Development of Irreversible Substrate Competitive Probes for PKA Activity

The current environment for drug discovery and disease treatment relies heavily on genomic analysis, structural biology and chemical biology techniques. With the enormous advances in genomic analysis and structural biology, the use of and desire for targeted therapies has increased. However, as more genomic data for cancer disease state pathology becomes available we must ask increasingly difficult questions and even produce new technologies, such as activity-based probes, to answer these questions. In particular, targeted kinase inhibitors for the treatment of cancer has become a mainstay for drug development for both industry and academia, but it is evident that the genomic data is not always indicative of protein expression. Additionally, protein expression alone does not completely characterize functional activity. Therefore, in order to more accurately validate drug targets and predict drug efficacy, we must not only identify possible targets but also determine their activity in vivo. The goal of this work was to develop a probe for Protein Kinase A that would act by alkylating a conserved cysteine in the substrate-binding pocket of the enzyme. We hypothesized that by targeting the substrate-binding pocket we could effectively utilize the natural substrate selectivity filters as well as take into account multiple endogenous regulatory mechanisms. We produced probes utilizing portions of the pseudosubstrate inhibitor PKI that demonstrate the ability to label the catalytic subunit of Protein Kinase A in an activity-dependent manner, thus making it an important first step in a new class of activity-based probes for the kinome