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

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,...

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Main Authors: Pratik Shriwas, Andre Revnew, Sarah Roo, Alex Bender, Kevin Miller, Christopher M. Hadad, Thomas R. Lane, Sean Ekins, Craig A. McElroy
Format: Article
Language:English
Published: MDPI AG 2025-05-01
Series:Molecules
Subjects:
CYP450
plate reader
kinetic assay
high throughput screening
fluorescent probes
metabolism
Online Access:https://www.mdpi.com/1420-3049/30/9/2032
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Summary: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<sub>M</sub>) and product formation rates (V<sub>max</sub>) 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.
ISSN:1420-3049

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