Development and Characterization of pFluor50, a Fluorogenic-Based Kinetic Assay System for High-Throughput Inhibition Screening and Characterization of Time-Dependent Inhibition and Inhibition Type for Six Human CYPs

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 (KM) and product formation rates (Vmax) 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.