Crystal Structure of the Genetic Variant of Human Cytochrome P450 2C9 (*27): Insights into the Effect of Single Nucleotide Polymorphism

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Issue Date
2022-12
Authors
Deodhar, Shruti '22
Degree
MS in Pharmaceutical Sciences
Advisor
Shah, Manish
Committee Members
Lee, Insong James
Musteata, Marcel
Journal Title
Journal ISSN
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Abstract
Cytochrome P450 (CYP) enzymes are heme-thiolate monooxygenases involved in the metabolism of xenobiotics. Most of the currently available drugs are bio-transformed by the CYP 1, 2, and 3 families. CYP2C9 is a major drug-metabolizing enzyme with more than 80 single nucleotide polymorphisms, several of which significantly affect drug metabolism. This study aims to characterize the CYP2C9*27 allele that represents an amino acid substitution Arg150Leu (arginine to leucine at position 150) in the presence or absence of various drug substrates. The change from Arg150Leu is predominantly found in the Japanese population. In this study, we elucidated the impact of distal variation on the binding of drug substrate in the active site. The CYP2C9*27 enzyme was expressed in E. coli, purified, and crystallized with the antihypertensive drug substrate, losartan. For data collection and structure determination, crystals were sent to Stanford Synchrotron Radiation Light Source. The structure of the CYP2C9*27 protein in complex with losartan is similar in conformation to the previously solved structure of the CYP2C9 wild-type-losartan complex but differed in the number of losartan bound. There were two molecules of losartan bound in the *27 complex, one in the active site and another at the periphery in an orientation identical to that observed in the wild-type complex. This was in contrast to the wild-type complex that demonstrated the binding of three molecules, with an additional losartan in the access channel that was not observed in the *27 structure. In a reconstituted enzymatic assay that also included the redox partner P450 reductase, the CYP2C9*27 showed lower consumption of NADPH than the wild type enzyme suggesting reduced capacity of the variant to turn-over losartan. This was further corroborated by isothermal titration calorimetry studies that illustrated significantly reduced binding affinity of losartan with the *27 variant than for the wildtype. Furthermore, various other angiotensin II receptor blockers were characterized to elucidate differences and role of substrate selectivity in binding to CYP2C9. Overall, the results obtained provide insights into the effect of single nucleotide polymorphism on losartan binding that could be clinically relevant in patients carrying the *27 allele.
Citation
Deodhar SR. Crystal structure of the genetic variant of human cytochrome p450 2c9 (*27): insights into the effect of single nucleotide polymorphism [thesis]. Ann Arbor (MI): Proquest LLC; 2022. 57 p.
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