| Summary: | 碩士 === 國立臺灣大學 === 毒理學研究所 === 96 === Benzodiazepine (BDZ) abuse in hospitals and by young people is a growing problem and carries serious risks to health and society. BDZ are a class of psychoactive drugs with varying hypnotic, sedative, anxiolytic, anticonvulsant, muscle relaxant and amnesic properties, which are mediated by slowing down the central nervous system. Flunitrazepam (FNZ) is a short-intermediate acting BDZ derivative, prescribed for the treatment of insomnia, marketed by Roche most commonly under the trade name Rohypnol, has a high affinity for the BDZ receptor within the gama-aminobutyric acid (GABA) complex. In humans, FNZ is oxidized to the major metabolites N-demethylflunitrazepam (NDF) and 3-hydroxyflunitrazepam (3HF) and reduced to 7-aminoflunitrazepam (7AF). Human CYP2C19 and CYP3A4 are the principal P-450 cytochromes involved in NDF and 3HF formation. In our laboratory previous researches, we know not only the liver NADPH-cytochrome P450 reductase, but also intestinal micorflora are involved in 7AF formation. Furthermore, we obtained the three microflora form intestine: Escherichia coli, Salmonella typhi, and Enterobacter cloacae under aerobic culture condition, and confirmed that Escherichia coli nitroreductase nfsB involved in FNZ reduction for 7AF formation. In order to figure out intestinal micorflora nitroreductase in FNZ reductive metabolism, it therefore seemed worth to investigate the role of E. coli nitroreductases in the reduction of FNZ.
In this study, first we confirmed the activities of FNZ reductive reaction by cytosol proteins of intestinal microflora Escherichia coli, Salmonella typhi, and Enterobacter cloacae, and then made sure the nitroreductase activities in cytosol proteins and messenger RNA express levels in homologous genes nfsA, nfsB, and ydjA. Secondary, used the purified E. coli nitroreductases nfsA, nfsB, and ydjA for FNZ reductive analysis. Finally, we did the amino acid sequence point mutations in nitroreductase nfsB for kinetic studies, and combined the protein structures to investigate the effects of point mutation for enzyme activity.
The following results were obtained: (I) the activities of 7-AF formation, protein nfsB, and mRNA of homologous genes nfsA, nfsB, ydjA could be observed in all the cytosol proteins of three intestinal microflora; (II) the E. coli nitroreductases nfsA and nfsB both could metabolite FNZ for 7-AF formation, but not to ydjA; (III) point mutations of Asn-71-Ser, and Phe-124-Trp could enhance the enzyme catalytic constant (Kcat/Km).
Therefore, these results suggested that the nitroreductases of intestinal microflora were involved in FNZ reduction, and supposed that they may have selective specificity for substrates. In addition, the kinetic data of point mutations showed the point mutants Asn-71-Ser, and Phe-124-Trp could enhance the enzyme activity for 7AF formation. In future, we expect to find the high efficiency nitroreductase would be applied to antidote for flunitrazepam abuse.
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