Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.
Int J Mol Sci. 2019 Jul 30;20(15):3724. doi: 10.3390/ijms20153724.
Duration of receptor antagonism, measured as the recovery of agonist responsiveness, is gaining attention as a method to evaluate the 'effective' target-residence for antagonists. These functional assays might be a good alternative for kinetic binding assays in competition with radiolabeled or fluorescent ligands, as they are performed on intact cells and better reflect consequences of dynamic cellular processes on duration of receptor antagonism. Here, we used a bioluminescence resonance energy transfer (BRET)-based assay that monitors heterotrimeric G protein activation via scavenging of released Venus-Gβγ by NanoLuc (Nluc)-tagged membrane-associated-C-terminal fragment of G protein-coupled receptor kinase 3 (masGRK3ct-Nluc) as a tool to probe duration of G protein-coupled receptor (GPCR) antagonism. The Gα-coupled histamine H receptor (HR) was used in this study as prolonged antagonism is associated with adverse events (e.g., insomnia) and consequently, short-residence time ligands might be preferred. Due to its fast and prolonged response, this assay can be used to determine the duration of functional antagonism by measuring the recovery of agonist responsiveness upon washout of pre-bound antagonist, and to assess antagonist re-equilibration time via Schild-plot analysis. Re-equilibration of pre-incubated antagonist with agonist and receptor could be followed in time to monitor the transition from insurmountable to surmountable antagonism. The BRET-based G protein activation assay can detect differences in the recovery of HR responsiveness and re-equilibration of pre-bound antagonists between the tested HR antagonists. Fast dissociation kinetics were observed for marketed drug pitolisant (Wakix) in this assay, which suggests that short residence time might be beneficial for therapeutic targeting of the HR.
受体拮抗作用的持续时间（以恢复激动剂反应性来衡量）作为评估拮抗剂“有效”靶标停留时间的方法正受到关注。这些功能测定方法可能是与放射性配体或荧光配体竞争的动力学结合测定的良好替代方法，因为它们在完整细胞上进行，并且更好地反映了动态细胞过程对受体拮抗作用持续时间的影响。在这里，我们使用了一种基于生物发光共振能量转移（BRET）的测定方法，该方法通过纳米荧光素（Nluc）标记的 G 蛋白偶联受体激酶 3（masGRK3ct-Nluc）膜相关 C 端片段来监测异三聚体 G 蛋白的激活，从而监测 G 蛋白偶联受体（GPCR）拮抗作用的持续时间。在这项研究中，使用了 Gα 偶联组胺 H 受体（HR），因为延长的拮抗作用与不良事件（例如失眠）相关，因此，短停留时间的配体可能更受青睐。由于其快速而持久的反应，该测定法可用于通过测量在预结合拮抗剂冲洗后激动剂反应性的恢复来确定功能拮抗作用的持续时间，并通过 Schild 图分析评估拮抗剂再平衡时间。可以通过监测从不可逾越到可逾越的拮抗作用的转变，来跟踪预孵育的拮抗剂与激动剂和受体的再平衡时间。基于 BRET 的 G 蛋白激活测定法可以检测出在测试的 HR 拮抗剂中 HR 反应性的恢复和预结合拮抗剂的再平衡之间的差异。在该测定法中，观察到市售药物哌唑嗪（Wakix）的快速解离动力学，这表明短停留时间可能有利于 HR 的治疗靶向。
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