A New Fluorogenic Small-Molecule Labeling Tool for Surface Diffusion Analysis and Advanced Fluorescence Imaging of β-site Amyloid Precursor Protein-Cleaving Enzyme 1 Based on Silicone Rhodamine: SiR-BACE1
A New Fluorogenic Small-Molecule Labeling Tool for Surface Diffusion Analysis and Advanced Fluorescence Imaging of β-site Amyloid Precursor Protein-Cleaving Enzyme 1 Based on Silicone Rhodamine: SiR-BACE1
复制标题
DOI：
10.1021/acs.jmedchem.8b00387
复制
发表时间：
2018-07-26
影响因子：
7.3
通讯作者：
Huth, Tobias
中科院分区：
文献类型：
--
作者：
Karch, Sandra;Broichhagen, Johannes;Huth, Tobias
β位点APP裂解酶1（BACE 1）是阿尔茨海默病发病机制中的主要参与者。结构和功能荧光显微镜提供了一个强大的方法来了解这种蛋白酶的生理和病理生理。然而，到目前为止，普通的标记技术需要遗传操作，使用大抗体，或者与活细胞成像不兼容。与目标蛋白特异性结合的荧光小分子可以克服这些限制。在此，我们引入SiR-BACE 1，BACE 1抑制剂S-39和SiR 647的缀合物，作为BACE 1的新型荧光、无标签和无抗体标记。我们介绍了它的化学发展，表征其生物物理和药理学特性，并评估其在溶液中的行为，在过表达系统，并在原生脑组织。我们证明了它的适用性，共聚焦，受激发射损耗和动态单分子显微镜。SiR-BACE 1对BACE 1表面迁移率的第一次功能研究揭示了一种明显受限的扩散模式。
beta-site APP-cleaving enzyme 1 (BACE1) is a major player in the pathogenesis of Alzheimer's disease. Structural and functional fluorescence microscopy offers a powerful approach to learn about the physiology and pathophysiology of this protease. Up to now, however, common labeling techniques require genetic manipulation, use large antibodies, or are not compatible with live cell imaging. Fluorescent small molecules that specifically bind to the protein of interest can overcome these limitations. Herein, we introduce SiR-BACEl, a conjugate of the BACE1 inhibitor S-39 and SiR647, as a novel fluorogenic, tag-free, and antibody-free label for BACE1. We present its chemical development, characterize its photophysical and pharmacologic properties, and evaluate its behavior in solution, in overexpression systems, and in native brain tissue. We demonstrate its applicability in confocal, stimulated emission depletion and dynamic single-molecule microscopy. The first functional studies with SiR-BACE1 on the surface mobility of BACE1 revealed a markedly confined diffusion pattern.