June 3, 2010

Protein’s structure may offer drug design insights

Featured Image

Joseph Deweese, Ph.D., left, and Neil Osheroff, Ph.D., with a structural model of part of topoisomerase II, a protein that untangles DNA and is a target of anti-cancer and anti-bacterial drugs. (photo by Susan Urmy)

Protein’s structure may offer drug design insights

A new view of a protein that “untangles” DNA could provide clues for designing better treatments for cancer and bacterial infections.

Researchers from Vanderbilt University Medical Center and the University of California, Berkeley, have determined the first structure of the protein topoisomerase II — a target for anti-cancer drugs and antibiotics — at a critical stage of its activity. The findings are reported in the journal Nature.

The DNA in our cells is essentially a very long double-stranded rope, explained Neil Osheroff, Ph.D., professor of Biochemistry and Medicine at VUMC. As it is “unwound” so that enzymes can copy it — to provide protein-making instructions or to generate new DNA during cell division — it can become knotted or tangled.

Topoisomerase II removes these knots and tangles from the genetic material. To do this, the protein cuts the DNA, passes one double helix through another, and stitches it back together.

“This enzyme is essential — if you can't remove knots and tangles from your DNA, bad things happen (like cancer-causing mutations or chromosomal translocations) — but the enzyme also is dangerous because it cuts both strands of the double helix,” Osheroff said.

Drugs that interfere with topoisomerase II activity take advantage of the dangerous moment after the DNA is cut. They insert themselves into the complex “like molecular doorstops” and prevent topoisomerase II from putting the DNA back together, leading to cell death.

Such drugs — called “topoisomerase II poisons” because they convert an essential enzyme to a lethal enzyme — are among the most widely prescribed anti-cancer drugs in the world (e.g., etoposide, adriamycin).
“Every form of cancer that we consider to be 'curable' by systemic chemotherapy is treated with drugs that target topoisomerase II,” Osheroff said.

Bacteria also contain topoisomerase II-equivalent enzymes, and these bacterial proteins are the targets of quinolones, a family of broad-spectrum antibacterial drugs that includes ciprofloxacin, the treatment of choice for anthrax infections.

Osheroff and his team have focused on how topoisomerase II works — how it cuts DNA, how it puts DNA back together, and how drugs affect its function. Because topoisomerase II rejoins the cleaved DNA very quickly, it has been difficult to study the enzyme just after it has cut the DNA, in a stage called the “cleavage complex.”

“Trying to capture the cleavage complex has been a sort of grail for the field,” Osheroff said.

Osheroff and Joseph Deweese, Ph.D., a graduate student at the time, were studying the role of metal ions in regulating topoisomerase II activity.

They realized that manipulating the types of metal ions available to the enzyme made it possible to trap the cleavage complex. Crystallographer James Berger, Ph.D., and his colleagues at the University of California, Berkeley, were able to use the method to generate enough of the cleavage complex for structural studies.

The resulting “first glimpse” of the topoisomerase II cleavage complex has revealed critical roles for metal ions in cutting the DNA and putting it back together, Osheroff said. The studies also have suggested how the enzyme opens and closes multiple “protein gates” to carry out its DNA-detangling steps.

“Being able to 'see' how all of these pieces fit together makes it possible to know where drugs can be modified to design better therapeutics — drugs that are more potent and that can overcome tumor and bacterial drug resistance,” Osheroff said.

Osheroff holds the John G. Coniglio Chair in Biochemistry. Deweese is an assistant professor of Pharmaceutical Sciences at Lipscomb University College of Pharmacy. The National Institutes of Health supported the research.