High-throughput methods of studying human cytochrome P450 activity in Saccharomyces cerevisiae

Abstract

Genetic variation in cytochrome P450 enzymes leads to inter-individual variability in drug metabolism, while lack of functional annotation prevents most variants from being used to inform drug choice and dosing. To measure P450 activity in a high-throughput manner, we developed a yeast-based activity assay. S. cerevisiae has a long history of use as a heterologous system of mammalian CYP expression, but we improved on this with targeted strain engineering and screening of natural isolates. In the process, we identified a sake strain with much higher human CYP expression levels than the laboratory strain. To measure P450 activity in yeast cells, we developed an assay using activity-based protein profiling with novel P450 activity-based probes to label a pooled population of variants in an activity-dependent manner. We extended this approach to a particularly important pharmacogene: CYP2C9, genetic variation in which affects the efficacy of warfarin, phenytoin, and other drugs. We coupled our yeast activity assay with fluorescence-activated cell sorting and high throughput sequencing, and generated activity scores for 6,142 single missense variants of CYP2C9. Strikingly, 65% of missense variants have significantly decreased activity suggesting altered drug metabolism in vivo. With collaborators, we also performed a second deep mutational scan of CYP2C9 in a human cell line, measuring variant abundance for 6,370 single missense variants, revealing that stability plays a large role in CYP2C9 function. Our yeast activity assay can be extended to other CYP enzymes, and will lead to advances in adverse drug response prevention by providing clinical guidance for patients carrying both currently known and yet-to-be discovered alleles of CYP2C9.