Cytoplasmic protein tyrosine kinases play critical roles in many signal transduction events in multiple cell types making them targets for drug design. Yet their precise roles in these pathways have not yet been defined. The engagement of the T cell receptor (TCR) by its ligand, the major histocompatibility complex in association with a peptide, has been one of the most intensely studied pathways because of its importance in regulating the immune response to foreign invaders such as HIV. The activation of TCR associated kinases (Lck, Fyn, Zap-70, Csk, Itk, and others) and their subsequent phosphorylation of each other and of downstream effectors likely results in the activation of several branched and interconnected pathways ultimately resulting in IL-2 gene transcription. A central question in T cell signal transduction concerns the unique functions of Lck. This kinase appears to be activated very early in the pathway and has been studied through both organismal and somatic cell knock-outs. While these approaches and many others have demonstrated that Lck is required for TCR signal transduction its specific roles have been hard to establish because of the presence of the closely related kinase, Fyn, in the same pathway. Much of our current understanding comes from experiments which rely on making drastic changes to the normal TCR; deletion of Lck, or Fyn, attachment of irrelevant domains to different kinases, or expression of selected PTKs in non-T cell lines in """"""""add-back"""""""" experiments. The key tool which has been lacking in the study of Lck's role in T cell signal transduction is a cell-permeable highly specific inhibitor of Lck. We have recently developed a method which allows for highly specific inhibitors of individual Src-family tyrosine kinases to be developed. The key to our method is the engineering of Lck's active site to contain a new pocket that does not naturally occur in any wild-type kinase in the cell. To complement the expanded pocket, a known kinase inhibitor was modified with bulky substituents making it inactive towards wild- type kinases. The important feature of this mutation to Lck is that it is designed to be completely functionally silent, in terms of the Lck's substrate specificity and its ability to replace wild-type Lck. Engineering Lck to be structurally distinct from all other kinases in the cell, yet functionally equivalent to the wild-type Lck is the key which allows for rapid discovery of uniquely specific kinase inhibitors. This proposal seeks to generate the first specific inhibitor of Lck which can be used to study the specific role of Lck in the TCR mediated production of IL-2, PLC gamma-1 activation, and Ca2+ mobilization. By determining the precise role of Lck in these downstream events, new drug design strategies can be designed to specifically interrupt abberant T cell signal cascades resulting from autoimmune diseases, viral infections, or other disease states.
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