Three mechanistically distinct kinase assays compared: Measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors
Three mechanistically distinct kinase assays compared: Measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors
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DOI：
10.1177/1087057106296047
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发表时间：
2007-02-01
影响因子：
--
通讯作者：
Jakes, Scott
中科院分区：
文献类型：
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作者：
Kashem, Mohammed A.;Nelson, Richard M.;Jakes, Scott
已经开发了许多测定方法来鉴定蛋白激酶的小分子效应物，但没有一种方法可以适用于所有分离的激酶。作者开发了一套3种高通量筛选（HTS）兼容的生化分析方法，可以测量激酶活性位点的3种不同的机械特性，目标是3种方法中至少有1种适用于任何被选为药物发现目标的激酶。两种测定催化活性酶的方法：解离增强镧系元素氟免疫测定（DELFIA）使用抗体来定量磷酸化底物的产生；第二种方法使用荧光素酶来测量三磷酸腺苷（ATP）在磷酸化转移到肽底物或转移到水（内在ATP酶活性）过程中的消耗。第三个试验不依赖于催化活性酶，测量抑制剂和荧光ATP结合位点探针之间与激酶结合的竞争。为了评估这些检测方法在药物发现中的适用性，作者比较了它们识别Tec家族非受体蛋白酪氨酸激酶抑制剂的能力，即白细胞介素-2诱导的T细胞激酶（ITK）。这三种检测方法对从10208个化合物文库中鉴定出的57%的联合确认命中集达成一致，这些化合物文库富含已知的激酶抑制剂和结构相似的分子。在这3种检测方法中，测量内在atp酶活性的方法产生了最多的唯一命中，最少的唯一未命中，最全面的命中集，只有2.7%的被其他2种检测方法确认的抑制剂缺失。基于这些数据，所有3种检测格式都可用于筛选，并根据目标激酶提供更多的检测设计选择。
Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.