Dataset Information

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.

SUBMITTER: Wilson BAP

PROVIDER: S-EPMC10111623 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Publications

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

Wilson Brice A P BAP Li Ning N Martinez Fiesco Juliana A JA Dalilian Masoumeh M Wang Dongdong D Smith Emily A EA Wamiru Antony A Shah Rohan R Goncharova Ekaterina I EI Beutler John A JA Grkovic Tanja T Zhang Ping P O'Keefe Barry R BR

ACS pharmacology & translational science 20230321 4

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 su ...[more]

PMID: 37082750

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Project description:BackgroundThe cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) signaling pathway is closely associated with myocardial infarction (MI). Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) is a key component of this pathway; however, there is currently a lack of clinical evidence linking plasma cGAMP levels to MI.MethodsThis study utilized clinical data from 270 patients diagnosed with coronary heart disease (CHD) at the Second Xiangya Hospital of Central South University. The outcomes included ST-segment elevation and non-ST-segment elevation MI. Univariate and multivariate logistic regression models were used to explore the relationships between plasma cGAMP levels and MI, while restricted cubic spline (RCS) using logistic regression to explore the dose-response relationship.ResultsAmong the 270 patients, the mean plasma cGAMP level was 1352.58 ± 106.02 ng/L and 89 (32.96%) patients were diagnosed with MI. The RCS curves indicated a U-shape association between the cGAMP levels and MI; the risk of MI was negatively correlated with the cGAMP until it hit bottoms at 1352 ng/L. When the cGAMP level exceeded 1352 ng/L, the risk of MI increased significantly (adjusted OR, 1.02; 95% CI: 1.01-1.03). When considering cGAMP as a categorical variable, patients in Tertile 1 and Tertile 3 had a 167% (adjusted OR: 2.67, 95% CI: 1.23-5.78) and 155% (adjusted OR: 2.55, 95% CI: 1.17-5.55) higher risk of MI compared to those in Tertile 2, respectively. These results were consistent across subgroup analyses, notably, a significant interaction by age category was observed in patients with cGAMP ≥ 1352 ng/L, where the positive association was pronounced in the elderly.ConclusionsA U-shaped association exists between cGAMP and MI in the CHD population, with a cutoff point at the cGAMP of 1352 ng/L. Both excessively high and low cGAMP levels are associated with an increased risk of MI, particularly among the elderly with cGAMP ≥ 1352 ng/L. This is the first clinical evidence of the cGAS-cGAMP-STING pathway in metabolic cardiovascular diseases.ClinicaltrialsGov identifierNCT03363035 (Registration date: 2018-01-15).

Project description:Background and objectivesResolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration.MethodsVSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.ResultsTreatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.ConclusionsOur results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

Dataset Information

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

Publications

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

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