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

陳怡帆(Chen, Yi Fan)

指導教授 ： 王雯靜

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2015年)

摘要

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。

關鍵字

幽門螺旋菌 ； 結核分枝桿菌 ； 莽草酸去氫酶

並列摘要

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.

並列關鍵字

Helicobacter pylori ； Mycobacterium tuberculosis ； shikimate dehydrogenase

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