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

21 February 2018, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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.

Keywords

Protease-Activated Receptor
PAR1
PAR2
GPCR
Calcium mobilization
Parmodulin
ML161
Vorapaxar
RWJ-58259
Negative allosteric modulator

Supplementary materials

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2018 02 20 Characterization of PAR ligands as inhibitors of Ca SI
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