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| 基于OSMAC-NMR-PCA 天然产物排重策略的新颖抗结核活性成分研究 | |
| 其他题名 | Chemical investigation of new anti-tuberculosis metabolites from microorganisms by applying the OSMAC-NMR-PCA dereplication strategy |
| 刘苗苗 | |
| 出版年 | 2017 |
| 学位类型 | 博士 |
| 导师 | 董治宝 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 摘 要 结核病(Tuberculosis，TB)是一种世界范围内的流行性致死疾病，每年新感染病人高达 100 万人。然而，多药耐药型结核(multi-drug resistant TB，MDR-TB) 的出现和流行严重地阻碍了结核的控制和治疗进程。目前仅有极少数的药物能用于MDR-TB 的治疗，所以我们的研究目的是从特殊生境微生物中发现能够对结核治疗， 特别是能用于耐药型结核控制的新型活性次级代谢产物。本论文从目前广泛应用的 不同次级代谢产物发现方法出发，构建了一个结合单菌多次级代谢产物(one strain many compounds，OSMAC)、NMR 指纹图谱以及主成分分析(principal component analysis， PCA)等多重策略的复合手段作为有效的从微生物中鉴定抗结 核代谢产物的研究手段。 本论文的研究基础是利用实验室构建的以牛型分枝杆菌 Mycobacteria bovisBCG 为细菌模型的高通量模型进行的活性筛选。首先，将 654 株放线菌进行天然产 物构建共形成 2562 个粗 物，对粗 物进行抗 BCG 活性筛选共获得 415 个活性样 品，经过对活性菌株的多方位评价最终挑选 14 株极端生境微生物进行后续研究。 论文以激活微生物沉默基因表达为目的，建立了一个有效的包含 OSMAC、NMR 指纹图谱和 PCA 等方法的针对微生物次级代谢产物的综合研究策略，并对 13株极端生境放线菌进行了相关研究。最终从 4160 个 fractions 中的 37 个潜力 fractions选择了 3 个最强活性的流份进行了后续研究，共得到 20 个靶标化合物。其中从沙 漠放线菌 LS120194 的相应 fraction 中获得一个具有罕见结构骨架的新化合物，且其 对 BCG 的抑制活性甚至高于阳性对照异烟肼，MIC 值为 0.14 μM，显示了该化合物 成为新一代抗结核候选药物的极高潜力。 在不同培养基条件的 OSMAC 策略指导下，本论文还对一株植物内生菌 Y3111进行了化学成分研究。最终从该菌种获得 6 个 pluramycin 类的蒽醌化合物，其中 4个化合物为新化合物。新化合物的结构鉴定是基于对其 NMR 和 MS 数据的分析完 成的，对化合物的活性测试发现其中 3 个化合物具有不同程度的抑菌活性。 对获得的 26 个化合物进行的 Rule of five 和 ChemGPS-NP 物理化学性质评价结 果显示大多数的化合物都具有优秀的成药性，且与目前的抗 TB 药物处于相似的理 化空间内。其中在物理化学性质方面极具优势的化合物将可能成为后续的抗结核药物研发重点。 |
| 英文摘要 | ABSTRACTTuberculosis (TB) is the leading cause of death from infectious diseases in the world, affecting more than ten million patients each year. However, multi-drug resistant (MDR- TB) threatens progress achieved in TB care and control, and there are few drugs available to treat MDR-TB. Our overall aim was to identify anti-TB natural products from microbes sourced from unique environments. This thesis presents efforts to achieve an effective approach to identify anti-TB microbial natural products with the combination of one strain many compounds (OSMAC) strategy, NMR fingerprint and principal component analysis. The thesis describes a cell-based HTS platform for discovering small molecules as well as crude extracts showing growth inhibition to Mycobacteria bovis Bacillus Calmette–Guérin (BCG). With the aim to identify potent anti-TB natural products from actinomycetes, a microbial extract library was generated from 654 strains and 2562 crude extracts were evaluated for their activity. 415 actives were identified as the Hit Library that was the basis for this TB-focus project.With the purpose of inducing the expression of the silent gene clusters in microbes, this thesis presents a method with combination of OSMAC, NMR fingerprint and PCA as an effective strategy for the discovery of novel anti-TB microbial natural products. Applying the potential strategy on 13 strains, 4160 fractions were generated. NMR fingerprinting and PCA of the data set revealed 37 outliers, 3 of which were selected for scale-up investigations and 20 natural products were identified. One new compound with a novel structure was identified from the desert actinomycete LS120194 under the effect of a specific medium. This compound showed extreme strong activity in terms of BCG inhibition with a MIC value of 0.14 μM, indicating its high potential being a new anti-TB drug candidate.We also demonstrate the chemical and biological investigation of an endophyte Y3111 under the guidance of OSMAC strategy. Chemical investigation of the biota sample resulted in the identification of 6 pluramycin- type compounds, of which four were new compounds. The structures of the isolated compounds were determined by the analysis of NMR and MS data. Anti-BCG screening suggested that 3 compounds had moderate effects on the inhibition of the bacteria.The thesis concluded with an analysis of the physico-chemical properties and ChemGPS analysis of the 26 isolated microbial natural products. Most of the compounds identified in the thesis had druggable properties and clustered in a chemical space with current drugs. The highlighted compounds can be potentially used as lead compounds in future anti-TB drug discovery. |
| 中文关键词 | 微生物天然产物 ; 化学排重 ; OSMAC-NMR-PCA ; 抗结核活性 |
| 英文关键词 | Microbial natural products Chemical dereplication OSMAC-NMR-PCA Anti-tuberculosis activity |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 生物化学与分子生物学 |
| 来源机构 | 中国科学院微生物研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287926 |
| 推荐引用方式 GB/T 7714 | 刘苗苗. 基于OSMAC-NMR-PCA 天然产物排重策略的新颖抗结核活性成分研究[D]. 中国科学院大学,2017. |
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