By David N. Leff

To paraphrase a prominent pundit: "It's the metastasis, stupid!"

That is to say, it's not the initially diagnosed malignancy that kills; it's when that primary tumor jumps its reservation and invades distant organs, typically, lung or bone. Medical chemist Karl Tryggvason, at Sweden's Karolinska Institute, in Stockholm, put it this way:

"In pathological states, such as cancer, the malignant cells must be able to move from the site of the primary tumor, for example, through the connective tissue, into the blood vessels and on to other sites, in order to form metastases. Metastasis is what really kills the cancer patient.

"So, in order to be able to make their way to a distant site by way of the blood vessels," Tryggvason said, "the connective tissues and walls of blood vessels have to be broken down by specialized enzymes, the metalloproteinases [MMPs]. These enzymes are highly associated with this tumor invasion process.

"At the moment," Tryggvason told BioWorld Today, "there is no way of inhibiting metastasis other than by drugs. Known anticancer drugs are cytotoxic or cytostatic. Drugs that inhibit the metastatic process itself don't exist. So, this is one major drug-discovery target, and most of the big pharmaceutical companies have projects going on, aiming at inhibitors of metalloproteinases as anticancer drugs."

Besides his Karolinska professorship, Tryggvason is vice-president of research and development at BioStratum Inc., in Research Triangle Park, N.C., a start-up biotechnology firm he co-founded in 1994. It owns the rights to intellectual property generated by Tryggvason's research.

BioStratum's executive vice president, J. Wesley Fox, traced the wreckage-strewn route by which tumor cells metastasize.

"When a metastasizing tumor cell must go from its primary site to lungs and bone," Fox explained, "the biggest, strongest, toughest barrier that it has to traverse is the basal lamina. To do so, it has these MMP enzymes, which are activated in the neoplastic phenotype. They chew up the extracellular matrix in general. But the first thing they have to chew up specifically is the basal lamina.

"With respect to the cancer context," Fox said, "the basal lamina is relevant to the blood vessel. It is basically a thin, membranous sheath, lining the blood vessel's lumen. It provides a site for the endothelial cells to adhere to. The basal lamina is more than a barrier; it's essentially the blood/tissue barrier, because any protein or cell that goes from the bloodstream into a tissue must cross the basal lamina. Nutrients and small peptides will diffuse across readily, but big proteins such as tumor cells can't break through, unless those cells have certain enzymatic properties that get activated. These enable them to penetrate the lamina, get into the bloodstream, travel to the distant tumor, then get out of the bloodstream, and into that tissue."

This metastatic break-and-enter mayhem involves enzymes of the numerous MMP family. One of its most active members is MMP-2, of which Tryggvason, after seven years of effort, has just elucidated the active site's crystal structure. His paper, in today's issue of Science, dated June 4, 1999, bears the title: "Structure of human pro-matrix metalloproteinase-2: Activation mechanism revealed."

"This structure," Tryggvason said, "will tell us exactly what the enzyme looks like in three dimensions. So, now one can start looking for anti-metastasis drugs in a new way. For example, several inhibitors have been developed against MMPs, and they can now be modified to fit better into the structure of the enzyme. Maybe one would be able to develop inhibitors that are specific for certain members of the MMP family."

From X-Ray Structure To Inhibitor Function

His laboratory has just purchased a Cray super-computer to model its search for new compounds that can fit into different sites of the enzyme's targets, for inhibition of enzyme activity.

"Several matrix metalloproteinase inhibitors that react with the active site are being tested in pharmaceutical companies," Tryggvason said. "Some of them have been going to clinical trials." He cited British Biotech plc, of Oxford, U.K., which has an ongoing Phase III clinical study of its oral agent, Marimastat, and Agouron Pharmaceuticals Inc., of La Jolla, Calif., with a Phase II/III study of its oral AG3340.

However, Tryggvason added a caveat: "These drug candidates have some side effects. It's important to emphasize that several compounds are being tested as inhibitors against different members of this MMP proteinase family. Most of them are against the same site, which is very conserved in all of these enzymes. Now, recent experiments from our laboratory and some others have shown that these enzymes have different functions, some of which are essential for life.

Too Much Inhibition Can Be Hazardous

Tryggvason warned that, "if one has broad inhibitors, to inhibit for example all of these proteinases, these MMP enzymes would clearly have bad side effects. They could literally dissolve you, so you wouldn't want them floating around freely. So, it's important, I think, that one starts to make inhibitors that are enzyme-specific. And the exact structures that we get from this MMP-2 enzyme and others in the future, hopefully, will help us design useful inhibitors that are specific for each member of this family."

On this score, Tryggvason recalled, "We have recently made knockout mice in which we inactivated one of these MMP genes. Those animals die a few weeks after birth. Other enzymes of this family have also been inactivated by this knockout technology, and have not had any problems. So, how important these enzymes are for the human body varies. Therefore, we would not want to make an inhibitor that would inhibit an MMP that is essential for life, while others, maybe, would be more important for cancer."