A functional in vitro assay for screening inhibitors of STAT5B phosphorylation
A functional in vitro assay for screening inhibitors of STAT5B phosphorylation
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DOI：
10.1016/j.jpba.2018.08.036
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发表时间：
2019-01-05
影响因子：
3.4
通讯作者：
Gunning, Patrick T.
中科院分区：
文献类型：
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作者：
de Araujo, Elvin D.;Manaswiyoungkul, Pimyupa;Gunning, Patrick T.
抑制STAT磷酸化被认为是一种可行的治疗策略，用于破坏肿瘤发生。在许多原发性肿瘤类型中发现了高频率的组成性STAT磷酸化，而非癌细胞表现出低基础活性，提供了可利用的治疗窗口。STAT激活涉及通过多种酪氨酸激酶磷酸化SH2结构域，随后STAT二聚化并易位至细胞核。通过阻断同源结合位点，STAT SH2结构域抑制剂可以阻止激酶介导的STAT从头磷酸化。STAT磷酸化抑制剂的评估以前仅在细胞中使用蛋白质印迹分析进行。然而，虽然提供了有用的纤维素功效，但不可能得出抑制是由于直接阻断STAT蛋白的结论。在这里，我们开发了一种功能性测定，直接报告磷酸化的阻断作为抑制剂与STAT蛋白相互作用的结果。我们已经优化了功能测定的反应条件，并针对已知的STAT5B配体（包括肽和小分子抑制剂）验证了测定。作为研究的一部分，我们还确定了由Abl激酶磷酸化STAT5B的几个位点。该试验将用于描述STAT结合剂的体外功能机制，并解卷积蛋白质印迹分析中观察到的磷酸化STAT抑制机制。(C)2018由Elsevier B.V.出版
Inhibition of STAT phosphorylation is recognized as a viable therapeutic strategy for disrupting tumorigenesis. Constitutive STAT phosphorylation is found with high frequency in a number of primary tumor types, while non-cancer cells exhibit low basal activity, providing an exploitable therapeutic window. STAT activation involves phosphorylation of the SH2 domain by a number of tyrosine kinases followed by STAT dimerization and translocation to the nucleus. By blocking the cognate binding site, STAT SH2-domain inhibitors can impede kinase-mediated de novo STAT phosphorylation. Assessing for inhibitors of STAT phosphorylation has previously been conducted exclusively in cellulo using Western blot analysis. However, while providing useful in cellulo efficacy, it is not possible to conclude that inhibition is due to a direct blockade of STAT protein. Here we developed a functional assay that directly reports the blockade of phosphorylation as a result of inhibitor interaction with STAT proteins. We have optimized reaction conditions for the functional assay and validated the assay against known STAT5B ligands, including peptides and small molecule inhibitors. As part of the study, we have also identified several sites of STAT5B phosphorylation by Abl kinase. This assay will serve to delineate the functional mechanism of STAT binders in vitro and deconvolute the mechanism of phospho-STAT inhibition observed in Western blot analysis. (C) 2018 Published by Elsevier B.V.