Probing functional interactions between cytochromes P450 with principal component analysis of substrate saturation profiles and targeted proteomics

Dangi, Bikash; Davydova, Nadezhda Y.; Maldonado, Marc A.; Ahire, Deepak; Prasad, Bhagwat; Davydov, Dmitri R.

doi:10.1016/j.abb.2021.108937

Cited by 8 publications

(6 citation statements)

References 44 publications

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“…The effects of interactions of CYP2E1 with other P450 enzymes provide the most likely explanation for the alcohol-induced increase in the metabolism of diazepam and doxycycline [17][18][19], the substrates of CYP3A, or phenytoin, tolbutamide, and warfarin [20][21] metabolized primarily by CYP2C9. Conclusive evidence of a direct cause-to-effect relationship between alcohol-dependent induction of CYP2E1 and the effects of this kind is provided by our studies of the impact of CYP2E1 on the activity of CYP3A4, CYP1A2, and CYP2C19 in HLM [22][23].…”

Section: Introductionmentioning

confidence: 84%

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry

Dr¹,

Dangi²,

et al. 2021

Preprint

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This study aimed on exploration of the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) in the human liver on drug metabolism. Using membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide (BPM) and 4-(N-succinimidylcarboxy)benzophenone (BPS), we explored the array of its protein-protein interactions (proteome) in human liver microsomes (HLM) with chemical cross-linking mass spectrometry (CXMS). Exposure of bait-incorporated HLM samples to light was followed by isolation of the His-tagged bait protein and its cross-linked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the cross-linked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively cross-linked partners of CYP2E1 are cytochromes P450 2A6, 3A4, 2C9, and 4A11. We also detected the conjugates of CYP2E1 with UDP-glucuronosyltransferases (UGTs) 1A6, 1A9, 2B4, 2B15, and 2B17. These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes. Of particular interest is the observation of efficient cross-linking of CYP2E1 with CYP4A11. This enzyme plays a central role in the synthesis of vasoactive eicosanoids and its interactions with alcohol-inducible CYP2E1 may shed light on the mechanisms of alcohol-induced hypertension.

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Section: Introductionmentioning

confidence: 84%

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry

Dr¹,

Dangi²,

et al. 2021

Preprint

Self Cite

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“…The method may considerably facilitate and streamline routine studies of drug metabolism in the development of new pharmaceuticals. Furthermore, its combination with the toolset of proteomics offers a potent way for an in-depth analysis of correlations between the profile of drug metabolism and the composition of the drug-metabolizing ensemble, which is necessary for further elaboration of the concept of functional integration in human drug metabolism [ 2 , 35 ].…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism

Davydova,

Hutner,

Gaither

et al. 2023

Biology

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In a search for a reliable, inexpensive, and versatile technique for high-throughput kinetic assays of drug metabolism, we elected to rehire an old-school approach based on the determination of formaldehyde (FA) formed in cytochrome P450-dependent demethylation reactions. After evaluating several fluorometric techniques for FA detection, we chose the method based on the Hantzsch reaction with acetoacetanilide as the most sensitive, robust, and adaptable to high-throughput implementation. Here we provide a detailed protocol for using our new technique for automatized assays of cytochrome P450-dependent drug demethylations and discuss its applicability for high-throughput scanning of drug metabolism pathways in the human liver. To probe our method further, we applied it to re-evaluating the pathways of metabolism of ketamine, a dissociative anesthetic and potent antidepressant increasingly used in the treatment of alcohol withdrawal syndrome. Probing the kinetic parameters of ketamine demethylation by ten major cytochrome P450 (CYP) enzymes, we demonstrate that in addition to CYP2B6 and CYP3A enzymes, which were initially recognized as the primary metabolizers of ketamine, an important role is also played by CYP2C19 and CYP2D6. At the same time, the involvement of CYP2C9 suggested in the previous reports was deemed insignificant.

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“…The method may considerably facilitate and streamline the routine studies of drug metabolism in the development of new pharmaceuticals. Furthermore, its combination with the toolset of proteomics offers a potent way for an in-depth analysis of correlations of the profile of drug metabolism with the composition of the drug-metabolizing ensemble, which is necessary for further elaboration of the concept of functional integration in human drug metabolism [2,34].…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-evaluate the Pathways of Ketamine Metabolism

Davydova,

Hutner,

Gaither

et al. 2023

Preprint

View full text Add to dashboard Cite

In a search for a reliable, inexpensive, and versatile technique for high-throughput kinetic assays of drug metabolism, we elected to rehire an old-school approach based on the determination of formaldehyde (FA) formed in cytochrome P450-dependent demethylation reactions. After evaluating several fluorometric techniques of FA detection, we choose the method based on the Hantzsch reaction with acetoacetanilide as the most sensitive, robust, and adaptable to high-throughput implementation. Here we provide a detailed protocol for the use of our new technique for automatized assays of cytochrome P450-dependent drug demethylations and discuss its applicability for high-throughput scanning of the pathways of drug metabolism in the human liver. To probe our method further, we applied it to re-evaluating the pathways of metabolism of ketamine, a dissociative anesthetic, and potent antidepressant increasingly used in the treatment of alcohol withdrawal syndrome. Probing the kinetic parameters of ketamine demethylation by 11 major cytochrome P450 (CYP) enzymes, we demonstrate that besides CYP2B6 and CYP3A enzymes, which were initially recognized as the primary metabolizers of ketamine, an important role is also played by CYP2C19, and CYP2D6, while the involvement of CYP2C9 suggested in the previous reports deemed insignificant.

show abstract

Probing functional interactions between cytochromes P450 with principal component analysis of substrate saturation profiles and targeted proteomics

Cited by 8 publications

References 44 publications

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry

High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism

High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-evaluate the Pathways of Ketamine Metabolism

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