School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin, 300072, China.
School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin, 300072, China.
Anal Biochem. 2020 Oct 15;607:113704. doi: 10.1016/j.ab.2020.113704. Epub 2020 Jul 19.
The development of convenient assays for the in vitro study of drug metabolizing enzymes (DMEs) such as cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) greatly facilitates metabolism studies of candidate drug compounds and other xenobiotics. We have developed and optimized an experimental approach that combines the advantages of recombinant expression in yeast with a microsomal-like biotransformation and thus allows for rapid and convenient enzymatic assays. Recombinant strains of the fission yeast Schizosaccharomyces pombe have previously been demonstrated to functionally express human CYPs and UGTs. Permeabilization of such cells with Triton X-100 results in the formation of enzyme bags, which can be used as biocatalysts. This protocol describes the preparation of such enzyme bags (3 h) and their application in enzyme activity assays (4 h) utilizing either pro-luminescent substrates and luminescence measurements or non-luminescent substrates and liquid chromatography coupled to mass spectrometry (LC-MS). Both applications provide practical tools for investigating CYP and UGT reactions in vitro without the need for additional sophisticated instrumentation or expertise.
开发用于体外研究药物代谢酶（DMEs）如细胞色素 P450（CYPs）和 UDP-葡糖醛酸基转移酶（UGTs）的便捷检测方法，极大地促进了候选药物化合物和其他外源化合物的代谢研究。我们开发并优化了一种实验方法，该方法结合了酵母重组表达的优势与类微粒体生物转化，从而可以快速方便地进行酶检测。裂殖酵母 Schizosaccharomyces pombe 的重组菌株已被证明能够功能性表达人类 CYPs 和 UGTs。用 Triton X-100 对这些细胞进行透化处理会导致酶囊的形成，可将其用作生物催化剂。本方案描述了这种酶囊的制备（3 小时）及其在酶活性检测中的应用（4 小时），可使用原发光底物和发光测量或非发光底物和液相色谱与质谱联用（LC-MS）。这两种应用都提供了在体外研究 CYP 和 UGT 反应的实用工具，而无需额外的复杂仪器或专业知识。
