Kevan Shokat

Bio-organic Chemistry — Organic chemistry is used to probe fundamental signal transduction pathways in cells and whole organisms.

Research in our laboratory is focussed on the development of novel chemical tools to decipher signal transduction pathways on a genome-wide scale. The cellular machinery responsible for integrating complex extracellular signals from other cells is very complex. Protein phosphorylation is at the heart of these signaling cascades and is thus the most common on/off switch used in our bodies. The enzymes that catalyze protein phosphorylation are protein kinases. The major focus of our laboratory is to understand in detail the role of each kinase in the body, and determine which kinases would be good candidates for drug development to cure a wide range of human diseases. Specific examples of diseases we are currently working on are: asthma, multiple forms of cancer, neurological disorders, bacterial infections, drug addiction, and chronic pain. The main advantage of our chemical approach to these problems is that the small organic molecules we make to inhibit single kinases in whole animals are much better models of how potential drugs which target the same proteins might actually work, hopefully saving time in avoiding failed clinical trials.

We believe that small-molecule based methods for decoding cell-biology could provide information not currently accessible through solely genetic and biochemical techniques. The key problem with using small-molecules to control processes inside cells is that most often these reagents inhibit closely related enzymes with equal potency, making dissection of each individual protein impossible. Currently, small-molecules which alter the enzymatic activity or cellular localization of key biological macromolecules are derived from two sources: natural product screening (eg. Taxol, FK506, staurosporine, and others) and drug development efforts (eg. Aspirin, Raloxifine, SKB203580, and others). These two approaches require large commitments of time and resources to find even one specific inhibitor.

Our lab has developed a third method for producing these valuable reagents using an approach combining protein design and chemical synthesis. We use protein design to engineer a functionally silent yet structurally significant mutation into the active site of the protein of interest. This mutation could be the substitution of a conserved large residue in the wild-type enzyme for a smaller residue thus creating a new "pocket" in the active site. The mutant enzyme is then tested in a relevant cellular system to ensure that it functions in all aspects like the wild-type enzyme.

The next step is the initiation of a chemical design and synthesis project to modify a non-specific inhibitor of the wild-type enzyme with substituents which specifically complement the mutation introduced into the active site of the protein of interest. Substituents with the appropriate chemical functionality that bind to the newly introduced pocket are chemically appended to the origininal inhibitor structure. Importantly, the new substituent is designed to preclude binding of the inhibitor to any wild-type enzymes because they would "bump" into the large residue in the wild-type enzyme. This makes choosing a residue which is conserved in the entire protein family critical for the success of the method.

A pyrazolopyrimidine based kinase inhibitor we have identified satisfies the criteria for an inhibitor which only inhibits mutated kinases and does not inhibit any wild-type kinases we have assayed. We have most clearly demonstrated the utility of our approach in several studies of the yeast kinases CDC28 (cyclin dependent kinase 1: cell cycle), Fus3 (Map Kinase: involved in mating), Ipl1 (centrosome associate kinase), CDC15 (kinase involved in the exit from mitosis), Cla4 (Pak kinase, bud emergence), Elm1 (control of bud emergence), Ark1 (actin associated kinase 1, vescicle fusion), and a number of others are currently in progress.

The variety of kinases that our approach has been applied to already suggests to us that over 70% of the protein kinase superfamily can be suitably engineered to be sensitive to the pyrazolopyrimidine based inhibitor we have identified. The exciting aspect of this is that the pyrazolopyrimidine has ideal pharmacological properties, including good bioavailability in mice, is able to cross the blood brain barrier, crosses the yeast cell wall, and is easy to synthesize in large amounts. The inhibitor is quite potent, in that it is a <5 nM inhibitor of every mutant kinase we have made. These features make our chemical genetic system very portable for studies of protein kinases in multiple model organisms including yeast, mouse, and soon C. Elegans, and the fly.