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Protein kinases regulate numerous cellular processes, including cell growth, metabolism, and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set out to develop an inhibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an in vitro Kd of ~50 nM and reducing cardiac injury IC50 of ~5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 μM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors. [ABSTRACT FROM AUTHOR]

The article describes a laboratory preparation of aspartame analogs using simultaneous multiple parallel synthesis methodology. Many biological purposes require using numerous peptides that slightly differ in amino acid composition. To synthesize peptides the carboxylic acid group of the amino acid must be coupled to the amino group of previous amino acid while protecting any functional side chains that are present by using pre-protected residues. The synthesis and characterization of three dipeptide analogs of aspartame is demonstrated using the tea bag methodology.

We present a new approach for the conversion of active sequences of proteins and peptides into small molecules. A library of macrocyclic disulfide molecules was made, in which the active pharmacophores of the parent peptide are preserved while the size of the macromolecular scaffold on which the pharmacophores are arranged is varied. This enables a systematic search for macromolecules in which the pharmacophores are in an appropriate conformation for biological activity. We developed two procedures for the synthesis of such libraries from building blocks that include commercial amino acids and functionalized aldehydes. Chemical synthesis using the "tea-bag" method gave a library with higher diversity, but low yields, compared to the manual synthesis of the library, in which the compounds were synthesized in individual vessels and the yield and purity improved dramatically. As a proof of concept, we synthesized a 34-member library derived from the sequence of the activation loop of insulin-like growth factor-1 receptor. Selected compounds were screened, and one was found to be biologically active in the low micromolar range. The concept presented here may prove particularly useful in cases where the pharmacophores are known but need to be systematically screened for a spatial arrangement that will enable biological activity.