Zhu Hu, Daly Harrison C, Yang Tae Gyun, Born Josh, Camacho-Hernandez Gisela Andrea, Yuan Mingyang, Inglese Mari, Chenniappan Vinoth Kumar, Zimmerman Kris, Hurst Robin, Hu Xin, Newman Amy Hauck, Ferré Sergi, Ohana Rachel Friedman, Hall Matthew D, Patnaik Samarjit
Division of Preclinical Innovation, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH), Rockville, Maryland 20850, United States.
Medicinal Chemistry Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, Maryland United States.
ACS Chem Biol. 2025 Jul 18;20(7):1594-1608. doi: 10.1021/acschembio.5c00166. Epub 2025 Jul 4.
Galanin is a neuroendocrine peptide that regulates a wide range of physiological functions, including feeding and energy homeostasis, mood and anxiety, and modulation of pain. The function of the galanin peptide is mediated through its three galanin receptors, namely, GALR1, GALR2, and GALR3, which belong to the G protein-coupled receptor family. To measure the interaction of ligands with galanin receptor 1 (GALR1) in living cells, we developed a novel HiBiT peptide-based NanoBRET ligand binding assay. We generated six bioluminescence resonance energy transfer (BRET) tracers composed of modified and truncated galanin peptide derivatives tagged with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) acceptor fluorophore. The fluorophore-tagged peptide tracers were evaluated in cells expressing GALR1 tagged with HiBiT, an 11-amino acid subunit of NanoLuc, which, upon high affinity complementation with the cell-impermeable subunit LgBiT, reconstituted a functional NanoLuc luciferase. Addition of the furimazine substrate induced BRET to the BODIPY fluorophore acceptor component of the galanin-derived peptide tracers and produced a fluorescence signal output. Using this BRET assay, we characterized the binding affinity and binding kinetics of tracers with GALR1 in both equilibrium and real time. To validate our assay, we evaluated the binding affinity and function of a panel of unmodified galanin-derived peptide ligands through the competitive displacement of bound fluorescent galanin tracers. Our data showed that the binding affinity of these galanin peptide ligands correlated well with their rank order in β-arrestin recruitment and internalization functional assays. This study demonstrates that the HiBiT peptide-based NanoBRET ligand binding assay is a valuable system for studying the ligand engagement of GALR1 in living cells, offering an alternative to neuropeptide radioligand binding assays.
甘丙肽是一种神经内分泌肽，可调节多种生理功能，包括进食与能量稳态、情绪与焦虑以及疼痛调节。甘丙肽的功能是通过其三种甘丙肽受体介导的，即GALR1、GALR2和GALR3，它们属于G蛋白偶联受体家族。为了测量配体与活细胞中甘丙肽受体1（GALR1）的相互作用，我们开发了一种基于新型HiBiT肽的纳米生物发光共振能量转移（NanoBRET）配体结合测定法。我们生成了六种生物发光共振能量转移（BRET）示踪剂，这些示踪剂由标记有4,4-二氟-4-硼-3a,4a-二氮杂-s-茚（BODIPY）受体荧光团的修饰和截短的甘丙肽衍生物组成。在表达用HiBiT标记的GALR1的细胞中评估了荧光团标记的肽示踪剂，HiBiT是纳米荧光素酶（NanoLuc）的一个11个氨基酸的亚基它与细胞不可渗透的亚基LgBiT进行高亲和力互补后，可重构功能性纳米荧光素酶。加入腔肠素底物会诱导BRET至甘丙肽衍生肽示踪剂的BODIPY荧光团受体成分，并产生荧光信号输出。使用这种BRET测定法，我们在平衡和实时条件下表征了示踪剂与GALR1的结合亲和力和结合动力学。为了验证我们的测定法，我们通过结合的荧光甘丙肽示踪剂的竞争性置换，评估了一组未修饰的甘丙肽衍生肽配体的结合亲和力和功能。我们的数据表明，这些甘丙肽配体的结合亲和力与其在β-抑制蛋白募集和内化功能测定中的排名顺序密切相关。这项研究表明，基于HiBiT肽的纳米生物发光共振能量转移（NanoBRET）配体结合测定法是研究活细胞中GALR1配体结合的有价值系统，为神经肽放射性配体结合测定法提供了一种替代方法。
