Department of Chemistry, Columbia University , New York, New York 10027, United States.
ACS Chem Biol. 2013 Sep 20;8(9):1947-54. doi: 10.1021/cb400259n. Epub 2013 Jul 16.
Vesicular monoamine transporter 2 (VMAT2) is an essential component of the monoaminergic neurotransmission system in the brain as it transports monoamine neurotransmitters from the neuronal cytosol into the synaptic vesicles and thus contributes to modulation of neurotransmitter release. Considering the continuing interest in VMAT2 as a drug target, as well as a target for the design of imaging probes, we have developed a fluorescent substrate well suited for the study of VMAT2 in cell culture. Herein, we report the synthesis and characterization of a new fluorescent probe, FFN206, as an excellent VMAT2 substrate capable of detecting VMAT2 activity in intact cells using fluorescence microscopy, with subcellular localization to VMAT2-expressing acidic compartments without apparent labeling of other organelles. VMAT2 activity can also be measured via microplate reader. The apparent Km of FFN206 at VMAT2 was found to be 1.16 ± 0.10 μM, similar to that of dopamine. We further report the development and validation of a cell-based fluorescence assay amenable to high-throughput screening (HTS) using VMAT2-transfected HEK cells (Z'-factor of 0.7-0.8), enabling rapid identification of VMAT2 inhibitors and measurement of their inhibition constants over a broad range of affinities. FFN206 thus represents a new tool for optical examination of VMAT2 function in cell culture.
囊泡单胺转运体 2（VMAT2）是脑内单胺能神经递质系统的重要组成部分，因为它将单胺神经递质从神经元胞质中转运到突触小泡中，从而有助于调节神经递质的释放。鉴于人们对 VMAT2 作为药物靶点以及设计成像探针的持续兴趣，我们开发了一种非常适合在细胞培养中研究 VMAT2 的荧光底物。在此，我们报告了一种新的荧光探针 FFN206 的合成和表征，它是一种出色的 VMAT2 底物，能够使用荧光显微镜检测完整细胞中的 VMAT2 活性，具有亚细胞定位到表达 VMAT2 的酸性隔室，而不会明显标记其他细胞器。还可以通过微孔板读数器测量 VMAT2 活性。发现 FFN206 在 VMAT2 上的表观 Km 为 1.16±0.10 μM，与多巴胺相似。我们进一步报告了一种基于细胞的荧光测定法的开发和验证，该方法适用于使用 VMAT2 转染的 HEK 细胞进行高通量筛选（HTS）（Z'-因子为 0.7-0.8），能够快速鉴定 VMAT2 抑制剂并测量其抑制常数在广泛的亲和力范围内。因此，FFN206 代表了一种用于在细胞培养中光学检查 VMAT2 功能的新工具。
