Non-additivity of the functional properties of P450 enzymes in the human drug-metabolizing ensemble revealed in a study with a series of individual microsomal preparations

Abstract ID 102638 Poster Board 040 Recent advances in studying the effects of hetero-association of multiple cytochrome P450 species reveal a tight functional integration in the P450 ensemble attained through physical interactions between the constituting enzymes. To explore these integrative interconnections in human liver microsomes (HLM), we implemented a high-throughput study of correlations between its composition and the substrate saturation profiles (SSP) of substrates metabolized by multiple P450 enzymes. The study was carried out with a set of 23 individual preparations of human liver microsomes from donors with a known history of alcohol consumption complemented with eight pooled HLM preparations from 50-100 donors (mixed gender). All HLM samples were characterized by the composition of the cytochrome P450 pool with a toolset of global proteomics. As representative substrates, we choose amitriptyline and ketamine, the drugs used as antidepressants in the treatment of alcohol withdrawal syndrome. Both substrates are metabolized by a set of several P450 species, where the predominant role is expected to be played by CYP3A4, CYP2C19, and CYP2B6 (ketamine) or CYP3A4, CYP2A6, and CYP2D6 (amitriptyline). The rate of oxidative demethylation of these substrates was accessed with a high-throughput technique based on fluorometric detection of formed formaldehyde [1]. The sets of SSP for all HLM samples in the series were subjected to global kinetic fitting assisted with Principal Component Analysis. It allowed us to approximate all SSP profiles with linear combinations of three (amitriptyline) or four (ketamine) Michaelis-Menten equations with globally optimized KM values. The square correlation coefficients for the approximations of each SSP were higher than 0.995. The resulting profiles of the total Vmax and the amplitudes of the individual Michaelis-Menten components were subjected to the analysis of their correlations with the profiles of the relative abundance of the individual P450 enzymes. The results of this analysis suggest that the pathways of metabolism of ketamine and amitriptyline are largely dictated by the abundances of the species not directly involved in their bioconversion. In particular, we found that the abundance of CYP3A4, which is expected to play a major role in the metabolism of both substrates, is not among the major determinants of the rate of their demethylation. Instead, for both substrates, this rate is positively correlated with the abundance of CYP4A11 and negatively correlated with the abundance of CYP2B6. These results demonstrate the limitations of the proportional projection approach for predicting the participation of the individual P450 enzymes in the metabolism of the drugs metabolized by multiple P450 species. Instead, the involvement of the individual P450s in the metabolism of such substrates is largely affected by the intermolecular interactions between multiple P450 enzymes and is dependent on the abundances of the P450 species that are not directly involved in their metabolism. [1] Davydova, N.Y.; Hutner, D.A.; Gaither, K.A.; Singh, D.K.; Prasad, B.; Davydov, D.R. (2023) High-throughput assay of cytochrome P450-dependent drug demethylation reactions and its use to re-evaluate the pathways of ketamine metabolism. MDPI Biology-Basel, 2023, 12 This research was supported by the National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health under Award R01AA030155.