Development of a NanoBRET assay for evaluation of 14-3-3σ molecular glues

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery Pub Date : 2024-07-01 DOI:10.1016/j.slasd.2024.100165

Holly R. Vickery , Johanna M. Virta , Markella Konstantinidou, Michelle R. Arkin

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引用次数: 0

Abstract

We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC₅₀ values between 100 nM and 1 μM in cells, which align with the EC₅₀ values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.

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开发用于评估 14-3-3σ 分子胶的 NanoBRET 分析法。

我们报告了一种 384 孔格式的 NanoBRET 检测方法的开发情况，该方法可鉴定活细胞中 14-3-3 与客户相互作用的分子胶。14-3-3 的七种异构体是二聚体枢纽蛋白，具有转录因子调控和信号转导等多种作用。14-3-3 与数百种客户蛋白相互作用，调节它们的功能，因此是一种理想的治疗靶标，可以实现客户选择性。我们开发的 NanoBRET 系统适用于三种 14-3-3σ 客户蛋白 CRAF、TAZ 和雌激素受体 α（ERα），它们代表了三种特定的结合模式。我们测量了分子胶对14-3-3σ/客户复合物的稳定作用，其在细胞中的EC50值介于100 nM和1 μM之间，这与体外荧光各向异性计算出的EC50值一致。为中心蛋白 14-3-3σ 开发这种 NanoBRET 系统可以简化方法，绕过其他 14-3-3σ 客户检测开发过程中的多个优化步骤。通过 NanoBRET 系统，可以使用全长蛋白质在更贴近生理、基于细胞的环境中评估 PPI 稳定性。该方法适用于各种蛋白质-蛋白质相互作用 (PPI)，为探索 PPI 稳定剂和抑制剂化合物库提供了一个强大的平台。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

SLAS Discovery Chemistry-Analytical Chemistry

CiteScore

7.00

自引率

3.20%

发文量

审稿时长

39 days

期刊介绍： Advancing Life Sciences R&D: SLAS Discovery reports how scientists develop and utilize novel technologies and/or approaches to provide and characterize chemical and biological tools to understand and treat human disease. SLAS Discovery is a peer-reviewed journal that publishes scientific reports that enable and improve target validation, evaluate current drug discovery technologies, provide novel research tools, and incorporate research approaches that enhance depth of knowledge and drug discovery success. SLAS Discovery emphasizes scientific and technical advances in target identification/validation (including chemical probes, RNA silencing, gene editing technologies); biomarker discovery; assay development; virtual, medium- or high-throughput screening (biochemical and biological, biophysical, phenotypic, toxicological, ADME); lead generation/optimization; chemical biology; and informatics (data analysis, image analysis, statistics, bio- and chemo-informatics). Review articles on target biology, new paradigms in drug discovery and advances in drug discovery technologies. SLAS Discovery is of particular interest to those involved in analytical chemistry, applied microbiology, automation, biochemistry, bioengineering, biomedical optics, biotechnology, bioinformatics, cell biology, DNA science and technology, genetics, information technology, medicinal chemistry, molecular biology, natural products chemistry, organic chemistry, pharmacology, spectroscopy, and toxicology. SLAS Discovery is a member of the Committee on Publication Ethics (COPE) and was published previously (1996-2016) as the Journal of Biomolecular Screening (JBS).