Faculty Collaboration Database

Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells. Bioorg Med Chem 2018 May 15;26(9):2514-2529

Date

04/25/2018

Pubmed ID

29685684

Pubmed Central ID

PMC5937995

DOI

10.1016/j.bmc.2018.04.016

Scopus ID

2-s2.0-85045909279 (requires institutional sign-in at Scopus site)   16 Citations

Abstract

Several classes of ligands for Protease-Activated Receptors (PARs) have shown impressive anti-inflammatory and cytoprotective activities, including PAR2 antagonists and the PAR1-targeting parmodulins. In order to support medicinal chemistry studies with hundreds of compounds and to perform detailed mode-of-action studies, it became important to develop a reliable PAR assay that is operational with endothelial cells, which mediate the cytoprotective effects of interest. 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 known and new 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 is also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. 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.

Author List

Gandhi DM, Majewski MW, Rosas R Jr, Kentala K, Foster TJ, Greve E, Dockendorff C

Author

Christopher Dockendorff PhD Assistant Professor, Organic and Medicinal Chemistry in the Chemistry department at Marquette University

MESH terms used to index this publication - Major topics in bold

Allosteric Regulation
Benzamides
Calcium
Cell Line
Endothelial Cells
Humans
Imines
Indazoles
Lactones
Ligands
Oligopeptides
Phenylenediamines
Platelet Aggregation Inhibitors
Pyridines
Receptor, PAR-1
Receptor, PAR-2
Technology, Pharmaceutical
Urea