ABSTRACT
As a new atypical antipsychotic, brexpiprazole is primarily metabolized by cytochrome P450 3A4 (CYP3A4). However, genetic polymorphisms in CYP3A4 cause wide variability in individuals' responses to brexpiprazole, leading to unpredictable adverse side effects or even therapeutic failure. The present study was designed to systematically study the effects of 26 recombinant CYP3A4 variants on the metabolism of brexpiprazole and investigate their enzymatic activity. Wild-type CYP3A4 and the 26 variants were incubated with the substrate brexpiprazole for 30 min at 37 °C. The metabolite DM-3411 was detected using ultraperformance liquid chromatography-tandem mass spectrometry. The activity of the wild-type CYP3A4 and 26 of its variants was analyzed. Then, the mechanism underlying the changes in enzyme function was observed using molecular dynamics simulations and molecular docking. Compared with CYP3A4.1, the enzymatic activities of CYP3A4.19, -.24, and -.28 were not significantly different (from 91.82% to 96.25%), but CYP3A4.14 and CYP3A4.15 exhibited higher enzyme activity (from 117.9 to 127.5%). The remaining 21 isoforms, including CYP3A4.2, -.3, -.4, -.5, -.7, -.8, -.9, -.10, -.11, -.12, -.13, -.16, -.17, -.18, -.20, -.23, -.29, -.31, -.32, -.33 and -.34, displayed lower enzymatic activities (from 2.90% to 75.72%). The results obtained from computer modeling indicated that weak binding affinity impaired the function of CYP3A4.32. Mutations that occur around the active site might lead to a loss of enzymatic activity, while the variants located far away from the active site perhaps had little effect on function of CYP3A4. These comprehensive data provide a reference and prediction for treatment strategies and risk assessments of brexpiprazole.
Subject(s)
Antipsychotic Agents/metabolism , Cytochrome P-450 CYP3A/metabolism , Dopamine Agonists/metabolism , Quinolones/metabolism , Serotonin Agents/metabolism , Thiophenes/metabolism , Cytochrome P-450 CYP3A/genetics , Molecular Docking Simulation , Molecular Dynamics Simulation , Recombinant Proteins/metabolismABSTRACT
Actin-depolymerizing factor (ADF) plays an important role in remodeling the actin cytoskeleton which contributes much to the invasion of host cells by the apicomplexan parasite. The gene encoding for Eimeria tenella ADF with one intron was cloned and identified by the E. tenella genome raw sequence data ( http://www.sanger.ac.uk/projects/E.tenella/ ). The deduced polypeptide sequence was only composed of 118 amino acids (13.14 kDa) without signal peptide and nuclear localization sequence. The amino acid sequence was most similar to the ADF of Toxoplasma gondii, 69.1%. Compared the putative three-dimensional structures between E. tenella and yeast, the actin filament binding sites on the segment from the alpha4-helices to the C-terminal were mostly missed in E. tenella. Real-time RT-PCR and dot blot both revealed that ADF expression was relatively stronger in the sporozoites and merozoites than in sporulated and unsporulated oocysts in both mRNA and protein levels. Northern blot analysis suggested that there was only one form of ADF transcripts in all different life stages of E. tenella. Actin-binding experiment showed that the recombinant fusion ADF protein could bind with actin, which indicated that ADF probably plays an important role in the invasion host of E. tenella.
