By David N. Leff

Science Editor

What substance would you nominate as "Molecule of the Millennium?"

A strong candidate is p53, a prominent protein with a molecular weight of 53. It's emblematic of the woes and wonders, the mayhem and the marvels, perpetrated by two-faced historic figures over the past thousand years. p53 is known as "The Guardian of the Genome" because it patrols the DNA of dividing cells, and either repairs damaged sequences on the spot, or causes the imperfect cell to commit apoptosis - suicide. This damage control prevents the cell from going berserk and dividing forever - that is, turning malignant. So the p53 protein's parent gene, p53, has been welcomed as a tumor-suppressor.

But that p53 gene, which encodes the protein, can itself suffer DNA damage, and so mutate. Then it spurns its beneficent task of tumor suppression, and assumes the murderous role of oncogene.

Molecular oncologists who discovered p53's double-dealing machinations in the 1980s found its mutant version infesting 52 kinds of cancer - in 50 percent of brain tumors, 35-60 percent of bladder cancers, 30-40 percent of breast, 30-60 percent of ovarian, and up to 70 percent in lung and colon cancers. So this mother of all malignancies has been the target ever since of research to neutralize the p53 oncogene, or rehabilitate the tumor suppressor.

The latest such effort appears in the Dec. 24, 1999, issue of Science. Its title: "Pharmacological rescue of mutant p53 conformation and function." The paper's senior author is cancer biologist Farzan Rastinejad, at Pfizer Central Research in Groton, Conn.

"The normal p53 protein," Rastinejad told BioWorld Today, "is expressed in every cell of the body, at very low levels. But when you look into tumor cells, there are huge levels. When something goes wrong in dividing cells," he continued," that's when p53 goes to work."

Likely Needles In Half-Million-Compound Haystack

Rastinejad pointed out that "p53 is an unusual gene. When most genes are mutated, they don't make protein at all," he noted. "In p53, it's usually just one point mutation, and that's expressed in the part of the protein that binds to DNA This DNA-binding domain is about 200 amino acids long - half the protein's total 390-amino-acid length. Point mutations can occur anywhere in that area. And at least 100 amino acids can be mutated in tumors. But the interesting thing," he added, "is that when you get these mutations, you still get this protein, despite that one amino acid difference."

As they reported in Science, Rastinejad and his co-authors discovered and tested a family of compounds that showed signs of reversing this malignant transformation in p53 - although weakly. "They are small molecules," he recounted, "with a molecular weight of 300 to 500, which is in the range where most drugs would be. Pfizer has been making compounds for tens of years," he went on, "so we have a collection on the order of 500,000 physical compounds, individually stored in small bottles.

"Usually," Rastinejad related, "when we find a target that we think is particularly important - in this case, p53 - we develop an assay that would tell us whether the compound could modulate this target. So, with inputs from OSI Pharmaceuticals Inc., of Uniondale, N.Y., we set up a high-throughput screen, where we tested tiny amounts of our most promising compounds. It was done with robots, so we were able to screen our entire library in a matter of weeks."

The team was looking in particular at the ability of its candidate drugs to maintain the structural conformation of the p53 target protein, so that even if it was mutated, it would still function as a tumor suppressor.

"I think the innovation in our paper," Rastinejad observed, "was showing that we could modulate the conformation of the protein. The p53 protein is like a coiled-up spring, and there's not much holding it together. It has to be folded up just right to be able to bind to the DNA and perform its function. And mutant p53, because you change one of its amino acids, is even less stable.

"Mutants may be originally folded up," he explained, "but they do not hold that structural shape very long. So the idea was if we could get a compound that would somehow interact with the protein, then it might make it a bit more stable. Then the mutant protein, or even the normal wild-type protein, would be able to hold the right form, and stay functional longer."

Tightening p53's 3-D Structure Is Key

"We were looking for compounds that would slow down, or even stop, this unfolding process," Rastinejad recalled. "Among those we tested was one, No. CP31398, which turned out to create a chemical leash that holds the mutated p53 together, so it can resume its normal tumor-suppressor activity."

After assaying this and other candidate compounds on tumor cells in vitro, "The obvious next step," Rastinejad pointed out, "was to take this compound into animals." The team conducted in vivo trials of CP31398 in nude mice carrying human colon carcinoma and melanoma tumor cells. These implanted tumors, they knew, harbored p53 mutations. "The upshot we reported," he said, "was that the compound could modulate the conformation of the p53 proteins that were inside the tumor cells. Also, that when we gave CP31398 to mice every day for a week, that slowed down the growth of their human tumors."

As for the future, Rastinejad concluded, "One of the things that we'd like to learn is how these compounds interact with the protein at the molecular level. Once we know that, it could help us understand how to get compounds that could fix mutant p53. Obviously that might be many years away, but there's a lot of promise for it."