Chemical phosphoproteomics and development of bisubstrate based inhibitors of protein kinase C isozymes

Protein phosphorylation is one of the most abundant post-translational modifications. Phosphorylation is important for the function of the protein, for example it regulates enzyme activity, signal transduction and cell division. Protein phosphorylation plays a central role in virtually all crucial cellular processes. Therefore methods have been developed to determine which proteins are phosphorylated, that is to unravel the phosphoproteome. One approach relies on beta-elimination of Ser-/Thr-phosphorylated peptides under basic conditions. This method for the identification of phosphorylated peptides is, however, not completely selective since O-glycosylated peptides are also modified and thus yielding false positives. Therefore, a reliable method for the selective analysis of phosphorylated peptides in the presence of O-glycosylated peptides had to be developed. In this thesis a model system is described containing both phosphorylated- and O-glycosylated peptides to investigate the optimal conditions for selective beta-elimination. By tuning the basic reaction conditions, selective beta-elimination was achieved and the phosphorylated peptide was selectively enriched in the presence of an O-glycosylated peptide. The enzymes responsible for creating the phosphoproteome are protein kinases. The protein kinase family comprise over 500 members and form another subproteome, the so called kinome. A subclass within this group is the Protein Kinase C (PKC) family which consists of 12 isozymes that are highly homologous at the amino acid level. PKC enzymes are involved in a wide range of cellular processes including gene expression and cell growth. Therefore, uncontrolled activation of PKC is related to several serious diseases like cancer and diabetes. With the realization of the importance of protein kinases in cellular processes, this class of enzymes has become a major drug target. However, the synthesis of selective kinase inhibitors has been proven to be difficult since these enzymes show high sequence homology, especially with respect to their catalytic domains. For inhibition of kinases in the catalytic domain several strategies can be used. This thesis describes the design and synthesis of bisubstrate-based inhibitors. These bisubstrate-based inhibitors target both the ATP-binding site and the peptide substrate binding site of the kinase. The ATP-binding site inhibitors were known ATP-competitive kinase inhibitors described in literature and modified with an acetylene functionality for bisubstrate formation. Peptide substrate binding site inhibitors were selected using dynamic microarray technology, developed by Pamgene B.V. ('s-Hertogenbosch, the Netherlands). A peptide sequence that was selectively phosphorylated by one of the kinases tested, was synthesized using solid phase peptide synthesis and functionalized with an azide functionality. Next, the azide-functionalized peptides were chemoselectively coupled to acetylene-functionalized ATP-competitive inhibitors by a Cu(I)-mediated click-reaction to give in total nine bisubstrate-based inhibitors. All synthesized compounds were biologically evaluated for their activity against PKC using flow-through microarrays. The best result obtained was a bisubstrate based inhibitor, that showed selective inhibition of PKC theta with an increased affinity in the low nanomolar concentrations (0.6 – 0.9 nM).