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

Abstract

1AbstractCytochrome P450 enzymes (CYPs) play an integral role in drug and xenobiotic metabolism in humans and thus understanding CYP inhibition 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 constant (KM) and product formation rate (Vmax) for each substrate-CYP pair. The substrate-CYP pairs were as follows: resorufin ethyl ether for CYP1A2 (KM= 0.8 μM), CYP2C9 (KM= 0.6 μM), and CYP2D6 (KM= 2.7 μM); resorufin benzyl ether for CYP2B6 (KM= 46 μM); 3-O-methyl fluorescein for CYP2C19 (KM= 3.0 μM); and dibenzyl fluorescein for CYP3A4 (KM= 2.9 μM). We then validated each assay using known inhibitors: α-naphthoflavone for CYP1A2 (IC50= 13.5 nM); sertraline for CYP2B6 (IC50= 410 nM) and CYP2D6 (IC50= 2.4 μM); sulfaphenazole for CYP2C9 (IC50= 1 μM); ticlopidine for CYP2C19 (IC50= 1.2 μM); and CYP3cide for CYP3A4 (IC50= 56 nM). pFluor50 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.