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

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 2ß-hydroxylation was diminished in CYP3A94217Asn and CYP3A95392Thr. Ketoconazole inhibited 2ß-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.

Testosterone metabolism of equine single CYPs of the 3A subfamily compared to the human CYP3A4.

Cytochrome P450 enzymes (CYPs) are responsible for the phase I metabolism of drugs, xenobiotics and endogenous substances. Knowledge of single CYPs and their substrates is important for drug metabolism, helps to predict adverse effects and may prevent reduced drug efficacy in polypharmacy. In this study, three equine isoenzymes of the 3A subfamily, the equine flavoprotein NADPH-P450 oxidoreductase (POR), and the cytochrome b5 (CYB5) were cloned, sequenced and heterologously expressed in a baculovirus expression system. Testosterone, the standard compound for characterization of the human CYP3A4, was used to characterize the newly expressed equine CYPs. The metabolite pattern was similar in equine and the human CYPs, but the amounts of metabolites were isoform-dependent. All equine CYPs produced 2-hydroxytestosterone (2-OH-TES), a metabolite never described in equines. The main metabolite of CYP3A4 6ß-hydroxytestosterone (6ß-OH-TES) was measured in CYPs 3A95 and 3A97 with levels close to the detection limit. Ketoconazole inhibited 2-OH-TES in the human CYP3A4 and the equine CYP3A94 and CYP3A97 completely, whereas a 70% inhibition was found in CYP3A95. Testosterone 6ß- and 2-hydroxylation was significantly different in the equine CYPs compared to CYP3A4. The expression of single equine CYPs allows characterizing drug metabolism and may allow prevention of drug-drug interactions.

Innate immune defenses induced by CpG do not promote vaccine-induced protection against foot-and-mouth disease virus in pigs.

Emergency vaccination as part of the control strategies against foot-and-mouth disease virus (FMDV) has the potential to limit virus spread and reduce large-scale culling. To reduce the time between vaccination and the onset of immunity, immunostimulatory CpG was tested for its capacity to promote early protection against FMDV challenge in pigs. To this end, CpG 2142, an efficient inducer of alpha interferon, was injected intramuscularly. Increased transcription of Mx1, OAS, and IRF-7 was identified as a sensitive measurement of CpG-induced innate immunity, with increased levels detectable to at least 4 days after injection of CpG formulated with Emulsigen. Despite this, CpG combined with an FMD vaccine did not promote protection. Pigs vaccinated 2 days before challenge had disease development, which was at least as acute as that of unvaccinated controls. All pigs vaccinated 7 days before challenge were protected without a noticeable effect of CpG. In summary, our results demonstrate the caution required when translating findings from mouse models to natural hosts of FMDV.