G-Protein coupled receptors (GPCRs) represent one of the largest classes of therapeutic targets. However, developing successful therapeutics to target GPCRs is a challenging endeavor with many molecules failing during in vivo clinical trials due to a lack of efficacy. The in vitro identification of drug targeted residence time (1/koff) has been suggested to improve prediction of in vivo success. Here, a ligand binding assay using fluorescence anisotropy was implemented to successfully determine on-rates (kon) and off-rates (koff) of labeled and unlabeled ligands binding to the adenosine A2A receptor (A2AR) purified into nanodiscs (A2AR-NDs). The kinetic assay was used to determine the optimal storage conditions of A2AR-NDs, where they were found to be stable for more than six months at -80oC. The binding assay was implemented to further understand receptor function by determining the effects of charged lipids on agonist binding kinetics, how sodium levels allosterically modulate A2AR function, and how A2AR protonation affects agonist binding.

中文翻译：

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配体结合动力学，用于评估 A2AR 在纳米盘中的功能和稳定性。


G 蛋白偶联受体 （GPCR） 是最大的治疗靶点之一。然而，开发靶向 GPCR 的成功疗法是一项具有挑战性的工作，由于缺乏疗效，许多分子在体内临床试验中失败。药物靶向停留时间 （1/koff） 的体外鉴定被认为可以提高体内成功的预测。在这里，使用荧光各向异性实施配体结合测定，以成功确定与纯化到纳米圆盘 （A2AR-NDs） 中的腺苷 A2A 受体 （A2AR） 结合的标记和未标记配体的开速率 （kon） 和关闭速率 （koff）。动力学测定用于确定 A2AR-NDs 的最佳储存条件，发现它们在 -80oC 下可稳定保存 6 个月以上。通过确定带电脂质对激动剂结合动力学的影响、钠水平如何变构调节 A2AR 功能以及 A2AR 质子化如何影响激动剂结合，实施结合测定以进一步了解受体功能。