Inhibitors for protein kinases may ultimately constitute a novel family of medicinally active agents. Unfortunately, the challenges associated with the acquisition of inhibitors for the enzyme targets are unlike any that have ever been encountered in medicinal chemistry. Protein kinases pose a variety of obstacles in terms of inhibitor design, nearly all of which deal with, in one fashion or another, the issue of specificity. The principal investigator has recently discovered that even closely related protein kinases can be distinguished by differences in their active site substrate specificity Based upon these differences a series of kinase specific/selective inactivators/inhibitors has been created. All of these substrates, inhibitors, and inactivators are peptide-based species that interact with the protein-binding region of the active site. As a consequence, these compounds d not suffer from the severe disadvantages associated with inhibitors directed t the ATP-binding region. The purpose of the present proposal is to translate th principal investigator's promising in vitro results into compounds that function under in vivo conditions. The focus of the present proposal continues to be the ACG subfamily of protein kinases. Is it possible to develop both selective and potent inhibitors for th members of this kinase family, including individual isoforms? The principal investigator has found that appending non-amino acid residues to active site-directed peptides can dramatically enhance PKA affinity. The principal investigator will employ an analogous approach using two different types of chemical libraries to identify compounds exhibiting the requisite inhibitory traits for all members of the ACG subfamily. Although the active site-directed peptide inhibitors can establish whether a particular protein kinase is a member of a specific signaling pathway, they will not provide information concerning when and where the kinase is active, so a series of caged inhibitor will be developed for that purpose. Can protein kinase activity be assayed in direct, continuous, and highly sensitive fashion? The principal investigator has developed such a continuous assay for PKC. Analogous assays will be developed for the other members of the ACG family. Can in vivo assays be developed for protein kinases? Issues related to the direct visualization of protein kinase activity in the cell, including fluorophore design and sensitivity to intracellular phsophatases and proteinases will be examined.
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