In silico and in vitro analysis of genetic variants of the equine CYP3A94, CYP3A95 and CYP3A97 isoenzymes

Abstract

Cytochrome P450 enzymes (CYPs) of the equine CYP3A subfamily are predominantly involved in drug metabolism. In this study, genetic variants of the equine CYP3A94, CYP3A95, and CYP3A97 were identified and characterized using in silico modeling and in vitro enzyme kinetics.The genomes of 81 horses were sequenced to obtain the genetic variants. Structural CYP modifications of the most frequent variants were analyzed in silico using the 3D-structures predicted by homology modeling. Enzyme kinetic analyses were performed using testosterone as substrate.Twenty genetic variants were found including five missense variants (CYP3A94:p.Asp217Asn, CYP3A95:p.Asp214His, CYP3A95:p.Ser392Thr, CYP3A97:p.Ile119Thr, CYP3A97:p.Met500Val) with a higher percentage of minor allele frequency (MAF) (range 0.2–0.4). A splice-site variant (c.798 + 1G > A) in CYP3A94, likely to generate a truncated protein, was found in 50% of the horses. CYP3A94:p.Asp217Asn and CYP3A95:p.Asp214His were localized on the CYP F-α-helix, an important region for the substrate interactions in the human CYP3A4. Testosterone 2β-hydroxylation was diminished in CYP3A94217Asn and CYP3A95392Thr. Ketoconazole inhibited 2β-hydroxylation differently in the five variants with the most pronounced inhibition obtained for CYP3A95392Thr.In vitro and in silico analyses of genetic variants allow unraveling structural features in equine CYPs that correlate with changes in the CYP activity.