scholarly journals Characterization of Protease-Activated Receptor (PAR) Ligands: Parmodulins Are Reversible Allosteric Inhibitors of PAR1-Driven Calcium Mobilization in Endothelial Cells

2018 ◽  
Author(s):  
Disha M. Gandhi ◽  
Mark W. Majewski ◽  
Ricardo Rosas ◽  
Kaitlin Kentala ◽  
Trevor J. Foster ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Calcium Mobilization ◽  
Response Curves ◽  
Allosteric Inhibitors ◽  
Liquid Handling ◽  
Intracellular Calcium Mobilization ◽  
Calcium Mobilization Assay ◽  
Multiwell Plates ◽  
Detailed Protocol

We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of different PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation. Detailed synthetic protocols are also provided for several known and novel PAR ligands.<br>

scholarly journals Characterization of Protease-Activated Receptor (PAR) Ligands: Parmodulins Are Reversible Allosteric Inhibitors of PAR1-Driven Calcium Mobilization in Endothelial Cells

2018 ◽  
Author(s):  
Disha M. Gandhi ◽  
Mark W. Majewski ◽  
Ricardo Rosas ◽  
Kaitlin Kentala ◽  
Trevor J. Foster ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Calcium Mobilization ◽  
Response Curves ◽  
Allosteric Inhibitors ◽  
Liquid Handling ◽  
Intracellular Calcium Mobilization ◽  
Calcium Mobilization Assay ◽  
Multiwell Plates ◽  
Detailed Protocol

We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of different PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation. Detailed synthetic protocols are also provided for several known and novel PAR ligands.<br>

scholarly journals Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells

2018 ◽  
Vol 26 (9) ◽  
pp. 2514-2529 ◽  
Author(s):  
Disha M. Gandhi ◽  
Mark W. Majewski ◽  
Ricardo Rosas ◽  
Kaitlin Kentala ◽  
Trevor J. Foster ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Calcium Mobilization ◽  
Allosteric Inhibitors

scholarly journals The Parmodulin NRD-21 is an Allosteric Inhibitor of PAR1 Gq Signaling with Improved Anti-Inflammatory Activity and Stability

2019 ◽  
Author(s):  
Disha Gandhi ◽  
Ricardo Rosas, Jr. ◽  
Eric Greve ◽  
Kaitlin Kentala ◽  
N'Guessan Diby ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Activity ◽  
Tnf Alpha ◽  
Human Platelet Aggregation ◽  
Intracellular Calcium Mobilization ◽  
Additional Detail ◽  
Negative Allosteric Modulator ◽  
Anti Inflammatory ◽  
Calcium Mobilization Assay ◽  
Western Portion

Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective negative allosteric modulator of PAR1 that inhibits human platelet aggregation. It showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications.

Intracellular Calcium Mobilization Suppresses the TNF-α–Stimulated Synthesis of PAI-1 in Human Endothelial Cells

1997 ◽  
Vol 17 (8) ◽  
pp. 1550-1560 ◽  
Author(s):  
Franck Peiretti ◽  
Marie-Christine Alessi ◽  
Mireille Henry ◽  
Francine Anfosso ◽  
Irène Juhan-Vague ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Intracellular Calcium ◽  
Calcium Mobilization ◽  
Human Endothelial Cells ◽  
Tnf Α ◽  
Intracellular Calcium Mobilization ◽  
Pai 1
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