Shriwas Pratik, Revnew Andre, Roo Sarah, Bender Alex, Miller Kevin, Hadad Christopher M, Lane Thomas R, Ekins Sean, McElroy Craig A
Division of Medical Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
Department of Chemistry and Biochemistry, College of Arts and Sciences, The Ohio State University, Columbus, OH 43210, USA.
Molecules. 2025 May 2;30(9):2032. doi: 10.3390/molecules30092032.
Cytochrome P450s (CYPs) play an integral role in drug and xenobiotic metabolism in humans, and thus, understanding CYP inhibition and/or activation by new therapeutic candidates is an important step in the drug development process. Ideally, CYP inhibition/activation assays should be high-throughput, use commercially available components, allow for analysis of metabolism by the majority of human CYPs, and allow for kinetic analysis of inhibition type and time-dependent inhibition. Here, we developed pFluor50, a 384-well microtiter plate-based fluorogenic kinetic enzyme assay system using substrates metabolized by six human CYPs to generate fluorescent products and determined the Michaelis-Menten kinetics constants (K) and product formation rates (V) for each substrate-CYP pair. The pFluor50 assay was also used to elucidate inhibition type and time-dependent inhibition for some inhibitors, demonstrating its utility for characterizing the observed inhibition, even mechanism-based inhibition. The pFluor50 assay system developed in this study using commercially available components should be very useful for high-throughput screening and further characterization of potential therapeutic candidates for inhibition/activation with the most prevalent human CYPs.
细胞色素P450（CYP）在人体药物和外源性物质代谢中发挥着不可或缺的作用，因此，了解新型治疗候选药物对CYP的抑制和/或激活作用是药物开发过程中的重要一步。理想情况下，CYP抑制/激活试验应具有高通量，使用市售组件，能够分析大多数人CYP的代谢情况，并能够对抑制类型和时间依赖性抑制进行动力学分析。在此，我们开发了pFluor50，这是一种基于384孔微量滴定板的荧光动力学酶分析系统，使用六种人CYP代谢的底物生成荧光产物，并确定了每个底物 - CYP对的米氏动力学常数（K）和产物形成速率（V）。pFluor50分析还用于阐明某些抑制剂的抑制类型和时间依赖性抑制，证明了其在表征观察到的抑制作用（甚至是基于机制的抑制）方面的实用性。本研究中使用市售组件开发的pFluor50分析系统对于高通量筛选以及进一步表征潜在治疗候选药物与最常见人CYP的抑制/激活作用应该非常有用。
