当前位置: X-MOL 学术Assay Drug Dev. Technol. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
A Genome-Edited ERα-HiBiT Fusion Reporter Cell Line for the Identification of ERα Modulators Via High-Throughput Screening and CETSA
ASSAY and Drug Development Technologies ( IF 1.6 ) Pub Date : 2021-12-08 , DOI: 10.1089/adt.2021.059
Hunter G Larson 1 , Alexey V Zakharov 1 , Sukumar Sarkar 2 , Shyh-Ming Yang 1 , Ganesha Rai 1 , James M Larner 2 , Anton Simeonov 1 , Natalia J Martinez 1
Affiliation  

The estrogen receptor α (ERα) is a target of intense pharmacological intervention and toxicological biomonitoring. Current methods to directly quantify cellular levels of ERα involve antibody-based assays, which are labor-intensive and of limited throughput. In this study, we generated a post-translational reporter cell line, referred to as MCF7-ERα-HiBiT, by fusing a small pro-luminescent nanoluciferase (NLuc) tag (HiBiT) to the C-terminus of endogenous ERα in MCF7 cells. The tag allows the luminescent detection and quantification of endogenous ERα protein by addition of the complementary NLuc enzyme fragment. This MCF7-ERα-HiBiT cell line was optimized for quantitative high-throughput screening (qHTS) to identify compounds that reduce ERα levels. In addition, the same cell line was optimized for a qHTS cellular thermal shift assay to identify compounds that bind and thermally stabilize ERα. Here, we interrogated the MCF7-ERα-HiBiT assay against the NCATS Pharmacological Collection (NPC) of 2,678 approved drugs and identified compounds that potently reduce and thermally stabilize ERα. Our novel post-translational reporter cell line provides a unique opportunity for profiling large pharmacological and toxicological compound libraries for their effect on ERα levels as well as for assessing direct compound binding to the receptor, thus facilitating mechanistic studies by which compounds exert their biological effects on ERα.

中文翻译:

用于通过高通量筛选和 CETSA 鉴定 ERα 调节剂的基因组编辑的 ERα-HiBiT 融合报告细胞系

雌激素受体 α (ERα) 是强烈药理学干预和毒理学生物监测的目标。目前直接量化 ERα 细胞水平的方法涉及基于抗体的测定,这些测定是劳动密集型的且通量有限。在这项研究中,我们通过将一个小的促发光纳米荧光素酶 (NLuc) 标签 (HiBiT) 融合到 MCF7 细胞内源性 ERα 的 C 末端,生成了一个翻译后报告细胞系,称为 MCF7-ERα-HiBiT。该标签允许通过添加互补的 NLuc 酶片段对内源性 ERα 蛋白进行发光检测和定量。这种 MCF7-ERα-HiBiT 细胞系针对定量高通量筛选 (qHTS) 进行了优化,以鉴定降低 ERα 水平的化合物。此外,同一细胞系针对 qHTS 细胞热转移分析进行了优化,以鉴定结合和热稳定 ERα 的化合物。在这里,我们针对 2,678 种已批准药物的 NCATS 药理学收集 (NPC) 询问了 MCF7-ERα-HiBiT 测定,并确定了有效减少和热稳定 ERα 的化合物。我们的新型翻译后报告细胞系为分析大型药理学和毒理学化合物库对 ERα 水平的影响以及评估化合物与受体的直接结合提供了独特的机会,从而促进了化合物对受体发挥生物学作用的机制研究。 ERα。678 种已获批准的药物和鉴定出的化合物可有效降低和热稳定 ERα。我们的新型翻译后报告细胞系为分析大型药理学和毒理学化合物库对 ERα 水平的影响以及评估化合物与受体的直接结合提供了独特的机会,从而促进了化合物对受体发挥生物学作用的机制研究。 ERα。678 种已获批准的药物和鉴定出的化合物可有效降低和热稳定 ERα。我们的新型翻译后报告细胞系为分析大型药理学和毒理学化合物库对 ERα 水平的影响以及评估化合物与受体的直接结合提供了独特的机会,从而促进了化合物对受体发挥生物学作用的机制研究。 ERα。
更新日期:2021-12-10
down
wechat
bug