Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, Monash University, Parkville, Victoria, Australia 3052.
ACS Chem Biol. 2010 Apr 16;5(4):365-75. doi: 10.1021/cb900276p.
"Functional selectivity", although new to many chemists and biologists only a few years ago, has now become a dominant theme in drug discovery. This concept posits that different ligands engender unique receptor conformations such that only a subset of signaling pathways linked to a given receptor are recruited. However, successful exploitation of the phenomenon to achieve pathway-based selectivity requires the ability to routinely detect it when assessing ligand behavior. We have utilized different strains of the yeast S. cerevisiae, each expressing a specific human Galpha/yeast Gpa1 protein chimera coupled to a MAP kinase-linked reporter gene readout, to investigate the signaling of the M(3) muscarinic receptor, a G protein-coupled receptor (GPCR) for which various antagonists are used clinically. Using this novel platform, we found that the "antagonists", atropine, N-methylscopolamine, and pirenzepine, were inverse agonists for Gpa1/Galpha(q) but low efficacy agonists for Gpa1/Galpha(12.) Subsequent studies with atropine performed in mammalian 3T3 cells validated these findings by demonstrating inverse agonism for G(q/11)-mediated calcium mobilization but positive agonism for G(12)-mediated membrane ruffling. This is the first study to utilize a yeast platform to discover pathway-biased functional selectivity in a GPCR. In addition to the likely applicability of this approach for identifying biased signaling by novel chemical entities, our findings also suggest that currently marketed medications may exhibit hitherto unappreciated functional selectivity.
“功能选择性”虽然对许多化学家和生物学家来说是几年前的新概念，但现在已经成为药物发现的主要主题。这个概念假定，不同的配体产生独特的受体构象，使得只有与特定受体相关的信号通路的一部分被招募。然而，要成功地利用这种现象实现基于通路的选择性，在评估配体行为时，需要有常规检测它的能力。我们利用不同的酵母 S. cerevisiae 菌株，每个菌株都表达一种特定的人 Galpha/酵母 Gpa1 蛋白嵌合体，与 MAP 激酶连接的报告基因读数相连接，来研究 M(3)毒蕈碱受体的信号转导，M(3)毒蕈碱受体是一种 G 蛋白偶联受体 (GPCR)，临床上有各种拮抗剂用于该受体。利用这个新的平台，我们发现“拮抗剂”阿托品、N-甲基东莨菪碱和哌仑西平对 Gpa1/Galpha(q)是反向激动剂，但对 Gpa1/Galpha(12)是低效能激动剂。随后在哺乳动物 3T3 细胞中用阿托品进行的后续研究通过证明 G(q/11)介导的钙动员的反向激动作用和 G(12)介导的细胞膜皱襞的正向激动作用验证了这些发现。这是第一项利用酵母平台在 GPCR 中发现偏向功能选择性的研究。除了这种方法可能适用于识别新型化学实体的偏向信号外，我们的研究结果还表明，目前市场上的药物可能表现出以前未被认识到的功能选择性。
