Institute of Physiology and Pathophysiology , Friedrich-Alexander-Universität Erlangen-Nürnberg , Universitaetsstrasse 17 , 91054 Erlangen , Germany.
Department of Chemical Biology , Max Planck Institute for Medical Research , Jahnstrasse 29 , 69120 Heidelberg , Germany.
J Med Chem. 2018 Jul 26;61(14):6121-6139. doi: 10.1021/acs.jmedchem.8b00387. Epub 2018 Jul 10.
β-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-BACE1, 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.
β-位点 APP 切割酶 1（BACE1）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种了解该蛋白酶生理学和病理生理学的强大方法。然而，到目前为止，常见的标记技术需要遗传操作、使用大型抗体，或者与活细胞成像不兼容。特异性结合靶蛋白的荧光小分子可以克服这些限制。本文中，我们介绍了 SiR-BACE1，它是 BACE1 抑制剂 S-39 和 SiR647 的缀合物，是一种新型的荧光、无标记和无抗体标签，用于 BACE1。我们介绍了它的化学发展，表征了它的光物理和药理特性，并评估了它在溶液、过表达系统和天然脑组织中的行为。我们证明了它在共聚焦、受激发射损耗和动态单分子显微镜中的适用性。使用 SiR-BACE1 进行的关于 BACE1 表面流动性的首次功能研究显示出明显受限的扩散模式。
