Massively parallel characterization of CYP2C9 variant enzyme activity and abundance

CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9...

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Bibliographic Details
Published in:American journal of human genetics 2021-09, Vol.108 (9), p.1735-1751
Main Authors: Amorosi, Clara J., Chiasson, Melissa A., McDonald, Matthew G., Wong, Lai Hong, Sitko, Katherine A., Boyle, Gabriel, Kowalski, John P., Rettie, Allan E., Fowler, Douglas M., Dunham, Maitreya J.
Format: Article
Language:English
Subjects:
Binding Sites
CYP2C9
Cytochrome P-450 CYP2C9 - chemistry
Cytochrome P-450 CYP2C9 - genetics
Cytochrome P-450 CYP2C9 - metabolism
deep mutational scanning
Enzyme Assays
Gene Library
High-Throughput Screening Assays
humanized yeast
Humans
Models, Molecular
Mutagenesis, Site-Directed
Mutation, Missense
pharmacogenomics
Phenytoin - chemistry
Polymorphism, Genetic
Prescription Drugs - chemistry
Prescription Drugs - metabolism
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
Transgenes
warfarin
Warfarin - chemistry
Warfarin - metabolism
Xenobiotics - chemistry
Xenobiotics - metabolism
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Summary:CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants. Using click-seq, we measured the activity of 6,142 missense variants in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants in a human cell line by using variant abundance by massively parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.
ISSN:0002-9297
1537-6605
DOI:10.1016/j.ajhg.2021.07.001