Scientists develop chemical switch for natural signaling molecules, opening new approach to biomedic

By Wallace Ravven

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

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.