新型布鲁顿酪氨酸激酶（BTK）底物的时间分辨荧光分析方法。
Novel Bruton's Tyrosine Kinase (BTK) Substrates for Time-Resolved Luminescence Assays.
机构信息
University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics, 420 Washington Avenue SE, Minneapolis, Minnesota 55455, United States.
出版信息
ACS Chem Biol. 2022 Jun 17;17(6):1328-1333. doi: 10.1021/acschembio.2c00106. Epub 2022 Jun 2.
Bruton's tyrosine kinase (BTK) is a well-documented target for cancer therapeutics due to its role in B-cell signaling pathways. However, inhibitor design is hindered by lack of tools to assess kinase activity. We used in vitro phosphoproteomics to determine BTK's substrate preferences and applied this information to our updated data processing pipeline, KINATEST-ID 2.1.0. This pipeline generates a position-specific scoring matrix for BTK and a list of candidate synthetic substrates, each given a score. Characterization of selected synthetic substrates demonstrated a correlation between KINATEST-ID 2.1.0 score and biochemical performance in in vitro kinase assays. Additionally, by incorporating a known terbium-chelation motif, we adapted synthetic substrates for use in an antibody-free time-resolved terbium luminescence assay. This assay has applications in high-throughput inhibitor screening.
布鲁顿酪氨酸激酶（BTK）是癌症治疗的一个明确靶点，因为它在 B 细胞信号通路中发挥作用。然而，由于缺乏评估激酶活性的工具，抑制剂的设计受到了阻碍。我们使用体外磷酸化蛋白质组学来确定 BTK 的底物偏好，并将这些信息应用于我们更新的数据分析管道 KINATEST-ID 2.1.0。该管道为 BTK 生成位置特异性评分矩阵和候选合成底物列表，每个底物都有一个评分。对选定的合成底物的特征分析表明，KINATEST-ID 2.1.0 评分与体外激酶测定中的生化性能之间存在相关性。此外，通过整合已知的铽螯合基序，我们改编了合成底物，用于无抗体的时间分辨铽发光测定法。该测定法可应用于高通量抑制剂筛选。
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