1. Massively parallel characterization of CYP2C9 variant enzyme activity and abundance
- Author
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Katherine A. Sitko, Lai Hong Wong, Allan E. Rettie, Gabriel Boyle, Clara J. Amorosi, John P. Kowalski, Melissa A Chiasson, Douglas M. Fowler, Maitreya J. Dunham, and Matthew G. McDonald
- Subjects
Models, Molecular ,Protein Conformation, alpha-Helical ,Prescription Drugs ,Mutation, Missense ,Saccharomyces cerevisiae ,Article ,Xenobiotics ,Abundance (ecology) ,Genetics ,Humans ,Missense mutation ,Protein Interaction Domains and Motifs ,Transgenes ,CYP2C9 ,Genetics (clinical) ,Cytochrome P-450 CYP2C9 ,Enzyme Assays ,Gene Library ,Binding Sites ,Polymorphism, Genetic ,Massive parallel sequencing ,biology ,Small molecule ,Enzyme assay ,Yeast ,High-Throughput Screening Assays ,Phenytoin ,Pharmacogenomics ,Mutagenesis, Site-Directed ,biology.protein ,Protein Conformation, beta-Strand ,Warfarin ,Function (biology) ,Protein Binding - Abstract
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 patient response leading 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 expressed in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants expressed in a human cell line 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.
- Published
- 2021
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