Fluorescence Resonance Energy Transfer-based Screening for Protein Kinase C Ligands Using 6-methoxynaphthalene-labeled 1,2-diacylglycerol-lactones
Fluorescence Resonance Energy Transfer-based Screening for Protein Kinase C Ligands Using 6-methoxynaphthalene-labeled 1,2-diacylglycerol-lactones
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使用 6-甲氧基萘标记的 1,2-二酰基甘油内酯进行基于荧光共振能量转移的蛋白激酶 C 配体筛选
DOI：
10.1039/d1ob00814e
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发表时间：
2021
影响因子：
3.2
通讯作者：
Hirokazu Tamamura
中科院分区：
文献类型：
--
作者：
Kohei Tsuji;Takahiro Ishii;Takuya Kobayakawa;Nami Ohashi;Wataru Nomura;Hirokazu Tamamura
蛋白激酶 C (PKC) 与中央细胞信号转导途径和癌症和阿尔茨海默型痴呆等疾病相关，因此是治疗这些疾病的靶点。开发适合高通量筛选的简单方法以寻找有效的 PKC 配体是可取的。我们开发了一种基于荧光淬灭筛选的检测方法，其中溶剂化变色荧光团连接到竞争性探针上，并且开发了一种基于福斯特/荧光共振能量转移 (FRET) 现象的替代方法。这里，开发了一种改进的基于 FRET 的 PKC 结合测定，使用用供体荧光染料 6-甲氧基萘 (6MN) 标记的二酰基甘油 (DAG) 内酯。与二乙氨基香豆素 (DEAC) 标记的 DAG-内酯相比，6MN 标记的 DAG-内酯对 PKCδ C1b 结构域和荧光素作为受体荧光染料 (Fl-δC1b) 标记的荧光 PKCδ C1b 结构域具有更高的结合亲和力。 6MN标记的DAG-内酯和Fl-δC1b的组合显示出比DEAC标记的DAG-内酯和Fl-δC1b更大的荧光响应变化。通过使用 6MN 标记的 DAG-内酯的当前基于 FRET 的方法计算的已知 PKC 配体的 IC50 值与通过传统的基于放射性同位素的测定获得的 Ki 值非常一致。一些通过先前基于溶剂化显色荧光团的方法鉴定的假阳性化合物，通过该方法被发现为阴性。目前基于 FRET 的 PKC 结合测定更灵敏并且可能更有用。
Protein kinase C (PKC) is associated with a central cellular signal transduction pathway and disorders such as cancer and Alzheimer-type dementia and is therefore a target for the treatment of these diseases. The development of simple methods suitable for high-throughput screening to find potent PKC ligands is desirable. We have developed an assay based on fluorescence-quenching screening with a solvatochromic fluorophore attached to a competitive probe and its alternative method based on Förster/fluorescence resonance energy transfer (FRET) phenomena. Here, an improved FRET-based PKC binding assay using a diacylglycerol (DAG) lactone labeled with a donor fluorescent dye, 6-methoxynaphthalene (6MN), was developed. The 6MN-labeled DAG-lactone has a higher binding affinity for the PKCδ C1b domain and the fluorescent PKCδ C1b domain labeled by fluorescein as an acceptor fluorescent dye (Fl-δC1b) than the diethylaminocoumarin (DEAC)-labeled DAG-lactone. The combination of the 6MN-labeled DAG-lactone and Fl-δC1b showed a change in fluorescence response larger than that of the DEAC-labeled DAG-lactone and Fl-δC1b. The IC50 values of known PKC ligands calculated by the present FRET-based method using 6MN-labeled DAG-lactone agree well with the Ki values obtained by the conventional radioisotope-based assays. Some false positive compounds, identified by the previous solvatochromic fluorophore-based method, were found to be negative by this method. The present FRET-based PKC binding assay is more sensitive and could be more useful.