Biochemical and Biophysical Characterization of Pharmacogenetic Variants of Human Cytochrome P450 2C9

Abstract

Cytochromes P450 (CYP) are a superfamily of heme-containing enzymes essential in the metabolism of various xenobiotics. The drug metabolizing CYP2C9 enzyme metabolizes up to 15-20% of the medications that include warfarin, losartan, tolbutamide, and glimepiride. Genetic variations in CYP2C9 gene that include the more prevalent *2, *3, and *8, as well as the less common *28 and *30 alleles, have resulted in reduced enzyme activity leading to altered metabolism of various drug substrates compared with the wild-type (WT). The CYP2C9*2 and *3 genetic variants represent an amino acid change from arginine to cysteine at position 144 (R144C) and isoleucine to leucine at position 159 (I359L), respectively. Whereas, the CYP2C9*8, *28 and *30 represents amino acid substitution from arginine to histidine at positions 150 (R150H), glutamine to leucine at 214 (Q214L) and alanine to threonine at 477 (A477T), respectively. These CYP2C9 variants were recombinantly expressed and purified with an aim to characterize the binding of the drug substrate losartan using enzymatic and thermodynamic approaches. The activity of the enzyme was measured in the reconstituted system containing P450 reductase and losartan by the consumption of NADPH or change in absorbance at 340 nm. The *3 variant demonstrated a significantly reduced ability to turn over the losartan substrate compared to the WT and the other variants. Furthermore, differential scanning fluorimetry confirmed lower thermal stability of the *2 and *3 variants in the presence or absence of losartan compared to the WT or *8 variant. This was consistent to the weaker binding affinity of losartan to these two variants as elucidated by isothermal titration calorimetry. The average apparent dissociation constant (KD) for losartan binding to CYP2C9*3 was more than three-fold lower than the WT enzyme. Whereas the KD value for losartan binding to CYP2C9*8 did not show any significant difference. Overall, functional characterization of these variants provide insights into the enzyme activity and binding of drug substrates to CYP2C9, and help improve our understanding of the effect of amino acid substitutions on drug metabolism.