Development of Novel Tools to Study Signal Transduction

Research in our laboratory is focused on the development of novel chemically based tools to decipher signal transduction pathways on a genome-wide scale. 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 chemicals (small-molecules) to control processes inside cells is that there are very few molecules that are highly specific for the most interesting proteins. Presently, small-molecules which alter the enzymatic activity or cellular localization of key biological macromolecules are derived from two sources: natural product screening (e.g. Taxol, FK506, staurosporine, and others) and drug development efforts (e.g. 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 original 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 that is conserved in the entire protein family critical for the success of the method. We first applied our approach to deciphering the functions of protein kinases. The protein kinases comprise the largest single gene family in the human genome and are components of the signaling apparatus of almost every key cellular response pathway. Using our combined chemical genetic approach we have developed the first method for identifying the direct downstream substrates of protein kinases. Additionally we have developed extremely potent inhibitors of individual protein kinases that are cell permeable and function in transgenic mice. These inhibitors can be used to inhibit any suitably engineered protein kinase to elucidate the downstream response pathway controlled by that kinase. We are also currently extending our efforts to other large superfamilies of enzymes involved in signal transduction pathways for which specific inhibitors are not available.