FTase inhibitors based on histidine-N (benzylglycinamides). These inhibitors showed in vivo activity against breast, colon and lung cancer cells in culture, he said.
- Ronald Doll, of Schering-Plough Research Institute, who disclosed for the first time the structure-activity data of a series of selective, orally active FTase inhbitors. These inhibitors, called trihalobenzocycloheptapyridine heterocycles, represent an advance because many FTase inhibitors are not potent in cells, or when given orally. One highly potent drug in this class is now undergoing early clinical trials, Doll said.
"The field of protein prenylation is a tremendously exciting one, not only because of its intrinsic scientific interest, but also because of the potential of prenylation inhibitors as anticancer drugs," said symposium organizer Richard Gibbs, professor of pharmaceutical sciences at Wayne State University. "This symposium demonstrates the breadth of work in the area, ranging from Dale Poulter and Pat Casey's talks on the basic mechanism of protein prenylation, to the presentations from Merck, Parke-Davis, and Schering-Plough on their development of prenylation inhibitors that have demonstrated in vivo anticancer activity."
Casey's report prepared for the ACS symposium detailed FTase studies by a trio of collaborating Duke laboratories -- his and those of Assistant Professor Lorena Beese and Associate Professor Carol Fierke, both in biochemistry. Casey is an associate professor of pharmacology and cancer biology.
In the collaboration, Beese has used X-ray crystallography to develop a detailed structure of the FTase enzyme. Fierke has used biochemical and biophysical techniques to understand the detailed mechanism of the enzyme, and Casey has performed kinetic and structure-function studies.
In Casey's studies, he and graduate student Ruth Fu have produced versions of FTase with selectively altered amino acids in the active site to test hypotheses about which amino acid residues are important in binding the farnesyl and Ras molecules and in carrying out the catalytic reaction to attach the two.
"We now have a very good idea of exactly how the chemistry of the reaction occurs," said Casey, "information that has eluded us for several years."
This chemical information comes primarily from the latest studies in Fierke's laboratory showing clearly that zinc plays an important role in catalyzing the reaction, Casey said. The studies by Fierke and graduate student Chih-chin Huang show that the zinc in the FTase enzyme interacts catalytically with the sulfur in the Ras protein in the process of joining the reactive farnesyl di-phosphate molecule with the Ras protein. In experiments in which they substituted for zinc in the enzyme other metals that more tightly bind sulfur, the biochemists were able to "freeze" the reaction to better understand the mechanism. The biochemists also tested the effects of altered farnesyl molecules -- with the two kinds of experiments revealing that the FTase reaction is a "carbo-cation nucleophile mixed mechanism."
Fierke and postdoctoral fellow Kendra Hightower -- along with postdoctoral fellow Becky Spense in the Casey lab -- are exploring how both FTase and a related enzyme called geranylgeranyl transferase bind different substrate molecules. This study is an attempt to understand differences between these two enzymes that might aid development of specific inhibitors.
However, still mysterious, say the biochemists, is why FTase requires magnesium to function, while geranylgeranyl transferase does not -- a difference that might also offer clues to new inhibitors.
In his prepared ACS presentation, Casey also cited advances by Beese and graduate student Steve Long in obtaining the first crystallographic structures of the FTase enzyme that include the farnesyl di-phosphate molecule bound at the enzyme's active site, where the reaction takes place. The new structure revealed how the pocket of the active site -- into which farnesyl nestles -- acts as a "molecular ruler" to discriminate between farnesyl and the longer geranylgeranyl molecule. Beese's next studies will aim at obtaining a structure of FTase with both the farnesyl molecule and the Ras protein bound at the active site.
"All these studies are giving us a much better definition of the active site and how the enzyme specifically recognizes the farnesyl molecule and the Ras protein," Casey said. "Also, it's clear now that the enzyme-farnesyl complex is the target for most of the inhibitors, so this information, along with insights from the ongoing studies in the three Duke laboratories, will be important for further development of inhibitory drugs."
In an interview before the symposium, Casey said that as many as eight U.S. pharmaceutical companies, as well as companies in Europe and Japan, are now developing FTase inhibitors. Results of the first clinical trials should emerge this spring, he said.
"The animal tests have been amazingly successful," he said. "Complete tumor regression is observed, with no sign of toxicity in many animal models." Also, Casey said, in vitro studies had revealed that FTase inhibitors worked on a far broader range of human cancer cells -- about 40 percent of those tested -- than was expected. Such broad action implies not only that Ras is central to many cancers, but probably that a broad range of cancer-triggering proteins are activated by the FTase enzyme's addition of a farnesyl group.
"However, it's always very difficult to translate such animal results into humans," Casey cautioned. "So, it will not be until the results of clinical trials are known that we will really know how effective these drugs are."
Casey also cautioned that, "in the animal studies, although the inhibitors caused almost complete tumor regression, the tumor is never completely killed. In the model systems, withdrawal of the drug caused the tumor to return, which means that cancer patients would have to remain on the drugs.
"Such long-term therapy means that cancers could become resistant to the inhibitors, which makes it especially advantageous that many companies are working on many different types of inhibitors," Casey said. He added that studies at other institutions are also underway on combinations of FTase inhibitors with such traditional anti-cancer drugs as taxol.
In addition to studies of farnesyl transferase, Casy also emphasized the importance of studying the related enzyme geranylgeranyl transferase, which adds a longer 20-carbon isoprenoid molecule to some proteins to activate them. According to Casey, who discovered geranylgeranyl transferase, the enzyme may also play an important role in many cancers, perhaps taking over a prenylation role should F Tase be inhibited.