A Miniaturized 1536-Well Format γ-Secretase Assay
A Miniaturized 1536-Well Format γ-Secretase Assay
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DOI：
10.1089/adt.2009.0202
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发表时间：
2009-10-01
影响因子：
1.8
通讯作者：
Li, Yue-Ming
中科院分区：
文献类型：
--
作者：
Shelton, Christopher C.;Tian, Yuan;Li, Yue-Ming
γ-分泌酶是一种乙酰基蛋白酶，其切割多种底物，包括淀粉样前体蛋白（APP）和Notch蛋白。APP的异常蛋白水解参与阿尔茨海默病（AD）的发病机制，并且过度活跃的Notch信号传导在多种癌症中起致癌作用。γ-分泌酶已经成为治疗AD和癌症的药物开发的有希望的靶标。具有增加的高通量筛选能力的测定将允许更快地筛选化学文库并促进抑制剂开发。我们已经开发了一种基于均相时间分辨荧光（HTRF）的检测方法，该方法使用新型生物素化重组APP底物和溶解膜制剂作为γ-分泌酶的来源。将该测定小型化为1536孔格式，并在针对类似于3，000种化合物的文库的中试筛选中进行验证。由于计算的Z'因子为0.74，且证明了其鉴别已知γ-分泌酶抑制剂（如胃酶抑素A）的能力，因此总体试验性能稳健。这种经过验证的测定法可容易地用于针对大型化学文库进行初步筛选，以寻找可能代表AD和各种肿瘤的潜在治疗剂的新型γ-分泌酶活性抑制剂。
gamma-Secretase is an aspartyl protease that cleaves multiple substrates including the amyloid precursor protein (APP) and the Notch proteins. Abnormal proteolysis of APP is involved in the pathogenesis of Alzheimer's disease (AD) and overactive Notch signaling plays an oncogenic role in a variety of cancers. gamma-Secretase has emerged as a promising target for drug development in the treatment of AD and cancer. Assays with increased capacity for high-throughput screening would allow for quicker screening of chemical libraries and facilitate inhibitor development. We have developed a homogeneous time-resolved fluorescence (HTRF)-based assay that makes use of a novel biotinylated recombinant APP substrate and solubilized membrane preparation as the source of the gamma-secretase enzyme. The assay was miniaturized to a 1536-well format and validated in a pilot screen against a library of similar to 3,000 compounds. The overall assay performance was robust due to a calculated Z' factor of 0.74 and its demonstrated ability to identify known gamma-secretase inhibitors such as pepstatin A. This validated assay can readily be used for primary screening against large chemical libraries searching for novel inhibitors of gamma-secretase activity that may represent potential therapeutics for AD and a variety of neoplasms.