Study Of Key Enzyme Sheds New Light On Programmed Cell Death And May Lead To New Drugs For Reducing The Severity Of Stroke

Scientists at Vanderbilt University and Northwestern University have determined the three-dimensional structure of a critical region of Death Associated Protein Kinase (DAPK) and created a quantitative assay capable of measuring its activity.

These results were published in the October issue of Nature Structural Biology and the October 19 issue of the Journal of Biological Chemistry and are generating considerable interest in the pharmaceutical industry because DAPK provides a new target for the development of drugs that could reduce cell damage following brain injuries and stroke.

The senior authors of the two papers are Martin Egli, associate professor of biological sciences at Vanderbilt University, and D. Martin Watterson, director of the Drug Discovery Program at Northwestern University. Egli’s group mapped the three-dimensional structure of DAPK’s kinase domain and Watterson’s group developed the quantitative assay. DAPK is a large protein with a number of distinct domains. It was discovered in 1995 by Adi Kimchi at the Weizmann Institute of Science in Israel while screening the entire genome for genes that promote a process called programmed cell death and inhibit tumor growth.

DAPK contains a "death domain" that can initiate a cascade of molecular events that cause a cell to commit suicide. This process, called programmed cell death or apoptosis, is programmed into all but the most primitive of cells. It causes the cell to shut down in an orderly manner so that its contents can be absorbed by surrounding cells without initiating an attack by the body’s internal self-defense systems. This is particularly important in enclosed areas like the brain. Previous research has implicated DAPK in a wide range of apoptotic systems and suggests that it is activated very early in the process, well before the cell becomes irreversibly committed to self-destruction.

Another region of DAPK has been labeled the kinase domain. Its role is to strip phosphates from adenosine triphosphate (ATP) – a molecule involved in enzyme regulation – and attach them to certain other proteins, called substrates. This process is called phosphorylation and it is a common method of turning cellular processes on and off.

Scientists have determined that DAPK’s kinase domain is intimately involved in triggering the process of programmed cell death, but they don’t know how. The determination of the domain’s structure and the ability to evaluate DAPK’s activity provide an important foundation for future investigations addressing this question.

"When we started this project, we didn’t think about it as a drug target," says Egli, "but we are getting a number of calls from drug company researchers."

This interest is based primarily on animal studies published in 1999 that showed significant increases in DAPK preceding episodes of neuron death. There is currently a "time window of unmet need" for therapeutics following a stroke or brain injury. During this period, which can last from hours to days, neurons continue to die, adding significantly to the initial damage.

The timing of DAPK’s increase in the animal studies combined with its established role in initiating cell death raise the possibility that DAPK inhibitors could reduce neuronal cell death during this critical period. "Currently, there is nothing that doctors can do to address the fundamental cause of neuronal death during this period," Watterson says. "So there is considerable interest in the possibility that administering a drug that inhibits DAPK activity during this period might reduce brain damage."

Before this idea can be tested, however, researchers must find small molecules that effectively inhibit DAPK activation. The determination of the structure of DAPK’s kinase domain and the development of a quantitative assay now make it possible for drug researchers to develop efficient methods for identifying candidate inhibitors and to employ structure-assisted design procedures to create them from scratch.

The description of a quantitative assay that can measure DAPK activity is the subject of the article in the Journal of Biological Chemistry. Watterson, working with Drug Discovery Program trainees Anastasia V. Velentza, Andrew M. Schumacher and Curtis Weiss, identified peptides that the kinase domain phosphorylates at a variety of different rates. This allowed them to create an assay that measures the activity level of DAPK quantitatively and provides insight into the localized features that DAPK prefers in such substrates.

The researchers are using the assay to screen large collections of chemical compounds for the ability to inhibit DAPK. The compounds represent a broad spectrum of small molecular structures with drug-like properties. Such screens are likely to find a small number of molecules that are weak inhibitors of DAPK.

Once such candidate inhibitors are found, their structures can be fine-tuned using the information about the structure of DAPK’s kinase domain that was reported in Nature Structural Biology by Egli, working with Valentina Tereshko and Marianna Teplova, former research associates in his laboratory who are now at the Memorial Sloan-Kettering Cancer Center. They produced a three-dimensional map of the kinase domain with the highest resolution obtained for any other known kinase. The higher the resolution with which the structure of a kinase is known, the easier it is for drug developers to use molecular modeling techniques to design "virtual" molecules that have the proper structure to bind with the active regions of the enzyme and inhibit its normal function.

DAPK’s involvement in cancer may also prove to be important. The view of cancer as a disease of uncontrolled cell growth is gradually being expanded by its additional characterization as a disease resulting from malfunctions in the process of cell death. Reductions in DAPK expression have been found in a variety of different types of human cancer. In this case, researchers will be searching for agents that can reactivate programmed cell death in tumor cells. The newly published research provides an important knowledge base for the search for DAPK substrates in normal and diseased tissue.

The initial phase of this inhibitor discovery research will be reported by Watterson, Egli and their collaborators at the Spring 2002 Experimental Biology Meeting in New Orleans.

The research was supported by the Alzheimer’s Association, the Institute for the Study of Aging and the National Institutes of Health.