of key enzyme sheds new light on programmed cell death and may lead
to new drugs for reducing the severity of stroke
By David F.
December 6, 2001
data on a complex enzyme that lies at the crossroad between cell
suicide and tumor suppression has opened a promising new front in
the battle to find effective treatments for stroke and cancer.
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.
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.
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
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.
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.
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."
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.
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."
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.
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.
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 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
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.
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.
was supported by the Alzheimer's Association, the Institute for
the Study of Aging and the National Institutes of Health.
Center for Structural Biology website
D. Martin Watterson