硝基苯二氮平(Nitrobenzodiazepine; NBDZ)是一種鎮靜催眠藥物，主要用於治療焦慮和睡眠問題。過量服用NBDZ可能會導致嚴重的神經影響，特別是藥物濫用或成癮者。不幸的是，臨床上對於NBDZ中毒的治療存在一些缺點，如處方劑量過高之不良影響和戒斷症狀等。本研究的目的在於發現和開發新的候選藥劑，以提供NBDZ中毒時治療上的其他選擇。
首先，我們探討大鼠腸道內容物對於NBDZ的硝基還原反應，並確認腸內菌硝基還原酶在還原途徑上的角色。在厭氧條件下，我們發現氟硝西泮(flunitrazepam; FZ)，一種NBDZ，可被空腸微菌叢顯著還原成尿液代謝物7-胺基氟硝西泮(7-aminoflunitrazepam; 7AFZ)。我們在大鼠空腸微菌叢中找到大腸桿菌第I型硝基還原酶NfsB (EC 1.5.1.34)，並證實其參與FZ的硝基還原反應。
其次，我們以經NfsB晶體結構所設計之各種突變體探討FZ的硝基還原反應，並確認其活體內效用。相較於野生型酵素，NfsB-N71S/F124W活性位置發生構形變化，包括Trp124和Phe70側鏈的翻轉，以及Ser71和FMN之間氫鍵網絡的擴展。因此，NfsB-N71S/F124W活性位置口袋明顯大於野生型，這可能與有氧和無氧條件下7AFZ產量的增加有關。小鼠抗催眠研究顯示，相較於溶液組，NfsB-N71S/F124W處理組的睡眠時間減少50%，並發現在24小時後小鼠血清中仍存在50%以上的該酵素。
再者，我們根據所解出的NfsB結構探討人類和腸內菌中含黃素還原酶(flavin-containing reductase)的蛋白質結構。我們發現這些還原酶的黃素區內催化位置相似，可能提供體內NBDZ還原一類似環境。
總而言之，我們首次發現NfsB-N71S/F124W可有效對抗FZ引發之催眠作用，並提供未來以NfsB及其類似蛋白進行合理化設計之分子基礎。

Nitrobenzodiazepine (NBDZ) is a sedative-hypnotic drug used in the treatment of anxiety and sleep problems. Overdose of NBDZ may cause severe neurological effects, especially for people in drug abuse or addiction. Unfortunately, the clinical intervention for NBDZ poisoning exists some disadvantages such as high prescribing dosage and withdraw symptom. The purpose of this study is to discover and develop new candidate that may provide alternative remedy for NBDZ poisoning.
First, we investigate NBDZ nitroreduction in rat enteric contents and characterize the role of enterobacterial nitroreductase in the reductive pathway. In the jejunal microflora, flunitrazepam (FZ), an NBDZ, was demonstrated to be significantly reduced to its urinary metabolite 7-aminoflunitrazepam (7AFZ) under anaerobic condition. Escherichia coli type I nitroreductase NfsB (EC 1.5.1.34) was found in rat jejunal microflora and demonstrated participating in FZ nitroreduction.
Second, we investigate FZ nitroreduction by various mutants of NfsB designed from the solved crystal structure and characterize the in vivo potency. Conformational changes occurred in the active site of NfsB-N71S/F124W in contrast to the wild-type enzyme, including the flipping of Trp124 and Phe70 side chains as well as the extended hydrogen bond network between Ser71 and FMN. Thus, the active site pocket of NfsB-N71S/F124W was significantly larger than that of the wild-type, which may be correlated with the increased 7AFZ production under both aerobic and anaerobic conditions. Mouse anti-hypnosis study showed that compared to the vehicle group, 50% decrease of sleeping time was observed in the NfsB-N71S/F124W group with over 50% of enzymes still remained in the mice sera after 24 hours.
Third, we investigate the protein structures of human and enterobacteria flavin-containing reductases based on the solved NfsB structure. These reductases share conserved catalytic site in the flavin domains that may provide a similar environment for the reduction of NBDZ in the body.
Taken together, we demonstrate that for the first time NfsB-N71S/F124W may be used as an effective therapeutic agent for FZ-induced hypnosis and provide the molecular basis for rational design of NfsB and the like in the future.

Abstract ……………………………………………………………………………….i
Abbreviations …………………………………………………………………………v
Chapter 1 Introduction ………………………………………………………………..1
1.1 Nitrobenzodiazepine ………………………………………………………….1
1.2 Metabolism and Toxicity of NBDZs …………………………………………3
1.2.1 Flunitrazepam
1.2.2 Clonazepam and Meclonazepam
1.2.3 Nitrazepam and Nimetazepam
1.3 Reductive Metabolism of NBDZ ……………………………………………9
1.3.1 Hepatic NADPH-Cytochrome P450 Reductase
1.3.2 Enterobacteria Reductase
1.4 Aim of Study ……………………………………………………….………12
Chapter 2 Materials and Methods ……………………………………….…………14
2.1 Chemicals and Reagents …………………………………………..………..14
2.2 Identification of Nitroreductase in Rat ……………………………..………14
2.2.1 Animals
2.2.2 Preparation of Microsomal Membrane Fractions of Rat Liver, Jejunum and Jejunal Microflora
2.2.3 PCR Amplification of Bacterial Genes
2.2.4 SDS-PAGE and Western Blot Analysis
2.2.5 Enterobacteriaceae Screening
2.3 Reductive Metabolism of NBDZ ………………………………………….19
2.3.1 Nitroreduction of NBDZ
2.3.2 HPLC Analysis
2.3.3 Chemical and Immunoinhibition
2.3.4 Kinetic Analysis
2.4 Protein Study of Bacterial Nitroreductase ………………………………….21
2.4.1 Cloning, Expression and Purification of Nitroreductase
2.4.2 UV/Visible Spectrophotometry
2.4.3 Crystallization, Data Collection and Processing
2.4.4 Structure Determination and Model Refinement
2.5 In Vivo Characterization of NfsB ………………………………………….25
2.5.1 Disk Assay for FZ Sensitivity
2.5.2 Sleep-time Study
2.5.3 Direct Competitive ELISA assay of NfsB
2.6 Statistical Analysis …………………………………………………………27
Chapter 3 Results …………………………………………………………………..28
3.1 Involvement of Enterobacteria Nitroreductase in the Metabolism of Nitrobenzodiazepine ………………………………………………………..28
3.1.1 Nitroreduction of FZ in Rat Liver, Jejunum and Jujenal Microflora
3.1.2 Identification of E. coli Nitroreductase NfsB in Rat Jejunal Microflora
3.1.3 Inhibition of NfsB Nitroreductase Activity in Rat Jejunal Microflora
3.1.4 Kinetic Study of NfsB on NBDZ
3.1.5 Nitroreductase from the Selected Coliform Enterobacteria
3.2 Modification of Escherichia coli Nitroreductase NfsB in Elevating Its Reductase Activity toward NBDZ …………………………………………33
3.2.1 Crystal Structure of NfsB
3.2.2 Prediction and Identification of NfsB Mutants with Increased 7AFZ
3.2.3 Enlarged Active Site of NfsB Mutants
3.2.4 Structural Analysis of NfsB-N71S/F124W
3.2.5 Substrate Binding of NfsB-N71S/F124W
3.2.6 In Vivo Potency of NfsB-N71S/F124W
3.3 Potential Flavin-containing Reductase in the Detoxification of NBDZ ……………………………………………..………………………...39
3.3.1 Investigation of Possible Nitroreductase in the Body
3.3.2 Structural Approach of Possible Nitroreductase in the Body
3.3.3 Reduction of FZ from E. coli Nitroreductase NfsA and YdjA
Chapter 4 Discussion ………………………………………………………………..43
4.1 NBDZ is Reduced by Enterobacterial Nitroreductase ………………………43
4.1.1 Nitroreductase Activity in Jejunal Microflora
4.1.2 Bacterial NfsB Nitroreductases
4.2 Enhancement of NfsB Reductase Activity toward FZ ………………………48
4.2.1 Modification of NfsB
4.2.2 Extended Active Site Pocket of NfsB
4.2.3 Improved Substrate Accessibility on NfsB
4.2.4 In Vivo Efficacy of NfsB
4.3 The Existing Nitroreductase in the Body ……………………………………54
4.3.1 Human Nitroreductase
4.3.2 Enterobacterial Nitroreductase
4.3.3 E. coli Nitroreductase
Reference …………………………………………………………………………….60
Figures ……………………………………………………………………………….77
Tables ……………………………………………………………………………….108
Appendix …………………………………………………………………………...116
Publications ……………………………………………..………………………… 118

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