High-throughput fluorescence polarization assay to identify inhibitors of Cbl(TKB)-protein tyrosine kinase interactions.
High-throughput fluorescence polarization assay to identify inhibitors of Cbl(TKB)-protein tyrosine kinase interactions.
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DOI：
10.1016/j.ab.2010.11.038
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发表时间：
2011-04-15
影响因子：
2.9
通讯作者：
Natarajan A
中科院分区：
文献类型：
--
作者：
Kumar EA;Charvet CD;Lokesh GL;Natarajan A
Casitas-B-lineage Lymphoma（Cbl）蛋白通过发挥E3-泛素连接酶的功能在调节信号转导途径中发挥重要作用。Cbl蛋白含有保守的酪氨酸激酶结合（TKB）结构域，其以磷酸化依赖性方式结合十几种蛋白，包括蛋白酪氨酸激酶（PTK）。细胞表面PTKs的表达水平受Cbl介导的泛素化、内化和降解的调节。这种信号级联中的功能障碍导致PTK的长期激活，因此与炎性疾病和各种癌症有关。由于这种负调节功能，Cbl作为治疗靶点在很大程度上被忽视。然而，最近的研究，如（a）在髓样癌亚群中功能获得c-Cbl突变和（B）c-Cbl作为与不良临床结果相关的前列腺基底细胞标志物的鉴定，表明情况并非如此。在这里，我们报告了一个竞争性的高通量荧光偏振分析的发展，在384孔格式，以确定抑制剂的Cbl（TKB）。通过筛选Prestwick化学文库®证明了测定的高通量筛选（HTS）就绪性。
The Casitas-B-lineage Lymphoma (Cbl) proteins play an important role in regulating signal transduction pathways by functioning as E3-ubiquitin ligases. The Cbl proteins contain a conserved tyrosine kinase binding (TKB) domain that bind over a dozen proteins, including protein tyrosine kinases (PTKs) in a phosphorylation dependent manner. The cell surface expression levels of the PTKs are regulated by Cbl-mediated ubiquitination, internalization, and degradation. Dysfunction in this signaling cascade has resulted in prolonged activation of the PTKs and therefore implicated in inflammatory diseases and various cancers. Due to this negative regulatory function, Cbl has been largely ignored as a therapeutic target. However recent studies such as the identification of (a) gain of function c-Cbl mutations in subsets of myeloid cancer and (b) c-Cbl as a prostate basal cell marker that correlates with poor clinical outcome, suggests otherwise. Here we report the development of a competitive high throughput fluorescence polarization assay in a 384-well format to identify inhibitors of Cbl(TKB). The high throughput screen (HTS) readiness of the assay was demonstrated by screening the Prestwick chemical library®.
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