ZINC

RONALD BRESLOW, COLUMBIA UNIVERSITY

Zinc is an important element, especially because it plays a key role in many enzymes that are essential to life. For this reason, we humans consume approximately 20 mg of Zn(II) in our daily diets.

I have also had a series of more special interactions with zinc. Some years ago, my wife, Esther, was writing her master's thesis on the enzyme carbonic anhydrase and proposed the correct mechanism of the enzyme--its zinc acts both as a Lewis acid, by coordinating to a developing oxyanion, and as the coordination site for a basic hydroxide ion, the nucleophile that adds to the carbon dioxide molecule. Later, my research group synthesized zinc-based mimics of carbonic anhydrase and of carboxypeptidase. We also showed that carboxypeptidase itself uses the carbonic anhydrase type of mechanism.

Another example of the special properties of zinc in enzyme catalyst systems was waiting for me, but I didn't know it for a while. In 1974, Paul Marks, then dean of Columbia University's medical school, came to me with a remarkable story and challenge. Charlotte Friend of Mount Sinai School of Medicine had discovered that certain cancer cells--pre-erythrocytes that were infected with a virus (MEL cells)--were induced to differentiate to normal red blood cells in the presence of high concentrations of dimethylsulfoxide. We set out to see if we could produce much more potent compounds for this remarkable "reform" of cancer cells, learn how they worked, and perhaps even find whether they were useful with other types of cancer.

FITTING IN An X-ray structure of SAHA bound to the zinc of an HDAC.

By a series of hypothesis-driven syntheses of novel compounds that were increasingly potent toward MEL cells, we eventually came to a compound with the acronym SAHA (suberoylanilide hydroxamic acid), with a hydrophobic group connected to a hydroxamic acid unit through a polymethylene chain. We then established that SAHA would induce other cancer cells to transform into normal cells or go into apoptosis--including the entire group of 60 human cancer cell types that are maintained for study at the National Cancer Institute. So we set out to determine the biological target of SAHA and some related compounds.

We created a radioactive photoaffinity compound based on SAHA to screen cell components, determining which ones bound the drug. At the same time, we became aware of studies by M. Yoshida in Japan concerning a natural product, trichostatin A (TSA), that also induced the differentiation of cancer cells. Yoshida had shown that TSA acted to inhibit the enzyme histone deacetylase (HDAC).

We tested our photoaffinity compound with HDAC and saw that, indeed, it was bound. Furthermore, SAHA inhibited HDAC, and the potency of our other drugs as inhibitors of HDAC ran parallel to their potency in inducing the differentiation of MEL cells. Our biological collaborators were able to clone and express the enzyme, so I suggested that they examine the purified enzyme for metals, specifically for Zn(II), using atomic absorption spectroscopy. They called back with the news that HDAC contained no metals, but I didn't believe it. SAHA, with its hydroxamic acid group, looked like a metal ligand, and the catalytic action of the enzyme HDAC looked like the kind of reaction that other zinc enzymes can perform. I received another telephone call with the news that the "purified" HDAC no longer had enzymatic activity, but that the addition of Zn(II) salts restored its activity. "Purification" had removed the zinc, and HDAC was indeed a zinc enzyme!

Our collaborator Nikola Pavletich was able to prepare crystals of SAHA bound to another HDAC and determine the structure of the enzyme/inhibitor complex by X-ray. We saw that the hydroxamic acid group was coordinated to the Zn(II) in the enzyme. Furthermore, the structure strongly supported the idea that this enzyme uses zinc in the same way that carbonic anhydrase does, binding the carbonyl oxygen of the acetyl group on lysine while delivering a bound hydroxide to the carbon of that group. Our inhibitor simply mimicked this structure.

Since that time, our consortium of chemists and biologists has been able to determine the pathway by which HDAC inhibition by SAHA causes important anticancer effects by regulating gene transcription. SAHA has been shown to be an effective anticancer agent in animal trials--and for the past three years, in human trials--against a variety of cancers. The results are very promising, and there is an excellent chance that SAHA or some related compound will prove to be an important tool in the fight against cancer. Zinc will once again turn out to be key in the biology that is central to life, as the element that permits HDAC to play its role in regulating the expression of genes.

Ronald Breslow is University Professor of Chemistry at Columbia University. He received the U.S. National Medal of Science in 1991 and the 2003 Welch Award.

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