Cecioni Samy, Ashmus Roger A, Gilormini Pierre-André, Zhu Sha, Chen Xi, Shan Xiaoyang, Gros Christina, Deen Matthew C, Wang Yang, Britton Robert, Vocadlo David J
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Department of Chemistry, Université de Montréal, Québec, Canada.
Nat Chem Biol. 2022 Mar;18(3):332-341. doi: 10.1038/s41589-021-00960-x. Epub 2022 Feb 24.
Understanding the function and regulation of enzymes within their physiologically relevant milieu requires quality tools that report on their cellular activities. Here we describe a strategy for glycoside hydrolases that overcomes several limitations in the field, enabling quantitative monitoring of their activities within live cells. We detail the design and synthesis of bright and modularly assembled bis-acetal-based (BAB) fluorescence-quenched substrates, illustrating this strategy for sensitive quantitation of disease-relevant human α-galactosidase and α-N-acetylgalactosaminidase activities. We show that these substrates can be used within live patient cells to precisely measure the engagement of target enzymes by inhibitors and the efficiency of pharmacological chaperones, and highlight the importance of quantifying activity within cells using chemical perturbogens of cellular trafficking and lysosomal homeostasis. These BAB substrates should prove widely useful for interrogating the regulation of glycosidases within cells as well as in facilitating the development of therapeutics and diagnostics for this important class of enzymes.
要了解酶在其生理相关环境中的功能和调节，需要能够报告其细胞活性的优质工具。在此，我们描述了一种针对糖苷水解酶的策略，该策略克服了该领域的几个局限性，能够对活细胞内的酶活性进行定量监测。我们详细介绍了基于双缩醛（BAB）的明亮且模块化组装的荧光淬灭底物的设计与合成，阐述了该策略用于灵敏定量与疾病相关的人类α-半乳糖苷酶和α-N-乙酰半乳糖胺酶活性的情况。我们表明，这些底物可用于活的患者细胞中，以精确测量抑制剂对靶酶的作用以及药理伴侣的效率，并强调了使用细胞转运和溶酶体稳态的化学扰动剂在细胞内定量活性的重要性。这些BAB底物对于研究细胞内糖苷酶的调节以及促进针对这类重要酶的治疗和诊断方法的开发应具有广泛的用途。
