海洋微生物沉默基因编码的新型抗阴性耐药菌先导分子的发现

The spread of multi-drug and extensive drug-resistance among Gram-negative bacteria has emerged as one of the most serious global public health threats. Infections caused by these pathogens pose severe challenges for clinical therapeutics due to the paucity of available antibiotics. It is urgently necessary to develop novel therapeutics against drug-resistant Gram-negative bacteria. Genome bioinformatic analyses revealled that microorganism generally has the genetic capacity to produce dozens of distinct secondary metabolites. Many biosynthetic genes are silent in which the secondary metabolites are not or expresssed at low levels under laboratory growth condition. The products encoded by the silent biosynthetic genes are import sources for novel molecules discovery. Acitvating the silent gene clusters attracted increasing attention in the field of natural products discovery in recent years. This project aims to discover novel antibiotics againt Gram-negative bacteria from marine microorganisms that did not produce any antibacterial metabolites under normal culture condition by activating the silent biosynthetic gene clusters guided by genome bioinformatics analysis. Several different and pleiotropic stragegies including co-culture and chemical epigenetic perturbation will be used to activate the silent genes. The dereplication of natural products are performed by the bioinformatics-predicted structure combined by LC-MS analysis. The project is expected to provide 1-2 lead molecules and lay foundation for the development of new drug with novel modes of action against multi-drug resistant Gram-negative bacteria.

多重耐药与泛耐药革兰氏阴性细菌不断出现和快速传播，已经成为全球公共健康最严重的威胁之一。耐药阴性菌感染的治疗面临严峻的挑战，亟需研发新型的抗阴性菌药物。基因组生物信息学分析表明，微生物携带多达数十种次级代谢生物合成基因簇。在常规实验条件下，大多数基因表达量很低或处于“沉默”状态。沉默基因编码产物丰富的结构多样性，是新药先导物发现具有潜力的资源。本项目以基因组生物信息学分析为指导，以 “超广谱”阴性耐药菌活性筛选为导向，对常规实验条件下不产抗阴性菌活性物质的海洋微生物开展次级代谢潜能挖掘研究。应用共培养、化学表观遗传修饰调控等多种策略，多向性激活菌株沉默基因，通过定向分离、结构确证和活性评价，发现由海洋微生物沉默基因编码的新颖抗阴性耐药菌活性物质，为新型抗阴性菌药物研发奠定基础。

当前，细菌、真菌等病原微生物耐药问题日益严重，不出现的新病毒导致的大流行，对全球人类生命健康造成了严重的威胁，急需发新型抗感染药物。海洋微生物蕴含着丰富的次级代谢潜能，是抗感染药物先导物发现的宝贵资源。本研究对多个海域采集的海洋生物或沉积物样品开展海洋微生物分离，获得海洋真菌739株，分布于34科45属，海洋细菌380株，分布于19科35属。利用共培养、化学表观遗传修饰调控、单菌多化合物等多种方法和策略，对海洋真菌和放线菌的沉默基因开展激活与抗阴性耐药菌活性筛选研究。利用基因组生物信息学分析和LC-MS分析对潜在的抗阴性微生物产物开展结构预测和化学排重。在此基础上，重点对10株海洋微生物的共培养和3株微生物的纯培养产物进行系统的分离纯化研究，分离并鉴定化合物134个，其中新化合物47个。对所得单体化合物进行抗耐药菌、抗真菌、抗病毒、抗炎、抗骨质疏松和细胞毒等活性评价，发现抗铜绿假单胞菌和幽门螺旋杆菌等阴性耐药菌活性新化合物6个，抗阳性菌新化合物4个，抗真菌活性新化合物9个，抗HCV、HIV和IAV等活性新化合物5个，抗炎和抗骨质疏松活性新化合物2个。经初步构效关系和作用机制研究，获得新型抗幽门螺旋杆菌活性先导分子2个，抗深部真菌感染先导分子1个，抗病毒先导分子3个，抗炎与抗骨质疏松先导分子1个。项目研究成果在化学和天然药物领域国际重要刊物发表高水平研究论文6篇，申请专利6项，其中4项已获授权。项目发现的先导分子为研发抗幽门螺旋杆菌、深部真菌、抗病毒等新型抗感染药物和抗炎、抗骨质疏松等新药研发提供了全新的化学实体分子，具有较好的研究价值和应用前景，为新型药物研发奠定基础。