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

Abstract

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.

Discovery and Synthesis of a Naturally Derived Protein Kinase Inhibitor that Selectively Inhibits Distinct Classes of Serine/Threonine Kinases

“…Briefly, in the case of J-PKAcα, the J-PKAcα holoenzyme was treated with both test compounds, ATP, and cAMP, which induced the dissociation of the regulatory units of the holoenzyme to release the activated catalytic unit (J-PKAcα). The kinase activity of the dissociated J-PKAcα was quantified by measuring the phosphorylation of a biotinylated peptide of the PKA substrate CREB (KRREILSRRPSYR) through an ELISA assay as reported previously . Compound 1 showed significant inhibition of the J-PKAcα activity with the IC 50 value at 0.99 μM.…”

“…The kinase activity of the dissociated J-PKAcα was quantified by measuring the phosphorylation of a biotinylated peptide of the PKA substrate CREB (KRREILSRRPSYR) through an ELISA assay as reported previously. 34 Compound 1 showed significant inhibition of the J-PKAcα activity with the IC 50 value at 0.99 μM. In contrast, the analog 2 only weakly inhibited J-PKAcα (IC 50 = 79 μM), ∼80-fold less potent than 1 (Figure 2a and b).…”

“…The reaction was allowed to proceed for 30 min prior to quenching with 15 μL of 0.5 M EDTA (pH 8.0). The quenched reaction solutions were then processed as previously described . The experiment was repeated three times, and the composite of all collected data was processed and fit into the Gaddum/Schild EC 50 shift equations using the GraphPad Prism 9.4.1 software to determine the dissociation equilibrium constant of 1 ( K B ).…”

“…The biochemical PKAcα (fusion or wild type) assay was adapted from the primary HTS screening assay as previously described . Briefly, the compound solution was made in 0.1 M Tris pH 7.5 buffer containing 50 μM ATP, 1 μM cAMP, 0–200 μM test compound, and 0.5% DMSO.…”

“…The reaction was allowed to proceed for 45 min prior to quenching with 0.5 M EDTA. Quenched reaction solutions were then transferred in quadruplicate to previously prepared Avidin-coated capture plates, which were processed as previously described for the ELISA assay . IC 50 values were obtained from the data of three independent experiments which were processed by nonlinear regression using the GraphPad Prism 9.4.1 software.…”