A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor
A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor
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DOI：
10.1007/s11302-019-09650-9
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发表时间：
2019-06-01
影响因子：
3.5
通讯作者：
Hill, Stephen J.
中科院分区：
文献类型：
--
作者：
Bouzo-Lorenzo, Monica;Stoddart, Leigh A.;Hill, Stephen J.
在将先导化合物进展到临床试验之前，人们对了解与G蛋白偶联受体结合的化合物的结合动力学越来越感兴趣。广泛表达的腺苷A（3）受体（A（3）AR）与包括免疫状况在内的一系列疾病有关，目前正在开发旨在选择性靶向该受体的化合物用于关节炎。使用放射性标记的拮抗剂进行了A（3）AR的动力学研究，但由于这种探针的动力学，它们在10 ℃下在膜制备物中进行。在这项研究中，我们已经开发了一种活细胞NanoBRET配体结合试验，使用荧光A（3）AR拮抗剂来测量生理温度下标记和未标记化合物在A（3）AR处的动力学参数。用动力学缔合法测定了四种荧光拮抗剂的动力学曲线，发现XAC-ser-tyr-X-BY 630在A（3）AR上的停留时间最长（RT=288 ± 62 min）。还使用两种荧光配体（XAC-ser-tyr-X-BY 630或AV 039，RT=6.8 +/-0.8min）作为标记探针测定了三种拮抗剂PSB-11、化合物5和LUF 7565的缔合和解离速率常数，并与使用放射性标记的拮抗剂（[H-3]PSB-11，RT=44.6 +/-3.9min）获得的那些进行了比较。用AV 039和[H-3]PSB-11测量的动力学参数非常一致，但与使用XAC-S-ser-S-tyr-X-BY 630获得的动力学参数存在显著差异。这些数据表明，选择具有适当动力学的探针对于准确测定具有显著不同动力学曲线的未标记配体的动力学是重要的。
There is a growing interest in understanding the binding kinetics of compounds that bind to G protein-coupled receptors prior to progressing a lead compound into clinical trials. The widely expressed adenosine A(3) receptor (A(3)AR) has been implicated in a range of diseases including immune conditions, and compounds that aim to selectively target this receptor are currently under development for arthritis. Kinetic studies at the A(3)AR have been performed using a radiolabelled antagonist, but due to the kinetics of this probe, they have been carried out at 10 degrees C in membrane preparations. In this study, we have developed a live cell NanoBRET ligand binding assay using fluorescent A(3)AR antagonists to measure kinetic parameters of labelled and unlabelled compounds at the A(3)AR at physiological temperatures. The kinetic profiles of four fluorescent antagonists were determined in kinetic association assays, and it was found that XAC-ser-tyr-X-BY630 had the longest residence time (RT=288 +/- 62min) at the A(3)AR. The association and dissociation rate constants of three antagonists PSB-11, compound 5, and LUF7565 were also determined using two fluorescent ligands (XAC-ser-tyr-X-BY630 or AV039, RT=6.8 +/- 0.8min) as the labelled probe and compared to those obtained using a radiolabelled antagonist ([H-3]PSB-11, RT=44.6 +/- 3.9min). There was close agreement in the kinetic parameters measured with AV039 and [H-3]PSB-11 but significant differences to those obtained using XAC-S-ser-S-tyr-X-BY630. These data indicate that selecting a probe with the appropriate kinetics is important to accurately determine the kinetics of unlabelled ligands with markedly different kinetic profiles.