Biochemical Discovery, Intracellular Evaluation, and Crystallographic Characterization of Synthetic and Natural Product Adenosine 3?,5?-Cyclic Monophosphate-Dependent Protein Kinase A (PKA) Inhibitors

The recent demonstration that adenosine 3 ',5 '- cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) plays an oncogenic role in a number of important cancers has led to a renaissance in drug development interest targeting this kinase. We therefore have established a suite of biochemical, cell-based, and structural biology assays for identifying and evaluating new pharmacophores for PKA inhibition. This discovery process started with a 384-well high-throughput screen of more than 200,000 substances, including fractionated natural product extracts. Identified active compounds were further prioritized in biochemical, biophysical, and cell-based assays. Priority lead compounds were assessed in detail to fully characterize several previously unrecognized PKA pharmacophores including the generation of new X-ray crystallography structures demonstrating unique interactions between PKA and bound inhibitor molecules.

Automated summary

The recent discovery of the oncogenic role of protein kinase A (PKA) in various cancers has revitalized the interest in developing drugs targeting this kinase. Therefore, a series of assays including biochemical, cell-based, and structural biology methods have been established to identify and evaluate new PKA inhibitors. The screening process involved a high-throughput screen of over 200,000 substances, followed by prioritization through biochemical, biophysical, and cell-based assays. Lead compounds with high priority were extensively characterized, which led to the identification of previously unrecognized PKA pharmacophores and the generation of new X-ray crystallography structures demonstrating unique interactions between PKA and inhibitor molecules.