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Carbonyl Reductases Role in the Biotransformation of Drug Bupropion
Tomanová, Radana ; Zemanová, Lucie (advisor) ; Malátková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Radana Tomanová Supervisor: RNDr. Lucie Škarydová, Ph.D. Title of thesis: Carbonyl reductases role in the biotransformation of drug bupropion Bupropion is a second-generation antidepressant that is also used at lower doses for smoking cessation. Structurally it is a monocyclic aminoketone, which is metabolized in human liver to three major pharmacologically active metabolites. Bupropion is hydroxylated to hydroxybupropion, reduction leads to formation of threohydrobupropion and also minor metabolite erythrohydrobupropion. The aim of this study was to determine in vitro the activity of human liver subcellular fractions and human carbonyl-reducing enzymes with bupropion and to discover the participation of enzymes in the liver metabolism of bupropion. Initially the role of subcellular fractions and recombinant carbonyl-reducing enzymes belonging to the superfamily AKR, family CBR and 11β-HSD 1 in in vitro biotransformation of bupropion was examined. Determination of formed metabolites was shown that all liver subcellular fractions and also 11β-HSD 1, AKR1C1, AKR1C2, AKR1C3 and CBR1 enzymes are active in the reduction of bupropion. The major in vitro liver metabolite was...
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Carbonyl Reductases Role in the Biotransformation of Drug Bupropion
Tomanová, Radana ; Zemanová, Lucie (advisor) ; Malátková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Radana Tomanová Supervisor: RNDr. Lucie Škarydová, Ph.D. Title of thesis: Carbonyl reductases role in the biotransformation of drug bupropion Bupropion is a second-generation antidepressant that is also used at lower doses for smoking cessation. Structurally it is a monocyclic aminoketone, which is metabolized in human liver to three major pharmacologically active metabolites. Bupropion is hydroxylated to hydroxybupropion, reduction leads to formation of threohydrobupropion and also minor metabolite erythrohydrobupropion. The aim of this study was to determine in vitro the activity of human liver subcellular fractions and human carbonyl-reducing enzymes with bupropion and to discover the participation of enzymes in the liver metabolism of bupropion. Initially the role of subcellular fractions and recombinant carbonyl-reducing enzymes belonging to the superfamily AKR, family CBR and 11β-HSD 1 in in vitro biotransformation of bupropion was examined. Determination of formed metabolites was shown that all liver subcellular fractions and also 11β-HSD 1, AKR1C1, AKR1C2, AKR1C3 and CBR1 enzymes are active in the reduction of bupropion. The major in vitro liver metabolite was...
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The use of differential scanning fluorimetry in characterization of selected carbonyl reductase
Tomanová, Radana ; Zemanová, Lucie (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Radana Tomanová Supervisor: RNDr. Lucie Škarydová, Ph.D. Title of diploma thesis: The use of differential scanning fluorimetry in characterization of selected carbonyl reductase Differential scanning fluorimetry (DSF) is simple, rapid method that enables to determine optimal conditions for stabilization of proteins and discover their ligands. DSF monitors thermal unfolding of a protein in the presence of fluorescent dye (e.g. SYPRO Orange, 2,6-ANS). The dye is highly fluorescent in non-polar environment such as hydrophobic sites of unfolded protein that appear on the surface in gradual increase of temperature. Melting temperature (Tm) of a protein expresses its stability. Ligand screening relies upon the fact that protein stability is enhanced upon ligand binding (ΔTm 0). The aim of this study was to introduce the DSF method on our department, use it for characterization of carbonyl reductase 1 (CBR1) and evaluate the obtained results with an independent methods and literature. First, the functionality of method was verified with citrate synthase and the conditions were optimized for CBR1. The group of several buffers pH 3-10 without additives or with NaCl or glycerol were tested...
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