blu0588  (MedChemExpress)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A PKA-selective inhibitor captures an open but more ordered conformation of the PKA catalytic subunit

doi: 10.1073/pnas.2536312123

Figure Lengend Snippet: Comparison of the AS and phosphate-organizing site of the BLU0588- vs. ATP-bound PKA-C. ( A ) Left panel: AS of the ATP-bound conformation (PDB ID: 1ATP). Right panel: The same AS of the BLU0588-bound conformation. The hinge residues (Met120-Glu127) as well as Thr183 are highlighted. All the H-bonds are shown with dashed lines, and the hydrophobic surfaces are shown as shells. The hinge point, G125, is marked as a red ball. ( B ) Left panel: Phosphate-organizing site of the ATP-bound conformation, displays the highly coordinated network of Mg 2+ 1 (magenta), Mg 2+ 2 (teal), phosphates (red/orange), and waters (gray spheres). The catalytic triad of Asp184, Lys72, and Glu91 is highlighted with a black circle. Right panel: The same Phosphate-organizing site of the BLU0588-bound conformation. The space that Mg 2+ 1 occupies when present is depicted as a white disc.

Article Snippet: The BLU0588 (MedChemExpress) concentration was varied in the range between 6.4 μM and 12.5 nM employing different ATP concentrations (50 μM to 5 mM) and measured at least in duplicates.

Techniques: Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A PKA-selective inhibitor captures an open but more ordered conformation of the PKA catalytic subunit

doi: 10.1073/pnas.2536312123

Figure Lengend Snippet: BLU0588 nucleates an extensive hydrophobic network of interactions. ( A ) The hydrophobic residues from the N-lobe (tan), the C-lobe (yellow), and the C-tail (red) surround BLU0588. The C-spine is shown in yellow. In the BLU0588 complex, the hydrophobic shell between Phe54 and Phe187, as well as the H-bond between H87 and pT197, that are present in the ATP-bound conformation, are now broken (marked by red arrow). ( B ) The hydrophobic surface of the N-lobe (gray) when bound with BLU0588. The hydrophobic residues, Phe54 from the G-loop, Val79 and Leu82 from the B-helix, as well as several residues from the C-tail (red) including Phe327, Tyr330, Phe347, and Phe350 (marked by black arrows) are highlighted.

Article Snippet: The BLU0588 (MedChemExpress) concentration was varied in the range between 6.4 μM and 12.5 nM employing different ATP concentrations (50 μM to 5 mM) and measured at least in duplicates.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A PKA-selective inhibitor captures an open but more ordered conformation of the PKA catalytic subunit

doi: 10.1073/pnas.2536312123

Figure Lengend Snippet: The conformational, dynamic, and kinetic differences between BLU0588 ( A ) and H89 ( B ). Left panels: Comparison of the PKA-C B-factor dynamics when bound to the respective inhibitor. Middle panels: Inhibitor-bound active site interactions and occupation of each PKA-C subsite. Right panels: SPR sensorgrams show the inhibitor’s association and dissociation for BLU0588 and for H89 using a concentration series of the respective inhibitors interacting with captured FSS-tagged PKA-C. Dashed lines are fits to determine the respective rate constants (k ass and k diss ) and the experimental R max for both inhibitors. Representative experiment of n = 2 experimental setups.

Article Snippet: The BLU0588 (MedChemExpress) concentration was varied in the range between 6.4 μM and 12.5 nM employing different ATP concentrations (50 μM to 5 mM) and measured at least in duplicates.

Techniques: Comparison, Concentration Assay

Journal: Molecular & cellular proteomics : MCP

Article Title: Integrative Proteomic Analysis Reveals the Cytoskeleton Regulation and Mitophagy Difference Between Ischemic Cardiomyopathy and Dilated Cardiomyopathy.

doi: 10.1016/j.mcpro.2023.100667

Figure Lengend Snippet: FIG. 6. Differences in cytoskeleton associated proteins following myocardial infraction and DOX-induced injury. A and B, expression of PRKACA and phosphorylation of Ser339, GSK3β, and phosphorylation of Ser9 in tBHP treatment detected by Western blotting analysis. C and D, expression of PRKACA and phosphorylation of Ser339, GSK3β, and phosphorylation of Ser9 in tBHP and BLU0588 treatment detected by Western blotting analysis. E, representative images of f-actin detected by phalloidin-Rhodamine (red) in tBHP and CHIR99021 treatment in cardiomyocytes. F, representative images of VE-cadherin (red) and β-catenin (green) detected by immunofluorescence in tBHP- and CHIR99021-treated CMECs. G, statistical analysis of f-actin in tBHP and CHIR99021 treatment in cardiomyocytes. H, statistical analysis of VE- cadherin and β-catenin in tBHP and CHIR99021 treatment in CMECs. I, statistical analysis of f-actin in site-mutant cardiomyocytes. J, statistical analysis of VE-cadherin and β-catenin in site-mutant CMECs. K, representative images of f-actin detected by phalloidin-Rhodamine (red) in site- mutant cardiomyocytes. L, representative images of VE-cadherin (red) and β-catenin (green) detected by immunofluorescence in site-mutant CMECs. Statistical significance was calculated using a two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. CMEC, cardiac microvascular endothelial cell; DOX, doxorubicin; tBHP, tert-butyl hydroperoxide.

Article Snippet: Kinase inhibitors BLU0588, CHIR99021 and ROCK-IN-1 were purchased from MCE.

Techniques: Expressing, Phospho-proteomics, Western Blot, Mutagenesis, Two Tailed Test