巴比妥酸衍生物之設計與合成應用於抗胃幽門螺旋菌藥物與莽草酸去氫酶抑制劑之開發

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研究生：	周仕堇 Chou, Shih-Ching
論文名稱：	巴比妥酸衍生物之設計與合成應用於抗胃幽門螺旋菌藥物與莽草酸去氫酶抑制劑之開發 Design, synthesis and biological evaluation of barbiturate-conjugated derivatives as anti-Helicobacter pylori toward the shikimate pathway
指導教授：	王雯靜 Wang, Wen-Ching
口試委員:	張大慈 Chang, Dah-Tsyr 許銘華 Hsu, Ming-Hua
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	142
中文關鍵詞：	胃幽門螺旋菌、莽草酸路徑、莽草酸去氫酶、藥物合成、巴比妥酸、分子模擬
外文關鍵詞：	Helicobacter pylori, shikimate pathway, shikimate dehydrogenase, chemical synthesis, barbituric acid, molecule docking
相關次數：	點閱：158 下載：0
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胃幽門螺旋桿菌 (Helicobacter pylori, H. pylori)，和許多腸胃道相關疾病有密不可分的關聯，如：胃潰瘍和胃腺癌的發生。1994年時國際癌症研究署(International Agency for Research on Cancer, IARC)和世界衛生組織(World health Organization, WHO)更宣布將H. pylori定為一級致癌的微生物，然而具有抗藥性的H. pylori臨床菌株比例隨著藥物濫用逐年激增。為了尋找更有效的抑菌劑，我們將目標放在shikimate pathway當中的一個酵素，莽草酸去氫酶(shikimate dehydrogenase, E.C. 1.1.1.25)，其為shikimate pathway中第四個酵素，以菸草醯胺腺嘌呤二核苷酸磷酸鹽(Nicotiamide adenine dinucleotide phosphate, NADPH)為輔，催化3-dehydroshikimate轉變為shikimate的過程。
本研究以先前篩出之藥物7a為基礎，設計一系列共66種不同的巴比多酸衍生物 (barbiturate derivatives)。實驗結果發現7b、7d、7e、7g、7i 與25a等藥物具有抑制H. pylori生長的活性，在對抗26695和臨床分離菌株(v574, v633, v1086, v1254, v1267, v1354 與 v2311)的生長測試(MBC assay)中，具有抑制效果(4 mg/L ~10 mg/L)。另外在針對胃幽門螺旋菌莽草酸去氫酶(H. pylori shikimate dehydrogenase, HpSDH)的酵素活性抑制測試亦呈現相同的趨勢。本實驗求得7b、7e、7i、17、25a等藥物對HpSDH的IC50 (10~30 μM) 和7e、7i、17、25a在較高濃度下對NADP+抑制型態為競爭型，常數(Ki)介於2~7 μM之間；而對shikimate則是non-competitive 和uncompetitive為主，Ki值也較大(15~25 μM)。
本研究進一步藉由生物資訊分析與分子模擬的方式探討分子結構與抑制活性之間的關聯(Structureactivity relationship, SAR)。利用Discovery Studio 3.5來建立有效分子和HpSDH 之間的docking 模型模擬並探討重要的鍵結和氨基酸。本研究發現能否和結合位置中兩個氨基酸(Lys69 和Arg71)形成cation-π對於抑制活性可能有重要的影響。最後我們預測並建立可能的分子最佳化模型。

Persistent infection of Helicabacter pylori (H. pylori) is associated with intestinal disease including duodenal ulcers and gastric adenocarcinoma. International Agency for Research on Cancer and WHO had claimed H. pylori as a group 1 carcinogen of human being in 1994. Resistant clinical H. pylori isolates have rapidly increased due to a high use of antibiotic reagents. In an aim to search for new antibacterial agents against resistant isolates, we have targeted shikimate dehydrogenase (SDH, E.C. 1.1.1.25) that catalyzes 3-dehydroshikimate into shikimate, using Nicotiamide adenine dinucleotide phosphate as cofactor.
In this study, we reported a structure-based approach to identify inhibitors containing barbiturate moiety against H. pylori shikimate dehydrogenase (HpSDH). Series of barbiturate-conjugated compounds have been synthesized. Results from bactericidal activity against reference strain (26695) as well as different clinically isolated strains (v574, v633, v1086, v1254, v1267, v1354 and v2311) showed that compounds 7b, 7d, 7e, 7g, 7h and 25a displayed bactericidal activity (4.0 mg/L 10 mg/L). In parallel, these compounds exhibited inhibitory effects toward HpSDH, 7b, 7e, 7i, 17 and 25a with IC50 values of 10 to 30 μM). Ki values and inhibition types of 7e, 7i, 17 and 25a were determined respective. Effective compounds showed mixed-inhibition toward NADPH with Ki values from 2 to 7 μM; while these compounds showed non-competitive or
III
uncompetitive toward shikimate with Ki values from 15 to 25 μM. HpSDH-inhibitor docking models are built by Discovery Studio 3.5 (DS 3.5), which reveals crucial binding residues Lys69 and Arg71. Results from this study provide a basis for new antibacterial development targeting HpSDH.

中 文 摘 要.................................................................................................................. I
Abstract .............................................................................................................................. II
致 謝 ............................................................................................................................... IV
Key words and abbreviations ...................................................................................... XIV
Introduction ................................................................................................................ 1
1 Helicobacter pylori. (H. pylori). ......................................................................... 1
1.1 History of Helicobacter pylori ................................................................. 1
1.2 Morphology and Microbiology of Helicobacter pylori ........................... 1
1.3 Epidemiology and disease association of Helicobacter pylori ................ 2
1.4 Treatments of Helicobacter pylori ........................................................... 4
2 Shikimate pathway .............................................................................................. 5
3 Shikimate Dehydrogenase .................................................................................. 6
4 Shikimate Kinase ................................................................................................ 7
5 Barbituric acid and barbiturate derivatives ......................................................... 8
Preliminary results and specific aims of this study .................................................. 9
1 Preliminary results of H. pylori study ................................................................. 9
2 Preliminary results of shikimate dehydrogenase ................................................ 9
3 Purposes and specific aims ............................................................................... 10
Chemistry ................................................................................................................... 11
1 Synthesis of barbiturate-conjugated derivatives ............................................... 11
2 Synthesis of hydantoin-conjugated derivatives ................................................ 13
3 Synthesis of Bis- barbiturate-conjugated derivatives ........................................ 14
4 Synthesis of complicate barbiturate-conjugated derivatives ............................. 16
Materials and experimental section ........................................................................ 18
1 General procedure for compound synthesis ...................................................... 18
2 General procedure A for the synthesis of barbiturate- conjugated derivatives . 18
2.1 Barbiturate- conjugated derivatives (Scheme 1) .................................... 19
2.2 Hydantoin- conjugated derivatives (Scheme 2) ..................................... 19
2.3 Bis- barbiturate- conjugated derivatives (Schemes 3 and 4) .................. 19
2.4 (E)-N-(4-(3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) prop-1- en-1- yl)phenyl) benzamide (Scheme 5) ............................................................ 19
2.5 N-phenyl-4-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) methyl) benzamide (Scheme 6) ....................................................................................... 20
2.6 1,3-dimethyl-5-(4-(2-(piperidin-1-yl)ethoxy)benzylidene)
VIII
pyrimidine-2,4,6 (1H,3H,5H)-trione (Scheme 7) ............................................... 20
2.7 (E)-1,3-diallyl-5-(3-phenylallylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (Scheme 8) .......................................................................................................... 21
2.8 (E)-1,3-diallyl-5-(3-(4-methoxyphenyl)allylidene)pyrimidine-2,4,6 (1H,3H,5H) -trione (Scheme 8) .......................................................................... 21
3 H. pylori strains and culture .............................................................................. 22
4 Minimum bactericidal concentration test (MBC Test) ..................................... 22
5 Synergistic combination .................................................................................... 22
6 HpSDH (aroE) expression, purification and condensation. ............................. 23
7 HpSDH relative enzyme activity determination (aroE assay) .......................... 24
8 Enzyme kinetic parameters of clinical isolate HpSDHs………………….......25
9 Sequences multi-alignment and Circular dichroism spectra ............................. 26
10 Determination of selected compounds IC50 toward HpSDH. ......................... 26
11 Inhibition constant (Ki) determination. ........................................................... 27
11.1 Inhibitor constant (Ki) determination toward shikimic acid .................. 27
11.2 Inhibitor constant (Ki) determination toward NADP+ ........................... 27
12 Molecular modeling and structure-activity relationship (SAR) analysis ........ 28
12.1 Inhibitor binding sites prediction of 26695 HpSDH ............................ 28
12.2 Preparation of the target cavity. ........................................................... 28
12.3 Molecular docking for structure-activity relationship analysis ........... 29
12.4 Compound optimization ....................................................................... 29
13 HpSK (aroK) expression, purification and condensation ............................... 30
14 Relative HpSK activity determination (aroK assay). ...................................... 31
Result .......................................................................................................................... 32
1 Chemical synthesis of barbiturate derivatives .................................................. 32
2 MBC value determination of 26695 and clinical isolated strains of H. pylori . 32
3 Synergistic effect of effective compounds toward wild type H. pylori ............ 34
4 H.pylori shikimate dehydrogenase (HpSDH) expresstion and purification ..... 35
5 Relative activity of 26695- HpSDH and clinical variant strains HpSDH ......... 35
6 Sequence alignment and secondary structure determination ............................ 35
7 Enzymatic kinetics parameters determination .................................................. 36
8 Potential inhibitor screening toward different HpSDHs ................................... 36
9 IC50 determination of effective compounds toward HpSDHs .......................... 37
10 Inhibition constant determination toward shikimate and NADP+ .................. 37
10.1 Inhibition constants and inhibition type toward NADP+ ..................... 38
10.2 Inhibition constants and inhibition type toward shikimate. ................. 38
11 3D compound binding pockets prediction ...................................................... 38
12 Molecular docking in the predicted HpSDH cavity ........................................ 39
IX
13 Lead optimization of compounds .................................................................... 40
14 Results for H. pylori shikimate kinase (HpSK) activity study........................ 42
14.1 HpSK expresstion and purification ...................................................... 42
14.2 HpSK preliminary screening (100 μM) ............................................... 42
Discussion and conclusion ........................................................................................ 43
1 Compounds design and synthesis ..................................................................... 43
2 Essential core structure and substituent affect inhibition activity against H.pylori… .................................................................................................................. 44
3 Secondary structure analysis by circular dichroism (CD) ................................ 45
4 Essential core structure and substituent affect the inhibition activity against HpSDH…. .................................................................................................................. 46
5 Structure-activity relationship (SAR) analysis ................................................. 47
6 Enzymatic kinetic analysis ................................................................................ 48
7 Interaction with amino acid in the cavity .......................................................... 49
7.1 Hydrogen bonds formation .................................................................... 49
7.2 Cation- π interaction formation .............................................................. 49
8 Lead optimizations of compounds .................................................................... 51
9 Discussion of HpSK screening ......................................................................... 52
10 Conclusions and Future works ........................................................................ 53
TABLES ............................................................................................................................ 54
FIGURES .......................................................................................................................... 67
---------------------------------------------------------------------------------------------------------------------
Appendix ........................................................................................................................... 97
中 文 摘 要................................................................................................................ 98
Abstract ............................................................................................................................. 99
Introduction ............................................................................................................ 100
1 Global burden of the cancer ............................................................................ 100
2 Common features of the cancer ...................................................................... 100
3 Strategies for cancer treatments ...................................................................... 102
4 Barbiturate derivatives as potential anti- cancer agents .................................. 103
5 Preliminary result of anticancer screening ...................................................... 104
Material and Methods ........................................................................................... 105
1 Chemicals and reagents ................................................................................... 105
2 Cancer cell lines and cell culture .................................................................... 105
3 Tetrazolium dye methylthiotetrazole assay (MTT assay) ............................... 105
X
4 IC50 determination ........................................................................................... 106
Results ..................................................................................................................... 107
1 Preliminary screening of anticancer activity. .................................................. 107
2 IC50 determination of effective compounds. ................................................... 107
Disscussions .......................................................................... 錯誤! 尚未定義書籤。
1 Preliminary screening of six cancer cell lines ................................................. 109
2 IC50 and structure- activity relationship of effective compounds ................... 109
Conclusion and future direction ............................................................................ 111
Appendix tables .............................................................................................................. 112
Appendix figures ............................................................................................................ 116
Reference........................................................................................................................ 121
Supporting Informations ...............................................................................................128
                                

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