Facile Synthesis of a Fluorescent Cyclosporin A Analogue To Study Cyclophilin 40 and Cyclophilin 18 Ligands
Facile Synthesis of a Fluorescent Cyclosporin A Analogue To Study Cyclophilin 40 and Cyclophilin 18 Ligands
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DOI：
10.1021/ml1001272
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发表时间：
2010-12-01
影响因子：
4.2
通讯作者：
Hausch, Felix
中科院分区：
文献类型：
--
作者：
Gaali, Steffen;Kozany, Christian;Hausch, Felix
有强烈的迹象表明亲环素40参与类固醇激素受体的失调引起的疾病，如前列腺癌或乳腺癌。为了鉴定这种亲免疫素的新抑制剂，我们开发了一种简化的荧光偏振测定法，该测定法基于荧光素标记示踪剂的合成。这种示踪剂是由一个简单的四步合成涉及格拉布斯复分解和标准酰胺键偶联，标记环孢菌素A与荧光素。通过分析各向异性变化，我们显示了这种示踪剂与Cyp 40和Cyp 18的结合，K-D值分别为106 +/- 13或12 +/- 1 nM，并证明了其与环孢菌素A的竞争。通过荧光偏振获得的结合数据通过酶活性测定证实。所描述的示踪剂允许以高通量形式进行稳健的测定，以支持新型Cyp 40配体的开发。
There are strong indications for the involvement of cyclophilin 40 in diseases caused by misregulation of steroid hormone receptors, like prostate or breast cancer To identify novel inhibitors for this immunophilin, we developed a simplified fluorescence polarization assay based on the synthesis Of a fluorescein labeled tracer. This tracer was produced by a facile four-step synthesis involving Grubbs metathesis and standard amide bond coupling, to label cyclosporin A with fluorescein. We show the binding of this tracer to Cyp40 and Cyp18 with K-D values of 106 +/- 13 or 12 +/- 1 nM, respectively, by analyzing the anisotropy change and demonstrate its competition with cyclosporin A. Binding data obtained by fluorescence polarization were corroborated by an enzymatic activity assay. The described tracer allows for a robust assay in a high-throughput format to support the development of novel Cyp40 ligands.