Dataset Information

Detection and Quantification of Drug-Protein Adducts in Human Liver

ABSTRACT: Protein adducts formed by drugs or their reactive metabolites are risk factors for adverse reactions, toxicity and inactivation of cytochrome P450 (CYP) enzymes. Characterization of drug-protein adducts is limited due to lack of methods capable of discovering the proteins and peptides adducted by reactive metabolites in complex matrices. The current study presents a proteomics workflow that achieves this task. This workflow combines data-dependent, and data independent acquisition (DDA and DIA) based LC-MS/MS and is sensitive enough to detect very low abundance adducts resulting from CYP mediated drug metabolism in human livers. Human liver microsomes (HLMs) or recombinant CYPs were incubated with raloxifene as a model compound. The resulting adducts were identified using this workflow. In HLMs, raloxifene adducts were detected in 78 proteins, including multiple adducts in CYP3A and CYP2C family enzymes. Experiments with recombinant CYP3A and CYP2C enzymes confirmed adduct formation in all CYPs tested, including CYPs not subject to time dependent inactivation (TDI) by raloxifene. These data suggest many adducts formed by reactive intermediates are benign. DIA analysis showed variable abundance of raloxifene adducts in many proteins between livers, but no concomitant decrease in abundance of unadducted peptides. The current study sets a new standard for adduct detection in complex samples, offering valuable insights into the human adductome resulting from exposure to reactive metabolites. The developed methodology forms a foundation for mechanistic studies to identify, quantify and differentiate between adducts that result in adverse drug reactions and drug-drug interactions and adducts that do not.

ORGANISM(S): Homo Sapiens Spodoptera Frugiperda

SUBMITTER: Alex Zelter

PROVIDER: PXD054246 | panorama | Fri Oct 25 00:00:00 BST 2024

REPOSITORIES: PanoramaPublic

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Publications

Detection and Quantification of Drug-Protein Adducts in Human Liver.

Zelter Alex A Riffle Michael M Shteynberg David D DD Zhong Guo G Riddle Ellen B EB Hoopmann Michael R MR Jaschob Daniel D Moritz Robert L RL Davis Trisha N TN MacCoss Michael J MJ Isoherranen Nina N

Journal of proteome research 20241023 11

Covalent protein adducts formed by drugs or their reactive metabolites are risk factors for adverse reactions, and inactivation of cytochrome P450 (CYP) enzymes. Characterization of drug-protein adducts is limited due to lack of methods identifying and quantifying covalent adducts in complex matrices. This study presents a workflow that combines data-dependent and data-independent acquisition (DDA and DIA) based liquid chromatography with tandem mass spectrometry (LC-MS/MS) to detect very low ab ...[more]

PMID: 39442081

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Project description:Cytochrome P450s (CYPs) are a family of enzymes responsible for metabolizing nearly 80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, which may result in improper dosing and severe adverse drug reactions. CYPs have low sequence conservation, making it difficult to anticipate whether variant effects measured in one CYP may extend to others based on sequence alone. Even closely related CYPs, like CYP2C9 and its closest homolog CYP2C19, have distinct phenotypic properties despite sharing 92% of their sequences. Thus, we used Variant Abundance by Massively Parallel sequencing (VAMP-seq) to measure the protein abundance of 7,660 missense variants in CYP2C19 expressed in cultured human cells. Our results confirmed positions and structural features critical for CYP function and revealed how variants at positions conserved across all eukaryotic CYPs influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core, and that substitutions in some regions reduced abundance in CYP2C19 but not CYP2C9. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. When nearby amino acids were exchanged in double and triple mutants, we found distinct interactions between the sites in CYP2C19 and CYP2C9, revealing a region that is partially responsible for the difference in thermodynamic stability between the two homologs. Since these positions are also important for determining substrate specificity, there may be an evolutionary tradeoff between stability and altered enzymatic function. Finally, we used our data to analyze 368 previously unannotated human variants, finding that 43% had decreased abundance. Thus, by comparing variant effects between two closely related, important human genes, we have uncovered regions underlying their functional differences and paved the way for a more complete understanding of one of the most versatile families of enzymes.

Dataset Information

Detection and Quantification of Drug-Protein Adducts in Human Liver

Publications

Detection and Quantification of Drug-Protein Adducts in Human Liver.

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