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Shokat, Kevan
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Genetic programming of a single protein kinase (c-Src) provides direct tagging of the substrates of this kinase - using an unnatural ATP analog that is not accepted by any natural protein kinase.
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| Research Statement | | 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 (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 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 ellucidate 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. |
Publications | | Ubersax JA, Woodbury EL, Quang PN, Paraz M, Blethrow JD, Shah K, Shokat KM, Morgan DO. “Targets of the cyclin-dependent kinase Cdk1.”
Nature. 2003, 425(6960):859-64.
Papa FR, Zhang C, Shokat K, Walter P. “Bypassing a kinase activity with an ATP-competitive drug.” Science. 2003, 302(5650):1533-7.
Knight ZA, Schilling B, Row RH, Kenski DM, Gibson BW, Shokat KM.
“Phosphospecific proteolysis for mapping sites of protein phosphorylation.” Nat Biotechnol. 2003, 21(9):1047-54.
Kraybill BC, Elkin LL, Blethrow JD, Morgan DO, Shokat KM. “Inhibitor scaffolds as new allele specific kinase substrates.” J Am Chem Soc. 2002, 124(41):12118-28.
Bishop AC, Ubersax JA, Petsch DT, Matheos DP, Gray NS, Blethrow J, Shimizu E, Tsien JZ, Schultz PG, Rose MD, Wood JL, Morgan DO, Shokat KM. “A chemical switch for inhibitor-sensitive alleles of any protein kinase.” Nature. 2000, 407(6802):395-401. |
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