Department of Chemistry and Pharmacy, Friedrich Alexander University, 91052 Erlangen, Germany; and.
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):10744-8. doi: 10.1073/pnas.1410415111. Epub 2014 Jul 8.
Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis.
结构研究提供了关于这些重要药物靶点的结构和功能的重要见解。然而，配体与受体的复合物处于活跃状态的 G 蛋白偶联受体（GPCRs）的结晶是特别具有挑战性的，需要形成稳定和构象均匀的配体-受体复合物。与低亲和力结合的天然激素、神经递质和合成激动剂在稳定活跃状态以促进结晶方面效果不佳。为了促进药理学上高度相关的类胺能 GPCR 的结构研究，我们在这里开发了共价结合的分子工具，激活 Gs、Gi 和 Gq 偶联受体。共价激动剂来源于单胺神经递质去甲肾上腺素、多巴胺、血清素和组胺，并使用通用且通用的合成策略来获取。我们证明了本文介绍的工具化合物具有有效的共价结合模式，并且相应的共价配体-受体复合物激活 G 蛋白的效率与天然神经递质相当。β2-肾上腺素受体与共价去甲肾上腺素类似物和构象选择性抗体（纳米抗体）的复合物的晶体结构证明这些激动剂可用于促进结晶。
