Revnew, A. | bioRxiv Science

bioRxiv2025

Human CYP2C9 metabolism of organophosphorus pesticides and nerve agent surrogates

Of the Cytochrome P450 enzymes, the CYP2C9 variant is very important and is the cytochrome P450 (CYP) involved in the metabolism of several human drugs where it acts as a natural bioscavenger. Previously, CYP2C9 was demonstrated to remove sulfur from different organophosphorus (OP) pesticides, such as dimethoate, diazinon and parathion. In this study, we have tested the ability of CYP2C9 to degrade other OP compounds. We investigated the metabolism of OP compounds by CYP2C9 using LC-MS/MS approaches as well as the time-dependent inhibition of CYP2C9 by OP compounds using the previously developed pFluor50 fluorogenic assay. We found that CYP2C9 metabolizes thions (P=S) preferentially over oxons (P=O) and there were many OP compounds that inhibited CYP2C9 activity in a time-dependent manner. Additionally, we performed molecular docking based on the Protein Data Bank (PDB)crystal structure (1OG5) of the CYP2C9 receptor in order to gain atomistic insight from the in vitro experimental results. We observed a positive though moderate correlation between the calculated binding energy and the CYP2C9 metabolism of various OP compounds (R = 0.61) as well as the time-dependent changes in inhibition of OPs with binding energy (R = 0.59). Although the R values were modest,59). These in vitro data combined with analysis of additional OP derivatives could be used to develop artificial intelligence (AI)machine learning models to predict the metabolism of specific OP compounds by CYP2C9. This type of approach could be particularly relevant for the prediction of the metabolism of current and emerging chemical warfare agents.

pharmacology and toxicology↗

bioRxiv2023

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

1Cytochrome 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 M), CYP2C9 (KM= 0.6 M), and CYP2D6 (KM= 2.7 M); resorufin benzyl ether for CYP2B6 (KM= 46 M); 3-O-methyl fluorescein for CYP2C19 (KM= 3.0 M); and dibenzyl fluorescein for CYP3A4 (KM= 2.9 M). We then validated each assay using known inhibitors: -naphthoflavone for CYP1A2 (IC50= 13.5 nM); sertraline for CYP2B6 (IC50= 410 nM) and CYP2D6 (IC50= 2.4 M); sulfaphenazole for CYP2C9 (IC50= 1 M); ticlopidine for CYP2C19 (IC50= 1.2 M); 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.

pharmacology and toxicology↗