【作者】 李书芬；

【导师】 武临专；

【作者基本信息】 北京协和医学院 ， 微生物与生化药学， 2014， 博士

【摘要】 微生物次级代谢产物具有多种多样的化学结构与生物活性,且类药性强,为创新药物的重要来源。迄今,已知大多数微生物药物来自放线菌,其中又以链霉菌为主。因此,对链霉菌产生的次级代谢产物进行挖掘是发现创新药物研究的一个重要途径。本论文分为两部分,第一部分为链霉菌产生的苯安莎类抗生素(GDM与除草霉素A)新组分的发现与鉴定,第二部分为链霉菌产生的新抗生素或新组分发现探索。GDM是由吸水链霉菌产生的苯安莎类抗生素,它具有显著的抗肿瘤活性,但是由于其严重的肝毒性及较差的水溶性,使其在抗癌药物研发中只能作为先导化合物；寻找高水溶性、低肝毒性的GDM衍生物成为开发此类药物的研究热点。在通过化学半合成得到的数百个GDM衍生物中,17-AAG和17-DMAG已进入临床试验,用于多种肿瘤治疗。微生物次级代谢产物结构的复杂性,往往导致次级代谢产物在生物合成过程中产生一组结构相似的类似物,此即抗生素的多组分现象。近年来,从不同的GDM产生菌中,也陆续有一些天然GDM类似物的发现报道,其中有些类似物在水溶性或抗肿瘤细胞活性方面相对于GDM有所改善或提高,显示了较好的抗肿瘤药物开发潜能。吸水链霉菌17997(Streptomyces hygroscopicus17997)是中国医学科学院医药生物技术研究所分离鉴定的一株GDM产生菌。我们通过TLC和LC-DAD-MS法对该菌株发酵产物进行跟踪分析,发现一个GDM新组分,经分离纯化后,利用NMR、Mosher法及ECD等确定其结构为19-[(1’-S,4’R)-4’-羟基-1’-甲氧基-2’-氧代戊基]格尔德霉素(化合物713)。此外,我们还在吸水链霉菌17997的gdmP基因阻断变株中发现并鉴定了化合物713的4,5双氢形式—19-[(1’S,4’R)-4’-羟基-1’-甲氧基-2’-氧代戊基]-4,5双氢格尔德霉素(化合物715)。与GDM相比,化合物713和715的抗肿瘤细胞活性分别降低了约50倍和80倍,但水溶性提高了数千倍。除草霉素A是一个在化学结构方面与GDM非常类似的苯安莎类抗生素,也是由吸水链霉菌产生。在抗肿瘤药物研究中,除草霉素A及其类似物也受到一定的重视。我们通过在培养基中补充甲硫氨酸,从除草霉素A产生菌—吸水链霉菌N02Z-0421(S. hygroscopicus N02Z-0421)中,分离纯化、NMR鉴定了一个除草霉素A新组分—17,19-二甲硫基除草霉素A。初步的抗肿瘤细胞活性测定显示,该新组分与除草霉素A基本相当。此外,17,19-二甲硫基除草霉素A的获得,也为本实验室阐明19-S-甲基格尔德霉素中的甲硫基化机制以及甲硫基的来源(甲硫氨酸分解代谢产生甲硫醇,甲硫醇与格尔德霉素经迈克尔加成反应生成19-S-甲基格尔德霉素),提供了重要的佐证。吸水链霉菌17997具有丰富的次级代谢产物合成能力,它不仅产生GDM及其类似物,还产生一些有待鉴定的抗革兰阳性菌化合物。在我们对链霉菌产生的新抗生素或新组分发现探索研究中,我们首先对该菌株产生的抗革兰阳性菌化合物进行了研究：从该菌株的次级代谢产物中,鉴定了洋橄榄叶素。洋橄榄叶素具有抗菌、抗肿瘤和抗原虫等多种生物学活性。在鉴定洋橄榄叶素的过程中,我们建立了一种快速鉴定洋橄榄叶素及其产生菌的方法,主要包括TLC硅胶板分离和NaOH溶液喷雾显色、HPLC和LC-DAD-MS分析,以及洋橄榄叶素生物合成保守基因的PCR检测与序列分析；其中,NaOH溶液喷雾显色为首次报道。在链霉菌新次级代谢产物的发现研究中,洋橄榄叶素是需要早期鉴别或排除的一个假阳性化合物。我们已经成功地将NaOH溶液喷雾显色用于洋橄榄叶素的早期快速鉴别。利用中国药用微生物菌种保藏中心丰富的菌种资源,我们在对多株从我国土壤中分离的链霉菌菌株进行初步分析的基础上,选出2株具有显著抗菌活性、产生丰富次级代谢产物的链霉菌菌株,进行新抗生素或新组分的挖掘探索。首先,链霉菌CPCC200002.我们通过TLC及LC-DAD-MS分析法,对该菌株产生的5个具有抗菌活性的次级代谢产物进行了鉴定,分别为硫藤黄菌素、硫藤黄菌素氧化物、金丝菌素及丁酰-pyrrothine或异丁酰-pyrrothine(002-1～002-5)。这5个化合物均属于dithiolopyrrolone类抗生素,该类抗生素最初以其显著的抗微生物活性受到关注,后来研究发现它们还具有较强的抗肿瘤活性。最近,发现新结构的该类化合物受到关注,以及阐明了该类抗生素生物合成与半胱氨酸紧密相关。硫藤黄菌素类化合物是两个半胱氨酸分子的缩合产物。作为半胱氨酸的类似物,我们在CPCC200002的培养过程中添加高半胱氨酸,希望硫藤黄菌素的生物合成酶系能够识别并利用高半胱氨酸,产生新的硫藤黄菌素类似物。遗憾的是,我们没有检测到新化合物,只是看到金丝菌素等上述5个化合物生物合成水平的提高。其次,链霉菌CPCC203577.我们通过TLC及LC-DAD-MS等方法对该菌株产生的具有显著抗菌活性的蓝色化合物进行了鉴定,为衣草花青菌素及其类似物(1032-A-F)。衣草花青菌素属于吩嗪类抗生素。氯苯吩嗪是已知的临床应用的抗麻风杆菌药物；天然或化学合成的一些吩嗪类抗生素是抗炎或麻风杆菌的药物开发候选物。经天然产物化合物库、SciFinder数据库及文献检索排重后,确定衣草花青菌素类似物1032-C、1032-D和1032-F为衣草花青菌素新组分,相关化合物的分离纯化和结构鉴定等正在进行中。更多还原

【Abstract】 Microbial secondary metabolites with various chemical structures and biological activities are important sources of new drugs. So far, most known microbial drugs come from actinomycetes, particularly from Streptomyces. New secondary metabolites from Streptomyces remain to be a significant origin for new drug development. This dissertation is divided into two parts. The first part describes the identification of three novel benzoquinone ansamycins from Streptomyces hygroscopicus17997or Streptomyces hygroscopicus N02Z-0421. The second part involves the exploration of novel secondary metabolites from strains of the genus Streptomyces.Geldanamycin (GDM) is a benzoquinone ansamycin produced by Streptomyces hygroscopicus. Although with exceptional potency against cancer cells, GDM acts only as a lead compound in anticancer drug development because of its severe hepatoxicity and poor water solubility. GDM analogues with increased water solubility and low hepatoxicity are promising candidates in anticancer drug development. Of the hundreds of GDM analogues obtained by semi-synthesis, two compounds,17-allylamino-17-demethoxygeldanamycin (17-AAG) and7-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), entered into clinical trials for the treatment of solid tumors.Secondary metabolites from actinomycetes are usually produced as groups of molecules with similar structures. This phenomenon is also known as multi-components of antibiotics. A number of novel GDM analogues from different GDM producers were reported recently. Some of these GDM analogues displayed higher water solubility or in vitro anti-tumor cell activity than GDM, showing the potential for further antitumor drug development.Streptomyces hygroscopicus17997was a soil isolate from Institute of Medicinal Biotechnology, China Academy Of Medical Sciences, and identified as a GDM producer. Its secondary metabolites were analyzed carefully by us. As a result of our detailed analysis or comparison of silica gel TLC and LC-MS profiles of GDM analogues produced by Streptomyces hygroscopicus17997, a novel natural GDM analogue was discovered. The structure of the analogue was determined to be19-[(1’S,4’R)-4’-hydroxy-1’-methoxy-2’-oxopentyl]-geldanamycin (compound713) by extensive spectroscopic data analysis, including2D NMR, modified Mosher’s method, and electronic circular dichroism (ECD). The4,5-dihydro form of the analogue,19-[(1’S,4’R)-4’-hydroxy-l’-methoxy-2’-oxopentyl]-4,5-dihydrogeldanamycin (compound715), was then discovered and identified in the gdmP gene disruption mutant of Streptomyces hygroscopicus17997. Compared to GDM, compounds713and715showed much increased water solubility but decreased cytotoxicity against HepG2cells.Streptomyces hygroscopicus N02Z-0421was a producer of herbimycin A, another benzoquinone ansamycin that shares a very similar structure to GDM. Herbimycin A possesses also severe cytotoxicity against cancer cells, and like GDM, is a lead compound in anticancer drug development. To obtain new herbimycin A analogue(s), we supplemented Met in the culture medium for S. hygroscopicus N02Z-0421. A red compound appeared in the silica gel TLC of secondary metabolites of the strain. The compound was then identified by us as a new herbimycin A analogue, whose structure was determined as17,19-dimethylthioherbimycin A, with methylthiol group(s) in the molecule as expected. Preliminary assays indicated that17,19-dimethylthioherbimycin A showed a potent cytotoxic activity against HepG2cancer cells, with an IC50value of18.7μM (for herbimycin A,14.0μM).The identification of17,19-dimethylthioherbimycin A from Streptomyces hygroscopicus N02Z-0421in culture medium supplemented with methionine (Met) provides an additional strong evidence to support the synthetic mechanism and the origin of methylthiol group in19-S-methylgeldanamycin, a new GDM analogue discovered earlier in our laboratory from Streptomyces hygroscopicus17997. The synthetic mechanism and the origin of methylthiol group in19-S-methylgeldanamycin was clarified recently in our laboratory as Met being first metabolized to methanethiol, and the latter molecule then acting as a nucleophilic agent in its attacking GDM to form19-S-methylgeldanamycin.Streptomyces hygroscopicus17997produced not only GDM and its analogues, but also some un-identified secondary metabolites with antibacterial activities. One compound with anti-G+bacterial activity was determined by us to be elaiophylin. Elaiophylin is a macrolide antibiotic with many biological activities such as antibacteria, antitumor and antiprotozoal, and often appears in the secondary metabolites of Streptomyces hygroscopicus. In our identification of elaiophylin, a fast procedure was established, which consisted of silica gel TLC followed by color reaction with NaOH and a simple and fast genetic analysis of biosynthetic potential of elaiophylin by PCR, etc. It is worthy to note that elaiophylin in silica gel TLC turned light red upon spraying NaOH, which is a feature of elaiophylin never reported before. This feature has been used successfully to fast identify elaiophylin producers several times in our laboratory.As mentioned in the beginning of the abstract, Streptomyces produces abundant secondary metabolites with various biological activities. To obtain new secondary metabolites from Streptomyces, we made an initial inspection of a dozen strains of Streptomyces from China Pharmaceutical Culture Collection (CPCC). Two strains with antibacterial activities and diversified patterns of secondary metabolites were chosen by us for further investigation of new compounds with biological activities.Streptomyces sp. CPCC200002is a soil isolate with very strong antibacterial activities collected from Guangdong Province, China. The EtOAc extracts of the culture of this strain was fractionated by silica gel TLC and then analyzed by LC-DAD-MS. A group of five similar compounds with antibacterial activity were identified as thiolutin, thiolutin oxide, aureothricin, butyryl-pyrrothine and isobutyryl-pyrrothine. All of them belonged to dithiolopyrrolone antibiotics. Dithiolopyrrolone antibiotics were known for their very strong antimicrobial activities, and new interests about these compounds aroused due to their anticancer and antitumor properties.The biosynthesis of dithiolopyrrolone antibiotics is closely related to cysteines, and each of dithiolopyrrolone antibiotics comes from the condensation of two cysteines. As a close analogue of cysteine, homocysteine was supplemented in the culture of Streptomyces sp. CPCC200002, with the expectation of new condensation product(s) of dithiolopyrrolone antibiotics produced. Higher production levels of the above identified five compounds were observed, but new compounds with homocysteines or homocysteine-cysteine hybrid were not detected by us.Streptomyces sp. CPCC203577is a soil isolate with significant antibacterial activities collected from Yunnan Province, China. The EtOAc extracts of this strain were analyzed by silica gel TLC and LC-DAD-MS, which led us to identifiy lavanducyanin. Lavanducyanin belongs to phenazines antibiotics, and natural and synthetic phenazines derivatives are promising candidates for drug development.A close analysis of minor components from Streptomyces sp. CPCC203577led us to discover four lavanducyanin analogues. Of them, one was deduced to be WS-9659B, which was reported by Nakayama O, the other three should be new lavanducyanin analogues after our search in SciFinder database. The separation, purification and structure determination of these new lavanducyanin analogues are still in progress.更多还原