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.
Journal
Nature