Biology subjects

Davydova, N.

Publications and source records attributed to Davydova, N..

3 recordsLinked to original sources

bioRxiv2026

A New Fluorogenic Substrate for CYP1A2 and Its Application in Studying the Effects of Alcohol Exposure on Liver Drug Metabolism

Aiming to develop a high-throughput fluorimetric assay for the activity CYP1A2, we introduced 6-Methoxy-2-naphthoic acid (MONA) as a new fluorogenic substrate for this important metabolizer of antidepressants and psychotropic drugs in human liver. We demonstrated that oxidative demethylation of MONA by liver microsomes results in a red shift and a substantial increase in fluorescence. This effect, which is exceptionally well pronounced at alkaline pH, allowed us to develop a sensitive and robust high-throughput assay of MONA metabolism. Probing the activity of 15 individual recombinant human P450 enzymes, we found that only two P450 species exhibited activity in MONA demethylation: CYP1A2 (kcat=11.9{+/-}2.2 min-1, KM=578{+/-}106 {micro}M) and CYP2A6 (kcat=0.48{+/-}0.07 min-1, KM=54{+/-}15 {micro}M). Since the KM values of the two enzymes are well resolved and the turnover rate observed with CYP2A6 is much lower than that of CYP1A2, this new fluorogenic substrate is useful as a specific probe for CYP1A2 activity in HLM. Importantly, MONA is not metabolized by CYP1A1 and CYP2C19, which distinguishes it from all known CYP1A2 fluorogenic substrates. We then used MONA to investigate the effects of chronic alcohol exposure on CYP1A2 activity using a series of 23 proteomically characterized individual HLM preparations from donors with various levels of alcohol consumption. The substrate saturation profiles (SSP) acquired with these preparations were subjected to global kinetic analysis by approximating them with combinations of two Michaelis-Menten equations with globally optimized KM values of 11 and 553 {micro}M. The amplitudes (Vmax values) of both components showed a pronounced increase with increasing alcohol exposure of the liver donors. The Vmax of the minor high-affinity component was best correlated with the abundance of alcohol-inducible CYP2E1 enzyme. The correlation was further improved by combining it with the abundances of CYP2A6 and CPR. This finding suggests that this minor component reflects the activity of CYP2A6 in the complex with alcohol-inducible CYP2E1 protein. In contrast, the Vmax of the predominant CYP1A2-catalyzed low-affinity component revealed a pronounced correlation with the abundances of CYP1A2 and NADPH cytochrome P450 reductase (CPR). These results suggest a considerable increase in the rate of metabolism of drug substrates of CYP1A2 by chronic alcohol exposure that takes place despite an alcohol-induced decrease in CYP1A2 expression.

biochemistry↗

bioRxiv2026

The role of CYP3A-CYP2E1 interactions in activation of CYP3A enzymes by chronic alcohol exposure

Aiming to examine the effect of chronic alcohol exposure on the activity of CYP3A enzymes in human liver, we studied the metabolism of two CYP3A-specific substrates, 7-benzyloxyquinoline (7-BQ) and ivermectin, in 23 preparations of human liver microsomes (HLM) obtained from donors with documented alcohol exposure, graded from non-drinkers to heavy alcoholics. All HLM samples were characterized for the composition of the cytochrome P450 pool and the abundances of other drug-metabolizing and endoplasmic reticulum-stress-related enzymes by global proteomics. Our studies revealed a striking increase in the activities of CYP3A enzymes caused by chronic alcohol exposure. This effect is not associated with CYP3A enzyme levels, which do not correlate with alcohol exposure. Instead, the rates of 7-BQ and ivermectin metabolism correlate with the content of alcohol-inducible CYP2E1. However, this enzyme does not metabolize ivermectin, and its activity with 7-BQ is negligible. These results suggest that the observed acceleration of the elimination of drugs metabolized by CYP3A enzymes by alcohol exposure is due to functional effects of the interaction between CYP3A and CYP2E1. To elucidate the potential mechanism of this effect, we studied the formation of CYP2E1-CYP3A4 complexes in CYP3A4-containing Supersomes with co-incorporated CYP2E1 using tag-transfer chemical crosslinking mass spectrometry (CX-MS). These experiments confirmed physical interactions between the proteins and allowed the identification of CYP3A4 residues at the sites of contact. This information was used to build structural models of the CYP2E1-CYP3A4 complex and to propose possible mechanisms for the observed effects.

biochemistry↗

bioRxiv2024

Non-additivity of the functional properties of individual P450 species and its manifestation in the effects of alcohol consumption on the metabolism of ketamine and amitriptyline

To explore functional interconnections between multiple P450 enzymes and their manifestation in alcohol-induced changes in drug metabolism, we implemented a high-throughput study of correlations between the composition of the P450 pool and the substrate saturation profiles (SSP) of amitriptyline and ketamine in a series of 23 individual human liver microsomes preparations from donors with a known history of alcohol consumption. The SSPs were approximated with linear combinations of three Michaelis-Menten equations with globally optimized KM (substrate affinity) values. This analysis revealed a strong correlation between the rate of ketamine metabolism and alcohol exposure. For both substrates, alcohol consumption caused a significant increase in the role of the low-affinity enzymes. The amplitudes of the kinetic components and the total rate were further analyzed for correlations with the abundance of 11 major P450 enzymes assessed by global proteomics. The maximal rate of metabolism of both substrates correlated with the abundance of CYP3A4, their predicted principal metabolizer. However, except for CYP2D6 and CYP2E1, responsible for the low-affinity metabolism of ketamine and amitriptyline, respectively, none of the other potent metabolizers of the drugs revealed a positive correlation. Instead, in the case of ketamine, we observed negative correlations with the abundances of CYP1A2, CYP2C9, and CYP3A5. For amitriptyline, the data suggest inhibitory effects of CYP1A2 and CYP2A6. Our results demonstrate the importance of functional interactions between multiple P450 species and their decisive role in the effects of alcohol exposure on drug metabolism.

biochemistry↗