CYP3A4-Mediated Stereoselective Metabolism Of Metconazole And Nuarimol

Triazoles and ancymidols, two kinds of widely used chiral fungicides, have been frequently detected in various environmental medium, causing some health accidents. Till now, there still exists a tremendous gap on stereoselective CYP3A4-mediated metabolism of triazoles and ancymidols fingicides. The underlying molecular mechanism remains unknown. In this study, metconazole and fenarimol/nuarimol were choosen as the research target and their stereoselevtive metabolism mediated by CYP3A4 in vitro were deeply studied. The molecular interaction mechanism between CYP3A4 and small molecules was illustrated at the molecular level. The main results and conclusions of this work were summerized as following:(1) Four single enantiomers of metconazole were successfully separated and prepared using high-performance liquid chromatography (HPLC). Their stereochemistry was fully characterized in order to assign the absolute configuration to each stereoisomer based on circular dichroism spectra (CD) and time-dependent density functional theory (TDDFT).(2) Stereoselectivity of HLM- and CYP3A4-mediated metabolism of cis-RS and cis-SR metconazole was further probed. Both enantiomers can be metabolized by HLM and CYP3A4 and the degradation rate follows cis-RS> cis-SR. The enantiomer fraction (EF) showed a moderate increase from 0.5 to 0.82 during the process of metabolism, indicating that cis-RS was preferentially metabolized by CYP3A4.(3) The in vitro CYP3A4-mediated metabolism of fenarimol and nuarimol was studied. Both fenarimol and nuarimol can be metabolized by CYP3A4, with the degradation of nuarimol faster than fenarimol. No atropisomeric enrichment was observed for fenarimol upon incubation of the racemate. However, nuarimol underwent a time-dependent degradation with obvious stereoselectivity. CYP3A4 preferentially biotransformed the R-atropisomers of nuarimol.(4) The CYP3A4 GloTM Assay was used to determine CYP3A4 enzymatic activity. The configuration change of CYP3A4 was analyzed by fluorescence spectroscopy after binding with metconazole at the molecular level. Stereoselective metabolism of metconazole by CYP3A4 was investigated via molecular interaction. Being a potent inhibitor of CYP3A4, metconazole caused concentration-dependent inhibition of CYP3 A4 and the inhibitory effect of metconazole was confirmed to follow cis-SR> cis-RS. Two enantiomers have different binding mode to CYP3A4 and thier binding induces micro-environmental changes of CYP3A4.(5) The mechanisms of the interaction of fenarimol and nuarimol with CYP3A4 were investigated at the molecular level. CYP3A4 GloTM assay revealed the inhibitory effects of both fenarimol and nuarimol to CYP3A4 with a ranking of S-nuarimol> R-fenarimol> S-fenarimol>R-nuarimol. Fluorescence spectroscopy combined with molecular docking indicated that fenarimol and nuarimol could cause changes in micro-structure of CYP3 A4. Hydrogen bonds and van der Waals force were revealed as the driving forces of the molecular interaction. Then interactions energies rank as R-fenarimol> S-fenarimol and S-nuarimol> R-nuarimol.(6) As revealed by the yeast two-hybrid assay experiments, metconazole and fenarimol/nuarimol are antagonists of androgen receptor (AR). The effect of CYP3A4-mediated metabolism on AR disruption was then investigated and the AR disrupting potency of cis-SR and cis-RS metconazole displayed metabolic activation and attenuation, respectively.