By David N. Leff

Criminologists argue over which penal system is better: incarceration and execution or rehabilitation and reformation.

Clinical oncologists over the years have practiced tumor excision by surgery, ablation by irradiation and execution by chemotherapy. With the recent rise of molecular and cell biology, they are increasingly turning to reforming tumors by getting their malignant cells to revert to a benign state.

When cell growth factors get out of hand, and don't know when to stop, cancer ensues. MAP kinases (MAPK) are ubiquitous enzymes expressed in almost all tissues of the body. They empower transcription factors controlling cellular growth. That empowerment takes the form of phosphorylation, a process that transfers a phosphate group from ATP (adenosine triphosphate) to the transcription protein.

"MAPK is the master regulator of mammalian cell growth," observed cell biologist Alan Saltiel. "Many different stimuli on the outer surface of a cell can jump-start MAPK to launch a cascade of phosphorylation. The kinase upstream from MAPK," Saltiel explained, "is called kinase kinase, also known as MEK. And upstream from MEK is another one, kinase kinase kinase."

Saltiel is director of cell biology at the Parke-Davis Division of Pharmacological Research in Ann Arbor, Michigan. He described how this phosphorylation phenomenon led his group to develop an oral anticancer compound that causes reversion of tumor cells, rather than their destruction. (See BioWorld Today, March 7, 1997, p. 1.)

"A complex series of phosphorylations initiated from the cell surface by receptor interaction," Saltiel explained, "requires a number of interactions from different proteins. Then signals transduced from the cell's surface to its nucleus activates this phosphorylation cascade. And the one thing about this particular cascade," he pointed out, "is that it seems to be common to a number of growth-promoting stimuli.

"So based on that information," Saltiel recalled, "we thought it might be an attractive target for cancer, and perhaps in the future for other diseases involving proliferation of cells."

Hunting A MEK Blocker To Stop A Tumor

"As far as I know," he observed, "the MAPK gene has not been found to be mutated in cancers. But the upstream proteins are frequently found to be mutated in tumor cells, and the one that's most notable is the ras oncogene. It's four steps upstream from MAPK. Ras is mutated in maybe 50 percent of all colon tumors, and frequently in other tumors as well. So the MAPK pathway is constitutively [always turned on] activated in cancer.

"In order to find an inhibitor of that pathway," Saltiel recounted," we set up a screen in a diverse chemical library that was a cascade of phosphorylation. The output of our screen was to look at the activity of MAP kinase, but it was assayed in a map kinase kinase-dependent manner. So we could find inhibitors of both MAP kinase and its upstream kinase kinase, namely MEK.

"Actually we discovered a sort of family of molecules in the screen, and then our chemists optimized the structure of one compound to increase its potency, and give us the best properties for inhibition. In this case also its bioavailability, so we were able to use these molecules orally in mice.

"We don't know precisely where on MEK the compound binds," Saltiel continued. "But we do know that for a protein kinase inhibitor it's unusual, in that it's not competitive with ATP. Many of the other protein kinase inhibitors that have been discovered - tyrosine kinase inhibitors, protein kinase C inhibitors, for example - all block the binding of ATP to the protein.

"And our kinase inhibitor has an allosteric mechanism of action, which means it binds to a site other than the ATP binding site," he continued. "That means it doesn't interfere with the substrate binding. This is important because it implies a greater degree of specificity for the kinase. So PD184352, as we now call it, is a very specific inhibitor of MEK, and highly specific to colon cancer cells.

"That specificity," Saltiel pointed out, "lies in its ability to recognize MEK. We predict it will be specific for cells that have a constitutive activation of this pathway, as in cells that have oncogenic - mutated - forms of ras. But it may also act against cells that have constitutive activation of receptors, like the erb-2 oncogene, for instance, which is a target of Genentech's anti-breast-cancer therapeutic, Herceptin. So our compound may also target the same tumors that Herceptin does. It may even provide synergy some day, if we ever test them together."

Saltiel is senior author of a report on PD184352 in the July 1999 issue of Nature Medicine. Its title is, "Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo."

In one key experiment, the co-authors implanted human colon carcinoma cell fragments, about 3 cubic millimeters in size, under the skin of nude mice. Then they administered their inhibiting compound orally every 12 hours for 14 days.

Compound Scores 79 Percent Inhibiting Colon Tumor

"Tumor growth was inhibited 53 to 79 percent," their paper reported, "with a wide dose range of the compound." Saltiel told BioWorld Today, "We were able to clock the growth of the tumors, and block them as long as the inhibitor was administered to the mice. Once we stopped treatment, the tumors came back. So this was a cytostatic, not a cytotoxic, effect. They tumor cells reverted, albeit temporarily, from malignant to nonmalignant." They lost their rounded, cancerous shape, and took on the flattened morphology typical of nontransformed epithelial cells.

To which Saltiel added, "What will happen in people is not clear. We hope to be able to start our first Phase I clinical trials sometime in the year 2000, if all goes well. That's unpredictable, of course."

Meanwhile, he said, "With respect to this compound, we're interested in evaluating what side effects it might have. So we're doing a series of experiments, in various animal species, to evaluate its toxicology. So far, things look good."

At the same time, he and his co-authors are "trying to make improved compounds. The main thing now is to test PD184352 against a panel of other tumors, to see how useful it may be against other cancers. The obvious ones to look at are those that have oncogenic ras." In vitro, the co-authors have already obtained substantial reduction of phosphorylating MAPK levels in pancreatic, breast, cervical, ovarian and colon carcinoma cells.