A fluorescence polarization-based assay for peptidyl prolyl cis/trans isomerase cyclophilin A
A fluorescence polarization-based assay for peptidyl prolyl cis/trans isomerase cyclophilin A
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DOI：
10.1016/j.ab.2006.04.040
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发表时间：
2006-09-01
影响因子：
2.9
通讯作者：
Kati, Warren M.
中科院分区：
文献类型：
--
作者：
Liu, Yaya;Jiang, Jianjun;Kati, Warren M.
肽基脯氨酰顺反异构酶亲环素A（CypA）是重要的免疫抑制剂环孢素A的细胞受体。此外，CypA及其酶家族已被发现在各种生物过程中发挥关键作用，包括蛋白质运输，HIV和HCV感染/复制，以及Ca 2+介导的细胞内信号传导。由于这些原因，亲环素已经成为几种疾病的潜在药物靶标。因此，筛选新的小分子亲环素抑制剂是非常重要的。不幸的是，迄今为止报道的生物化学测定不适用于高通量筛选形式。在这里，我们报告了一种基于荧光偏振的检测人类CypA，可以适应高通量筛选药物发现。该技术是基于竞争，并使用荧光素标记的环孢菌素A类似物和纯化的人CypA定量测量未标记的抑制剂的结合能力。通过荧光偏振检测允许实时测量结合率而无需分离步骤。所获得的结果证明了测定程序之间的显著相关性，表明荧光偏振与CypA组合的应用对于准确评估抑制剂结合是非常有利的。(c)2006爱思唯尔公司All rights reserved.
Peptidyl prolyl cisltrans isomerase cyclophilin A (CypA) serves as a cellular receptor for the important immunosuppressant drug, cyclosporin A. In addition, CypA and its enzyme family have been found to play critical roles in a variety of biological processes, including protein trafficking, HIV and HCV infection/replication, and Ca2+-mediated intracellular signaling. For these reasons, cyclophilins have emerged as potential drug targets for several diseases. Therefore, it is extremely important to screen for novel small molecule cyclophilin inhibitors. Unfortunately, the biochemical assays reported so far are not adaptable to a high-throughput screening format. Here, we report a fluorescence polarization-based assay for human CypA that can be adapted to high-throughput screening for drug discovery. The technique is based on competition and uses a fluorescein-labeled cyclosporin A analog and purified human CypA to quantitatively measure the binding capacity of unlabeled inhibitors. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with CypA is highly advantageous for the accurate assessment of inhibitor binding. (c) 2006 Elsevier Inc. All rights reserved.