LC-MS和基因序列分析介导的海洋来源放线菌抗耐药菌活性物质的发现研究

New antibiotics are necessary to treat microbial pathogens that are becoming increasingly resistant to available treatment. The actinomycetes have been important sources of antibiotics. However, the rate of discovery of novel antibiotics from traditional soil actinomycetes has significantly decreased in the recent years.To improve th probability of success for the discovery of novel antibiotics, new sources of actinomycetes and new screening and dereplication technologies are needed. Since marine environmental conditions are extremely different from terrestrial ones, it is surmised that marine actinomycetes have developed different metabolic pathways from their terrestrial counterparts and therefore, might produce diferent types of bioactive compounds. Indeed, marine actinomycetes are a prolific but untapped source for novel antibiotics. Depreplication of already known compounds and their potential analogues is a vital part of the discovery process that saves time and resources. Dereplication processes typically combines chromatographic and spectroscopic methods with database searching. PCR-based genetic screening that targets marker genes from biosynthetic gene clusters provides a bioinformatic assessment of secondary metabolite biosynthetic potential. The rapid identification of strains that possess the greatest potential to produce new secondary metabolites along with those that produce known compounds can be used to improve the process of natural product discovery by providing a method to prioritize strains for fermentation studies and chemical analysis. To screen novel antibiotics from marine actinomycetes, 1215 strains of marine actinomycets were isolated from the sea sediments collected in Bohai bay，Dailian in our prious work and over 40 strains showed siginificant antimicrobial activity against drug-resistant pathogen. This project is going to discover novel halogenated agents against antibiotic-resistant pathogen from marine rare actinomycetes by application of the improved dereplication strategy based on a combination of the genetic screening, antibiotic-resistant pathogen screening models, HPLC-DAD-ELSD-HRMS, as well as natural product database searching technology.

当前，细菌耐药性日益严重，研发新的抗生素迫在眉睫。放线菌是天然抗生素的重要来源，经过长期广泛的研究，从传统的土壤微放线菌中发现新抗生素已十分困难。海洋放线菌由于特殊的生存环境和独特的次生代谢途径，能够产生结构特异的次生代谢产物，是尚未充分发掘的新资源。面对大量已知化合物的重复发现，如何利用新资源、新技术、新模型筛选发现新的活性物质，成为天然药物研究中亟待解决的重要课题。本项目以抗耐药菌活性为导向，综合应用发酵产物LC-DAD-ELSD-MS分析、天然产物数据库检索以及菌株次级代谢途径相关基因序列分析等多种技术，准确快速地识别并排除已知化合物的干扰，进而筛选出能够产生新结构抗耐药菌活性物质的潜在菌株。在此基础上，遴选2～4株海洋稀有放线菌开展系统的分离纯化和构效关系研究，以期通过有效排重，快速发现新结构抗耐药菌活性物质，为新型抗生素研发打下基础。

当前，细菌耐药性日益严重，研发新的抗生素迫在眉睫。放线菌是天然抗生素的重要来源，经过长期广泛的研究，从传统的土壤微放线菌中发现新抗生素已十分困难。海洋放线菌由于特殊的生存环境和独特的次生代谢途径，能够产生结构特异的次生代谢产物，是尚未充分发掘的新资源。本研究借助生物信息学以及液质联用排重技术，旨在发现海洋放线菌产生的抗耐药菌活性新结构化合物。研究内容：对1000株海洋放线菌进行了抗耐药菌活性筛选，对其中300株海洋放线菌进行生物合成酶基因筛选，包括I型聚酮合酶、II型聚酮合酶、非核糖体肽合酶、dTDP-葡萄糖-4,6-脱水酶（dTDG）和FADH2依赖型卤化酶等。探查菌株的I、II型聚酮类、肽类、糖苷类和卤代化合物次级代谢能力。基于生物信息学分析和活性筛选结果，对50种菌株的次级产物进行了LC-MS分析和代谢产物排重。在此基础上，对7株重点菌株进行活性物质的分离纯化，化合物结构鉴定、活性评价与构效关系研究。研究结果：从7株海洋放线菌中分离得到了61个单体化合物，发现新结构天然产物20个（其中新骨架结构化合物8个），合成2个新结构衍生物。发现具有良好抗甲氧西林耐药金黄色葡萄球菌（MRSA）、万古霉素耐药屎肠球菌(VRE)活性的化合物18个（其中11个为新结构，MIC:0.25～16ug/mL），具有良好抗结核或耐药结核分枝杆菌的活性化合物9个（3个为新结构，MIC:1～2ug/mL），具有强抗肿瘤细胞活性化合物5个（2个为新结构，IC50：0.0025-1,000 nM）。共发表研究论文9篇，其中在天然产物和药物发现领域国际重要刊物Journal of Natural Products、RSC Advance等期刊上发表SCI研究论文5篇(1篇正在审稿中)。获专利授权1项，另有1项专利在申请中。通过本项研究，发现1个抗菌药物先导化合物和1个抗肿瘤药物先导化合物，为进一步抗耐药菌、抗结核和抗肿瘤药物研发奠定基础。