Data_Sheet_1_Surface Plasmon Resonance as a Tool for Ligand Binding Investigation of Engineered GPR17 Receptor, a G Protein Coupled Receptor Involved in Myelination.PDF

The aim of this study was to investigate the potential of surface plasmon resonance (SPR) spectroscopy for the measurement of real-time ligand-binding affinities and kinetic parameters for GPR17, a G protein-coupled receptor (GPCR) of major interest in medicinal chemistry as potential target in demyelinating diseases. The receptor was directly captured, in a single-step, from solubilized membrane extracts on the sensor chip through a covalently bound anti-6x-His-antibody and retained its ligand binding activity for over 24 h. Furthermore, our experimental setup made possible, after a mild regeneration step, to remove the bound receptor without damaging the antibody, and thus to reuse many times the same chip. Two engineered variants of GPR17, designed for crystallographic studies, were expressed in insect cells, extracted from crude membranes and analyzed for their binding with two high affinity ligands: the antagonist Cangrelor and the agonist Asinex 1. The calculated kinetic parameters and binding constants of ligands were in good agreement with those reported from activity assays and highlighted a possible functional role of the N-terminal residues of the receptor in ligand recognition and binding. Validation of SPR results was obtained by docking and molecular dynamics of GPR17-ligands interactions and by functional in vitro studies. The latter allowed us to confirm that Asinex 1 behaves as GPR17 receptor agonist, inhibits forskolin-stimulated adenylyl cyclase pathway and promotes oligodendrocyte precursor cell maturation and myelinating ability.

本研究旨在探究表面等离子体共振（surface plasmon resonance, SPR）光谱学用于实时检测配体结合亲和力及动力学参数的潜力，研究对象为GPR17——一种在药物化学领域具有重要研究价值、可作为脱髓鞘疾病潜在治疗靶点的G蛋白偶联受体（G protein-coupled receptor, GPCR）。该受体可通过共价结合的抗6x-His抗体，一步法从可溶性膜提取物中直接捕获于传感器芯片表面，且其配体结合活性可维持超过24小时。此外，本实验体系在经过温和的再生步骤后，可在不损伤抗体的情况下移除结合的受体，从而实现同一芯片的多次重复使用。两种为晶体学研究设计的GPR17工程变体在昆虫细胞中表达，从粗制膜中提取后，针对其与两种高亲和力配体——拮抗剂坎格雷洛（Cangrelor）和激动剂Asinex 1——的结合情况进行了分析。计算得到的配体动力学参数及结合常数与活性检测实验的报道结果吻合良好，同时揭示了受体N端残基在配体识别与结合过程中可能发挥的功能作用。SPR实验结果通过GPR17-配体相互作用的分子对接（docking）与分子动力学（molecular dynamics）模拟，以及体外功能实验得到了验证。后者证实Asinex 1可作为GPR17受体的激动剂，能够抑制福斯高林（forskolin）激活的腺苷酸环化酶通路，并促进少突胶质前体细胞的成熟及髓鞘形成能力。