Streamlining cellular thermal shift assay for ultra-high throughput screening

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery Pub Date : 2026-02-01 Epub Date: 2025-11-29 DOI:10.1016/j.slasd.2025.100293

Pascal Lambertz, Loretta Hamacher, Jana Flegel, Philipp Pflüger, Torsten Feller, Mike Küster, Tom Stockter, Tommaso Mari, Yousef Morcos, Martin Adamczewski

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引用次数: 0

Abstract

The Cellular Thermal Shift Assay (CETSA) has emerged as a powerful tool for evaluating drug-target interactions in live cells, yet its application in ultra-high throughput screening (uHTS) has been limited by technical constraints. In this study, we present significant advancements in CETSA methodology, focusing on the development of an innovative isothermal CETSA platform for primary uHTS screen in 1536 well plates and a Gradient Peltier Device (GPD) for retesting hits in full melting curve CETSA. Our optimized isothermal CETSA allows for the evaluation of adherent cells in their physiological state, enhancing assay performance through a controlled thermal ramp-up instead of traditional heat shock methods and utilizing highly sensitive luminescence detection. The GPD enables all steps of a full melt curve CETSA to be conducted in one single flat bottom microtiter plate, improving data quality by reducing handling and pipetting steps and improving temperature control. We benchmarked both methods against an established fluorescence polarization assay using the androgen receptor as a model target. Results demonstrated a strong correlation between both CETSA methods and the fluorescence polarization assay, indicating the potential for identifying true binders while minimizing false positives. Our findings highlight the utility of this optimized CETSA platform for high throughput drug discovery, paving the way for more effective screening of true binders in live cells.

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精简细胞热移分析超高通量筛选。

细胞热移测定（CETSA）已成为评估活细胞中药物-靶标相互作用的有力工具，但其在超高通量筛选（uHTS）中的应用受到技术限制。在这项研究中，我们介绍了CETSA方法的重大进展，重点是开发了一种创新的等温CETSA平台，用于1536个井板的初级uHTS筛检，以及一种梯度Peltier设备（GPD），用于重新测试全熔化曲线CETSA中的hit。我们优化的等温CETSA允许在其生理状态下评估贴壁细胞，通过控制热上升而不是传统的热休克方法和利用高灵敏度的发光检测来提高检测性能。GPD使全熔体曲线CETSA的所有步骤都能在一个平底微量滴定板上进行，通过减少处理和移液步骤以及改善温度控制来提高数据质量。我们用雄激素受体作为模型靶标，对这两种方法进行基准测试。结果表明，在CETSA方法和荧光偏振法之间存在很强的相关性，表明在最大限度地减少假阳性的同时识别真正的结合物的潜力。我们的发现强调了这种优化的CETSA平台在高通量药物发现中的实用性，为更有效地筛选活细胞中的真正结合物铺平了道路。

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).