Sharma Sangeeta Shrestha, Sharma Shishir, Bureik Matthias
School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin, 300072, China.
J Pharm Anal. 2020 Jun;10(3):271-276. doi: 10.1016/j.jpha.2020.05.003. Epub 2020 May 26.
We have previously introduced the use of permeabilized fission yeast cells (enzyme bags) that recombinantly express full-length CYPs for drug metabolism studies. Such enzyme bags are cells with pores that function as enzymes . They can easily be prepared without a need for ultracentrifugation and may be used in similar protocols as microsomes. In this study we report the preparation of enzyme bag cocktails that permit the testing of multiple CYPs in a single enzyme bag reaction. Moreover, we established a convenient testing scheme that permits a rapid screen of all human CYPs for activity towards any given candidate substrate. An important aspect of this approach is the reduction of individual CYP test assays. If a cocktail containing many CYPs tests negative, it follows that all CYPs included in that cocktail need not be tested individually, thus saving time and resources. The new protocol was validated using two probe substrates.
我们之前介绍了使用重组表达全长细胞色素P450（CYPs）的通透化裂殖酵母细胞（酶袋）进行药物代谢研究。这种酶袋是具有孔的细胞，其功能如同酶。它们无需超速离心即可轻松制备，并且可用于与微粒体类似的实验方案。在本研究中，我们报告了酶袋混合物的制备方法，该方法允许在单个酶袋反应中检测多种CYPs。此外，我们建立了一种便捷的检测方案，可快速筛选所有人类CYPs对任何给定候选底物的活性。该方法的一个重要方面是减少单个CYP测试分析。如果含有多种CYPs的混合物测试结果为阴性，那么该混合物中包含的所有CYPs无需单独测试，从而节省时间和资源。使用两种探针底物对新方案进行了验证。
