Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom .
J Pharmacol Exp Ther. 2010 Sep 1;334(3):795-808. doi: 10.1124/jpet.110.166009. Epub 2010 May 27.
Glucagon-like peptide-1 (GLP-1) mediates antidiabetogenic effects through the GLP-1 receptor (GLP-1R), which is targeted for the treatment of type 2 diabetes. Small-molecule GLP-1R agonists have been sought due to difficulties with peptide therapeutics. Recently, 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (compound 2) has been described as a GLP-1R allosteric modulator and agonist. Using human embryonic kidney-293 cells expressing human GLP-1Rs, we extended this work to consider the impact of compound 2 on G protein activation, Ca(2+) signaling and receptor internalization and particularly to compare compound 2 and GLP-1 across a range of functional assays in intact cells. GLP-1 and compound 2 activated Galpha(s) in cell membranes and increased cellular cAMP in intact cells, with compound 2 being a partial and almost full agonist, respectively. GLP-1 increased intracellular [Ca(2+)] by release from intracellular stores, which was mimicked by compound 2, with slower kinetics. In either intact cells or membranes, the orthosteric antagonist exendin-(9-39), inhibited GLP-1 cAMP generation but increased the efficacy of compound 2. GLP-1 internalized enhanced green fluorescent protein-tagged GLP-1Rs, but the speed and magnitude evoked by compound 2 were less. Exendin-(9-39) inhibited internalization by GLP-1 and also surprisingly that by compound 2. Compound 2 displays GLP-1R agonism consistent with action at an allosteric site, although an orthosteric antagonist increased its efficacy on cAMP and blocked compound 2-mediated receptor internalization. Full assessment of the properties of compound 2 was potentially hampered by damaging effects that were particularly manifest in either longer term assays with intact cells or in acute assays with membranes.
胰高血糖素样肽-1（GLP-1）通过 GLP-1 受体（GLP-1R）发挥抗糖尿病作用，该受体是 2 型糖尿病治疗的靶点。由于肽类治疗药物存在困难，因此一直在寻找小分子 GLP-1R 激动剂。最近，6,7-二氯-2-甲基磺酰基-3-N-叔丁基氨基喹喔啉（化合物 2）被描述为 GLP-1R 别构调节剂和激动剂。我们用人肾 293 细胞表达人 GLP-1R，扩展了这项工作，以研究化合物 2 对 G 蛋白激活、Ca(2+)信号转导和受体内化的影响，特别是比较化合物 2 和 GLP-1 在完整细胞中的一系列功能测定中的作用。GLP-1 和化合物 2 在细胞膜中激活 Galpha(s)并增加细胞内 cAMP，化合物 2 分别为部分激动剂和完全激动剂。GLP-1 通过从细胞内储存中释放来增加细胞内[Ca(2+)]，化合物 2 模拟了这一过程，但其动力学较慢。无论是在完整细胞还是在膜中，正构拮抗剂 exendin-(9-39)抑制 GLP-1 cAMP 的产生，但增加了化合物 2 的效力。GLP-1 内化增强型绿色荧光蛋白标记的 GLP-1R，但化合物 2 引起的速度和幅度较小。exendin-(9-39) 抑制 GLP-1 和化合物 2 引起的内化。化合物 2 显示出与变构部位作用一致的 GLP-1R 激动作用，尽管正构拮抗剂增加了其 cAMP 的效力并阻止了化合物 2 介导的受体内化。化合物 2 的特性的全面评估可能受到损害，这种损害在完整细胞的长期测定中或在膜的急性测定中表现得尤为明显。
