Robust Array-Based Coregulator Binding Assay Predicting ERα-Agonist Potency and Generating Binding Profiles Reflecting Ligand Structure
Robust Array-Based Coregulator Binding Assay Predicting ERα-Agonist Potency and Generating Binding Profiles Reflecting Ligand Structure
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DOI：
10.1021/tx300463b
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发表时间：
2013-03-01
影响因子：
4.1
通讯作者：
Bovee, Toine F. H.
中科院分区：
文献类型：
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作者：
Aarts, Jac M. M. J. G.;Wang, Si;Bovee, Toine F. H.
测试化学品的内分泌干扰潜力，包括干扰雌激素受体（ER）信号，是化学品安全测试的一个重要方面。由于动物试验的实际缺点，开发子宫增重试验和其他体内（抗）雌激素试验的体外替代品具有高度优先性。以前证明，一个在体外测定，配置文件配体诱导的结合ER α的辅调节衍生肽的微阵列可能是一个有价值的候选人在体外试验的一个小组，目的是最终取代子宫滋养测定。在本研究中，该辅助调节因子结合试验的重现性和稳健性通过测量预防、农药和有毒物质办公室推荐的用于测试雌激素受体反式激活试验实验室能力的14种模型化合物的结合概况来确定。在重复测量之间具有5.0%的中位变异系数和良好的相关性（R-2 = 0.993），ER α-辅调节因子结合测定的再现性优于其他常用的体外ER功能测定的再现性。此外，辅调节因子结合试验正确预测了14种测试化合物中13种的雌激素活性。当将ER激动剂诱导ER α-辅调节因子结合的效力与其ER结合亲和力进行比较时，它们的排名相似，并且EC 50值之间的相关性极好（R-2 = 0.96），与它们在反式激活测定中的效力的相关性也是如此（R-2 = 0.94）。此外，当ER α-辅调节因子结合谱使用欧几里德簇距离进行分级聚类时，发现结构相关的化合物聚集在一起，而具有芳香族A环的类固醇测试化合物与具有环己烯A环的化合物分离。我们的结论是，该测定能够区分ER α激动剂和拮抗剂，并且它甚至反映了ER α激动剂的结构相似性，这表明有可能以高保真度实现ER α内分泌干扰物的鉴定和分类。
Testing chemicals for their endocrine-disrupting potential, including interference with estrogen receptor (ER) signaling, is an important aspect of chemical safety testing. Because of the practical drawbacks of animal testing, the development of in vitro alternatives for the uterotrophic assay and other in vivo (anti)estrogenicity tests has high priority. It was previously demonstrated that an in vitro assay that profiles ligand-induced binding of ER alpha to a microarray of coregulator-derived peptides might be a valuable candidate for a panel of in vitro assays aiming at an ultimate replacement of the uterotrophic assay. In the present study, the reproducibility and robustness of this coregulator binding assay was determined by measuring the binding profiles of 14 model compounds that are recommended by the Office of Prevention, Pesticides and Toxic Substances for testing laboratory proficiency in estrogen receptor transactivation assays. With a median coefficient of variation of 5.0% and excellent correlation (R-2 = 0.993) between duplicate measurements, the reproducibility of the ER alpha-coregulator binding assay was better than the reproducibility of other commonly used in vitro ER functional assays. In addition, the coregulator binding assay is correctly predicting the estrogenicity for 13 out of 14 compounds tested. When the potency of the ER-agonists to induce ER alpha-coregulator binding was compared to their ER binding affinity, their ranking was similar, and the correlation between the EC50 values was excellent (R-2 = 0.96), as was the correlation with their potency in a transactivation assay (R-2 = 0.94). Moreover, when the ER alpha-coregulator binding profiles were hierarchically clustered using Euclidian cluster distance, the structurally related compounds were found to cluster together, whereas the steroid test compounds having an aromatic A-ring were separated from those with a cyclohexene A-ring. We concluded that this assay is capable of distinguishing ER alpha agonists and antagonists and that it even reflects the structural similarity of ER alpha agonists, indicating a potential to achieve identification and classification of ER alpha endocrine disruptors with high fidelity.