Scientists Develop Chemical Switch For Natural Signaling Molecules,
Opening New Approach To Biomedical Research And Drug Design
Applying the tools of chemistry where modern genetic techniques have so far
fallen short, a team led by a University of California, San Francisco scientist
has developed drug-like inhibitors to study vital signaling molecules essential
for almost all cell activity. The research opens the way to identify the functions
of hundreds of these molecules, called kinases, crucial to signal transmission in
all cells and, in the same step, identify precisely how drugs can inhibit kinases
when they go awry and cause disease.
In the current (September 21) issue of the journal Nature, the scientists
reported first using genetic techniques to carve out a small part of a kinase
molecule - a constituent common to the many hundred known kinases. They
then searched for small molecules that fit precisely into the pocket created by
this structural change. The added molecule inhibited, but did not destroy
kinase function -- just what is needed to tease apart the unique role of one
kinase from all others.
The new technique can chemically switch on or off individual kinases among
many hundreds found in every cell. Until now, changing the structure of
proteins to study their function has been the hallmark of modern molecular
genetics, leading to fundamental leaps of understanding, including the
identification of tumor-suppressor genes and their role in cancer. But essential
as they are to nearly all life processes, kinases have proven resistant to genetic
approaches to study them.
"Instead of trying to figure out what was unique to each of the hundreds of
kinases, we looked for what was common to all of them," explained Kevan
Shokat, PhD, UCSF associate professor of cellular and molecular
pharmacology and senior author on the research paper. "We then used a
genetic mutation to carve a new hole in this common, active site of the kinase
and introduced a new molecule which specifically bound to the pocket we
created."
Kinase molecules are active in nearly all signaling within and between cells,
and are required for everything from cell division and development to learning
and memory. Genetic approaches that inhibit one kinase usually induce
compensatory responses such as over-expression of related kinases. Efforts
to get around this problem have led to other roadblocks. Overall, the genetic
approach has not been so successful in studying kinases without disrupting
overall cell function, Shokat said.
The new research succeeds precisely in this arena. The chemicals inhibit just
the catalytic effect of the specific kinase under study and do not alter its other
functions. More importantly, the function of other kinsases remains unaffected,
keeping the cell functioning and allowing experiments to determine the specific
function of the kinase.
In experiments with yeast -- done in collaboration with another UCSF
scientist, David Morgan -- the resultant "mutant kinase" was able to perform
its normal function in the organism but was clearly distinguishable from all
other kinases, establishing that the technique is a potent new research tool.
The research success demonstrates for the first time that a precise knowledge
of the composition and structure of kinases and their chemical inhibitors can
be used to design new kinase/inhibitor pairs by combining the tools of organic
chemistry and protein engineering. This offers a powerful alternative to using
genetic mutations to study proteins and determine their biological roles.
The researchers demonstrated the successful technique on five large families of
kinases. The broad-ranging success is expected to allow scientists for the first
time to isolate the function of hundreds of kinases. The research demonstrates
the power of combining genetic and chemical approaches.
The research goal, Shokat said, has been to find a chemically-based approach
to study all kinases in as rapid a manner as possible -- a basic challenge in
functional genomics.
Among the 800 known human kinases, all share a chemical site where they
bind to the energy-providing molecule ATP. Researchers now recognize that
kinases also appear to serve as scaffolds for other proteins, stabilizing a
network of molecules which together regulate vital signaling -- speeding up or
slowing down message transmission as the needs of the cells dictate. The ideal
research probe, Shokat points out, would be able to isolate just one of these
functions, while leaving the others intact, and that is just what the new
approach allows.
Shokat calls the new technique a "chemical switch" since the cell is able to
reverse the inhibiting effect over time and switch back on the kinase if the
chemical is withdrawn. This allows precise experiments to study the temporal
activity of individual kinases in animals.
With the success of the experiments in yeast, Shokat and his colleagues are
turning to mammalian cells, eyeing human drug development applications.
Shokat has already shown that the synthesized molecules work in mammalian
cell cultures and in mice. Pharmaceutical companies have a keen interest in
kinase inhibitors that could treat cancer by dampening overactive enzyme
activity often involved in uncontrolled tumor growth, Shokat says. Shokat is
confident that mutant kinases in animals will prove useful for screening such
drugs.
"In our experiments, we essentially identify potential drugs to inhibit kinases as
we are proceeding in our basic research. Drug companies can see the effect of
specific inhibitors of a single kinase without having to launch a more general
search," he says. Shokat and his colleagues have filed for patents on this
potent research approach and the patent has been licensed to a private
genomics company.
First author on the Nature paper is Anthony Bishop, PhD, a post-doctoral
researcher at Scripps Research Institute and formerly Shokat's graduate
student graduate at Princeton University where much of this research was
undertaken.
Co-authors and collaborators on the research with Shokat, Bishop and
Morgan are Jeffrey Ubersax, a graduate student and Justin Blethrow, research
associate, in physiology and biochemistry at UCSF; graduate student Dejah
Petsch and John Wood, PhD, professor, both in chemistry at Yale University;
Mark Rose, PhD and Joe Tsien, PhD, both professors; graduate student Dina
Matheos and post-doctoral researcher Elji Shimizu, all in molecular biology at
Princeton University; Nathanael Gray and Peter Schultz at the Genomics
Institute of the Novartis Foundation.
The research reported in Nature focused on the kinase Cdc28, needed for
normal cell division. Trying to study such a kinase using genetic techniques that
create a "knockout" yeast lacking the gene would not be possible, since
disrupting cell division would be lethal, Shokat points out. The results from the
collaboration between Morgan and Shokat revealed that the kinase activity of
Cdc28 was most critical before cell separation. In contrast, genetic studies
with temperature sensitive forms of Cdc28 suggested the most critical role
was before the DNA duplication stage of the cell cycle. These results suggest
that kinase activity is sensed during the cell cycle and inhibitors can precisely
regulate this function, leading to different arrest points than those revealed by
genetic studies.
The research was funded by the National Science Foundation, the National
Institutes of Health and the Glaxo-Wellcome pharmaceutical company. |
