Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI 53201-1881, USA.
Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI 53201-1881, USA.
Bioorg Med Chem. 2018 May 15;26(9):2514-2529. doi: 10.1016/j.bmc.2018.04.016. Epub 2018 Apr 6.
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.
几种蛋白酶激活受体（PARs）配体已显示出令人印象深刻的抗炎和细胞保护活性，包括 PAR2 拮抗剂和靶向 PAR1 的 parmodulins。为了支持数百种化合物的药物化学研究，并进行详细的作用机制研究，开发一种可靠的 PAR 测定法变得很重要，该测定法可与内皮细胞（介导感兴趣的细胞保护作用）一起使用。我们报告了一种详细的细胞内钙动员测定协议，用于多孔板中的贴壁内皮细胞，该测定法用于研究许多已知和新型的 PAR1 和 PAR2 配体，包括适合标记或缀合化合物制备的 PAR1 拮抗剂 RWJ-58259 的炔基化版本。使用 EA.hy926 细胞系，需要使用自动化液体处理设备进行培养基交换，以获得最佳和可重复的拮抗剂浓度-反应曲线。该测定法也适用于 PAR2 配体的研究；Fairlie 报道的一种肽拮抗剂被合成，并发现其以与使用上皮细胞报告一致的方式抑制 PAR2。该测定法用于确认沃拉帕沙在血管内皮细胞中作为 PAR1 的不可逆拮抗剂，并且发现 parmodulin 2（ML161）和相关的 parmodulin RR-90 可逆地抑制 PAR1，与负变构调节一致。
