Insights into the Effect of Genetic Variation in CYP2C9 (*11) on Drug Binding.

Abstract

Cytochrome P450s (CYP) are a family of membrane associated heme‐containing enzymes involved in xenobiotic metabolism with recent relevance in pharmacogenomics. The human CYP2C9 enzyme metabolizes up to 15–20% of drugs that include warfarin, losartan, ibuprofen, and tolbutamide. It is highly polymorphic with several genetic variations associated with significantly altered activity of the enzyme. The CYP2C9*11 allele occurs predominantly in African Americans but also in Caucasian and other ethnic populations with a frequency of around 0.05. The *11 genetic variation represents a substitution of amino acid residue at 335 from arginine to tryptophan (R335W) that has demonstrated decreased catalytic activity towards various substrates including S‐warfarin. The R335W was created in CYP2C9 wild‐type (WT) construct using site‐directed mutagenesis and co‐expressed in E. Coli alongside chaperone proteins (GroEL/ES). Generation of purified CYP2C9*11 protein enabled catalytic studies and crystallization in complex with the drug substrate losartan. The enzymatic assays revealed marked reduction in losartan turn‐over by CYP2C9*11 compared to the WT enzyme. Additionally, comparison of the amino acid sequence revealed that R335 is conserved across the CYP2C subfamily of enzymes. The R335 is located on the distal J‐J’ loop and interacts with multiple amino acid side chains on the J and J’ helices. The computational analysis suggests that the substitution with hydrophobic and bulkier tryptophan located on the surface region may result in destabilizing interactions with several helices including those located near the active site heme, affecting protein stability and function. Overall, these observations establish the effect of distal variation prompting further evaluation by structural and biophysical studies.Support or Funding InformationThe research was funded by the start‐up funds from the Albany College of Pharmacy and Health Sciences.