Structure-guided identification of 7a-based analogues against shikimate dehydrogenase from Helicobacter pylori and Mycobacterium tuberculosis.

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研究生：	陳怡帆 Chen, Yi Fan
論文名稱：	Structure-guided identification of 7a-based analogues against shikimate dehydrogenase from Helicobacter pylori and Mycobacterium tuberculosis. 以7a衍生物針對幽門螺旋菌及結核分枝桿菌之莽草酸去氫酶蛋白質結構為標準之抑制劑研究
指導教授：	王雯靜 Wang, Wen Ching
口試委員:	許宗雄王慧菁張溫良姚振文
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：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	81
中文關鍵詞：	幽門螺旋菌、結核分枝桿菌、莽草酸去氫酶
外文關鍵詞：	Helicobacter pylori, Mycobacterium tuberculosis, shikimate dehydrogenase
相關次數：	點閱：4 下載：0
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胃幽門螺旋菌 (Helicobacter pylori, H.pylori)，其為一種致病性微生物，在全球人口當中有超過80%的人感染過此微生物，經過眾多醫學期刊指出幽門螺旋菌與腸胃道相關疾病之影響極大。另外，結核分枝桿菌 (Mycobacterium tuberulosis)，為結核病的病原菌體，在2014年世界衛生組織發表中指出，每年超過一百四十萬人口死於此病。然而在世界衛生組織報告當中得知，由於近幾年發現藥物濫用的問題，對於胃幽門螺旋菌以及結核分枝桿菌的治療上有極大的影響，且發現病人對於抗生素的抗藥性的人數比例增加，也衍生出許多抗藥性的臨床菌株。為此以尋找更有效的抑菌劑對抗幽門螺旋菌與結合分枝桿菌，我們將目標放在莽草酸代謝路徑(shikimate pathway)之第四個酵素：莽草酸去氫酶 (shikimate dehydrogenase, SDH)以作為探討的目標，其酵素反應是以NADPH作為輔酶，將反應物3-dehydroshikimate轉變為shikimate。
為針對胃幽門螺旋菌以及結核分枝桿菌找到更為有效的抑制劑。因此我們以先前實驗室所測出的有效的抑制劑7a當作化學結構指標，利用化學結構資料庫以及iGEMDOCKv2.1軟體去篩選出前78個可能有抑制效果的衍生物，經過酵素動力學結果，發現7m化合物具有抑制胃幽門螺旋菌以及結核分枝桿菌的莽草酸去氫酶的蛋白質活性 (IC50: 11.7 µM for HpSDH和32.2 µM for MtSDH)，而對於NADP+與Shikimate抑制型態分別為競爭型(competitive)與不競爭型(uncompetitve)。此外，我們也將其有效的抑制劑7m去針對胃幽門螺旋菌以及恥垢分枝桿菌做抑菌效果的體外測試(MBC value: 25 µM for H. pylori 和 4 mM for M. tuberculosis)。
研究最後利用Discovery Studio 3.5針對HpSDH-7m進行蛋白質結構模擬探討，其發現HpSDH中的Lys69、Glu70 和Ser129為重要的胺基酸交互作用位置，因此將此胺基酸進行點突變測試其與抑制劑酵素活性是否改變，發現到S129A和E70D蛋白質活性變差(IC50 = 17.8 for S129A and 22.89 µM for E70D)。因此，
最後結論得到針對SDH新型抑制劑(7m)的發展且延伸新的化學構型可否對蛋白活性抑制差異。

Helicobacter pylori (H. pylori) is associated with gastrointestinal diseases including duodenal ulcers and gastric adenocarcinoma. Over 80% of the population is infected with H. pylori. Traditional triple therapy has become less effective because of drug resistance. Mycobacterium tuberculosis (M. tuberculosis) causes over 1.4 million deaths per year estimating from The World Health Organization (WHO) and is difficult to treat which leads to multi-drug resistance. In order to develop new antimicrobial agents against H. pylori and M. tuberculosis, we target the shikimate biosynthesis pathway that consists of seven-step enzymatic processes in microbial and parasites but absent in mammals. In this study, we focus on the fourth enzyme, shikimate dehydrogenase (SDH), uses NADPH as a cofactor to catalyze 3-dehydroshikimate into shikimate. Dr. Wang’s prior works have identified a potent SDH inhibitor (7a) that blocked the growth of H. pylori.
In this work, we have screened for new inhibitors based on the 7a skeleton using a structure-guided approach. Of 7a similar compounds (n =78), 7m was identified to block both HpSDH and MtSDH (IC50: 11.7 µM for HpSDH and 32.2 µM for MtSDH). Kinetic analysis revealed that 7m displays uncompetitive and competitive inhibition pattern toward shikimate and NADP+, respectively. Moreover, 7m reduced H. pylori growth (MBC value: 25 µM). The HpSDH-7m complex model is built by Discovery Studio 3.0, which reveals that Lys69, Glu70 and Ser129 are crucial binding residues. Site-directed mutagenesis analysis revealed that S129A and E70D were less sensitive to 7m (IC50 = 17.8 for S129A and 22.89 µM for E70D). Together, our results suggest that the SDH inhibitor 7m is a new inhibitor for antibiotics development and provides a new skeleton for further antibiotic development.

中文摘要    i
Abstract    iii
誌謝    iv
Table content    ix
Figure content    x
   Introduction    1
1.    Helicobacter pylori (H. pylori)    1
1.1.    History, morphology and microbiology of Helicobacter pylori.    1
1.2.    The epidemiology and treatment of Helicobacter pylori.    2
2.    Mycobacterium tuberculosis (M. tuberculosis)    3
2.1.    History, morphology and microbiology of Mycobacterium tuberculosis.    3
2.2.    The epidemiology and treatment of Mycobacterium tuberculosis.    4
3.    Shikimate pathway    5
4.    Shikimate dehydrogenase    6
   The preliminary results and specific aim of this study.    7
1.    The preliminary results on enzyme HpSDH inhibitor – 7a.    7
2.    Purpose and specific aim    7
   Material and methods    9
1.    H. pylori – wild type strain culture.    9
2.    Minimum Bactericidal Concentration (MBC) Test toward H. pylori.    9
3.    Minimum Bactericidal Concentration (MBC) Test toward M. smegmatis.    10
4.    Wild type HpSDH (aroE) expression and purification.    10
5.    MtSDH (aroE) expression, purification and condensation.    12
6.    HpSDH and MtSDH relative enzyme activity determination.    13
7.    Enzyme kinetic parameters of HpSDH and MtSDH toward shikimate and NADP+.    14
8.    Determination of novel inhibitor IC50 value to HpSDH.    14
9.    Determination of novel inhibitor IC50 value to MtSDH.    15
10.    Determination of inhibition constant (Ki) toward shikimate.    15
11.    Determination of inhibition constant (Ki) toward NADP+.    15
12.    HpSDH cloning and site-directed mutagenesis.    16
13.    The docking virtual screening strategies.    17
   Results    18
1.    Virtual screening for 7a analogues    18
1.1.    Generating protein (HpSDH, 3PHI)-compound interaction from docking virtual screening.    18
1.2.    Simulation of MtSDH homology model.    19
1.3.    H. pylori shikimate dehydrogenase (HpSDH, aroE) and M. tuberculosis shikimate dehydrogenase (MtSDH, aroE) expression and purification.    19
1.4.    NCI compounds screening toward wild type HpSDH and MtSDH.    20
2.    Analysis of enzyme kinetic activity.    21
2.1.    IC50 determination of efficient compounds toward HpSDH.    21
2.2.    IC50 determination of efficient compounds toward MtSDH.    21
2.3.    Enzyme kinetic parameters determination.    21
2.4.    Inhibition mode and inhibition constant of 7m toward shikimate of HpSDH.    22
2.5.    Inhibition mode and inhibition constant of 7m toward NADP+ of HpSDH.    22
3.    Characterization of anti-H. pylori and anti-M. tuberculosis activity.    23
3.1.    The MBC values of H. pylori.    23
3.2.    The MBC values of Mycobacterium smegmatis.    23
3.3.    Synergistic effect of effective compounds toward antibiotic resistant strains of H. pylori.    23
4.    Structure analysis relationship (SAR) analysis.    25
4.1.    To predict the inhibitor binding residues in HpSDH (3PHI) cavity.    25
4.2.    To establish the pharmacophore models of target-compounds.    25
4.3.    Expression and purification of the site-directed mutagenesis of H. pylori shikimate dehydrogenase (HpSDH, aroE).    26
4.4.    Relative enzyme activity of HpSDH and site-directed mutagenesis HpSDH.    26
4.5.    The IC50 values of S129A and E70D HpSDH treated with the compound 7m.    26
   Discussion    28
1.    Investigation of the effective inhibitor’s structure and substituent affect the inhibited activity toward HpSDH.    28
2.    Investigation of the potential inhibitor structure and substituent affect the inhibited activity against H. pylori.    30
3.    Enzyme kinetic analysis.    31
4.    Investigation of the pharmacophore model 7a, 7m, B-85 and B-84 interacted with amino acid in the HpSDH (3PHI) cavity.    32
4.1.    Hydrophobic interaction conformation.    32
4.2.    Hydrogen bond interaction conformation.    32
4.3.    Ionic bond conformation    33
5.    The site-directed mutagenesis HpSDHs were treated with 7m of efficient effects. 123    33
6.    Discussion of enzyme kinetic activity and microbial inhibition of MtSDH. 12344568    34
   Conclusion and future direction    35
   Reference    36

                                

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