Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University.
Chem Pharm Bull (Tokyo). 2020;68(10):954-961. doi: 10.1248/cpb.c20-00349.
Binding assays are widely used to study the estrogenic activity of compounds targeting the estrogen receptor (ER). The fluorescence properties of benzofurazan (BD), an environmentally sensitive fluorophore, are affected by solvent polarity. In this study, we synthesized BD-labeled estradiol (E2) derivatives hoping to develop a fluorescent ligand to be used in ER binding assays, without the separation of free- from bound-ligand. Three fluorescent ligands with a BD skeleton were obtained and their fluorescence properties were investigated. Analysis of the fluorescent ligands and human recombinant ERα (hr-ERα) interactions revealed that the fluorescence intensity increased in hydrophobic environments, such as the receptor-binding site. In saturation binding assays, ABD-E2 derivative 2c showed positive cooperative binding, and its dissociation constant (K) and Hill coefficient were 23.4 nM and 1.34, respectively. The estrogenic compounds affinity, assessed by competitive binding assays was well correlated with the results obtained by conventional studies, using the fluorescence polarization method. Overall, the developed assay using BD-labeled ligands was a simple, rapid, and reliable method for the evaluation of ER binding affinity.
结合测定法被广泛用于研究针对雌激素受体 (ER) 的化合物的雌激素活性。苯并呋咱 (BD) 是一种对环境敏感的荧光团，其荧光性质受溶剂极性的影响。在这项研究中，我们合成了 BD 标记的雌二醇 (E2) 衍生物，希望开发一种荧光配体用于 ER 结合测定法，而无需分离游离配体和结合配体。得到了三个带有 BD 骨架的荧光配体，并研究了它们的荧光性质。对荧光配体与人类重组 ERα (hr-ERα) 相互作用的分析表明，荧光强度在疏水环境中增加，例如受体结合部位。在饱和结合测定法中，ABD-E2 衍生物 2c 表现出正协同结合，其解离常数 (K) 和 Hill 系数分别为 23.4 nM 和 1.34。通过竞争性结合测定法评估的雌激素化合物亲和力与使用荧光偏振法进行的常规研究结果高度相关。总的来说，使用 BD 标记配体的开发测定法是一种简单、快速和可靠的方法，用于评估 ER 结合亲和力。
