Department of Physiology, Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, AZ 85724-5030, USA.
Bioconjug Chem. 2012 Oct 17;23(10):2098-104. doi: 10.1021/bc300300q. Epub 2012 Oct 8.
Protease activated receptor-2 (PAR(2)) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR(2) can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR(2) to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR(2) peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH(2) (2-f-LIGRLO-dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO-dtpa is a potent and specific PAR(2) agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO-dtpa can be utilized in an affinity binding assay to evaluate the ligand-receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH(2), 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH(2), 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH(2), 6-an-LIGRL) and PAR(2). A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH(2), 3-ia-LIGRL) that does not activate PAR(2) signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR(2) binding. In summary, we have developed a europium-containing PAR(2) agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR(2) ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR(2) ligands.
蛋白酶激活受体 2（PAR2）是四种 G 蛋白偶联受体（GPCR）之一，可被外源性或内源性蛋白酶激活，这些蛋白酶可裂解细胞外氨基末端以暴露连接的配体，随后引发 G 蛋白信号转导。或者，PAR2 可以被天然连接配体衍生的肽或拟肽配体激活。由于缺乏灵敏、高通量的亲和力结合测定法，直接与 PAR2 结合以激动或拮抗受体的新型配体筛选受到阻碍。在本报告中，我们描述了一种改良的 PAR2 拟肽激动剂 2-呋喃酰基-LIGRLO-（二亚乙基三胺五乙酸）-NH2（2-f-LIGRLO-dtpa）的合成和应用，该激动剂设计用于基于镧系元素的时间分辨荧光筛选。我们首先证明，2-f-LIGRLO-dtpa 是一种在各种体外测定中均具有强大且特异性的 PAR2 激动剂。然后，我们证明 2-f-LIGRLO-dtpa 可用于亲和力结合测定，以评估已知高活性拟肽激动剂（2-呋喃酰基-LIGRLO-NH2，2-f-LIGRLO；2-氨基噻唑-4-基-LIGRL-NH2，2-at-LIGRL；6-氨基烟酰基-LIGRL-NH2，6-an-LIGRL）与 PAR2 之间的配体-受体相互作用。另一种不激活 PAR2 信号的单独 N 端拟肽修饰（3-吲哚乙酰基-LIGRL-NH2，3-ia-LIGRL）被用作阴性对照。所有三种拟肽激动剂均表现出 S 形竞争性结合曲线，其中更有效的激动剂（2-f-LIGRLO 和 2-at-LIGRL）表现出增强的竞争。相比之下，对照肽（3-ia-LIGRL）对 PAR2 结合的竞争有限。总之，我们开发了一种含有镧的 PAR2 激动剂，可用于高灵敏度的亲和力结合测定，以高通量格式筛选新型 PAR2 配体。这种配体可以作为筛选和开发 PAR2 配体的重要工具。
