Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery Pub Date : 2024-03-01 DOI:10.1016/j.slasd.2023.12.007

Shitalben Patel , Marie Karlsson , Joseph T. Klahn , Frank Gambino Jr. , Helena Costa , Kathleen A. McGuire , Christina K. Baumgartner , Jon Williams , Sarah Sandoz , James E. Kath

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引用次数: 0

Abstract

The cellular thermal shift assay (CETSA®) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA® has been used for semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based readouts using cell lines. However, there has been no quantitative evaluation of CETSA® in unprocessed human whole blood, which is preferred for clinical applications. Here we report two separate assay formats – Alpha CETSA® and MSD CETSA® – that require less than 100 μL of whole blood per sample without PBMC isolation. We chose RIPK1 as a proof-of-concept target and, by measuring engagement of seven different inhibitors, demonstrate high assay sensitivity and robustness. These quantitative CETSA® platforms enable possible applications in preclinical pharmacokinetic-pharmacodynamic studies, and direct target engagement with small molecules in clinical trials.

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通过细胞热转移检测法定量检测人体全血中 RIPK1 的目标参与情况，以实现潜在的临床前和临床应用

细胞热转移分析法 (CETSA®) 是一种广泛用于小分子化合物临床前表征的目标参与方法。CETSA® 已用于分离 PBMC 的全血半定量读数，以及使用细胞系的平板定量读数。然而，CETSA® 还没有在未经处理的人体全血中进行定量评估，而临床应用更倾向于使用这种方法。在此，我们报告了两种不同的检测方法--Alpha CETSA® 和 MSD CETSA®，每份样本只需不到 100 μL 的全血，无需分离 PBMC。我们选择 RIPK1 作为概念验证靶点，并通过测量七种不同抑制剂的参与情况，证明了检测的高灵敏度和稳健性。这些定量 CETSA® 平台可应用于临床前药代动力学-药效学研究，并在临床试验中直接与小分子靶标结合。

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).