Structure-Function Studies of Human Cytochrome P450 2C9*30 Genetic Variant
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Issue Date
2020-07
Authors
Cho, Sunghwan '20
Degree
MS in Pharmaceutical Sciences
Advisor
Shah, Manish
Committee Members
Voigt, Jeffrey M.
Shi, Binshan
Shi, Binshan
Journal Title
Journal ISSN
Volume Title
Abstract
Cytochrome P450 (CYP) enzymes are a superfamily of heme-containing proteins found on the membrane of the endoplasmic reticulum. CYPs constitute the major enzyme family in drug metabolism with increasing importance in pharmacogenetics. The human CYP2C9 enzyme, which is responsible for the metabolism of over 20% of available clinical drugs, is highly polymorphic. More than 80 genetic variants of this enzyme have been identified, with many exhibiting significantly altered activities toward various medications compared to the wild type (WT). Despite the extensive functional and structural studies of various CYPs from different species, the fundamental basis of genetic polymorphisms is scant. The CYP2C9*30 is a rare allelic variant, which is found in Japanese population. It represents an amino acid substitution at position 477 from alanine to threonine that has resulted in significantly altered activity of the enzyme towards various substrates compared to the WT. We have expressed and purified the CYP2C9*30 variant in E.coli to the optimal quality. The in-vitro enzymatic assays revealed reduced activity and substrate turn-over by CYP2C9*30 compared to the WT. The previously solved crystal structure of the CYP2C9*30-losartan complex demonstrated the reorientation of amino acid side-chain of glutamine at 214 or Q214 that interacted with the polar substitution threonine at 477 or A477T in the *30 structure. The goal is to investigate the activity of CYP2C9*30, using losartan as a substrate, and determine the role of Q214 side chain in the binding of the drug substrates near the heme in the active site using computational docking. A total of 5 important substrates (Glyburide, Irbesartan, Tolbutamide, S-warfarin, and Losartan) of CYP2C9 were studied via molecular docking to characterize the effect of variation to threonine at 477 on drug metabolism. The data suggest that the reorientation of Q214 side chain to interact with T477 in CYP2C9*30 may affect binding of drugs near the heme in the active site, which can lead to altered metabolism profile compared to the wild-type. Overall, the results yield insights into the reduced catalytic activity of the CYP2C9*30 and may help understand the differences in drug metabolism.
Citation
Cho S. Structure-function studies of human cytochrome P450 2C9*30 genetic variant [thesis]. Ann Arbor (MI): Proquest LLC; 2020. 58p.
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