Implementing Innovative Approaches to Access the Hidden Metabolomes of Bacteria

实施创新方法来获取细菌隐藏的代谢组

• 批准号: 8955195
• 负责人: Mohammad R Seyedsayamdost
• 金额: $ 234.49万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8955195
• 关键词: Address; Anti-Infective Agents; Antibiotic Resistance; Antibiotics; Bacteria; Biological Assay; Biological Factors; Communicable Diseases; Complex; Coupled; Data; Development; Gene Cluster; Genes; Genetic; Growth; Health; High Pressure Liquid Chromatography; Housing; Human; Mass Spectrum Analysis; Mediating; Methods; Pharmaceutical Preparations; Process; Reading; Regulatory Pathway; Reporter; Scheme; Soil; Source; Therapeutic; United States National Institutes of Health; bacterial resistance; base; drug discovery; genome sequencing; high throughput screening; innovation; metabolomics; microbial; novel; pathogen; public health relevance; response; screening; small molecule; small molecule libraries

 DESCRIPTION (provided by applicant): Microbial natural products, also referred to as secondary metabolites, have traditionally served as a significant source of drug leads, especially in the development of antibiotics. Recent years, however, have witnessed a decline in the rate of discovery of natural products-based anti-infectives. The growing resistance of bacterial pathogens to our existing arsenal of drugs coupled with our inability to generate novel antibiotics between the 1960s and the early 2000s have catapulted infectious diseases to one of the key health challenges of the 21st century. In this application, the roadblocks for finding new bioactive molecules from microbial sources are discussed and innovative, multi-disciplinary approaches for their rapid discovery are proposed. The major challenge in finding new natural products is that most microbial biosynthetic gene clusters - the sets of genes that synthesize complex secondary metabolites from simple building blocks - are not expressed under normal growth conditions. These are referred to as `silent' or `cryptic' gene clusters and methods that activate them would have a major impact on drug discovery. A second challenge is that activation of silent gene clusters and structural elucidation of the resulting products are low-throughput processes by existing approaches. To address these challenges, we propose several new and high-throughput strategies for awakening silent gene clusters. In one approach, we propose the use of multiple genetic reporters to provide a simultaneous read-out for the expression of 4-5 silent gene clusters. High-throughput screening of small molecule libraries is then used to identify elicitors or activators of these clusters. In a second strategy,we propose methods based on mass spectrometry (MS) as a read-out for activation of cryptic clusters, while elicitor screening would provide the requisite small molecules. Both approaches, multiple genetic reporters and MS-based methods, enable high-throughput discovery of cryptic metabolites. To accelerate identification and structural elucidation of these molecules, we propose application of HPLC-MS- based differential metabolomics in concert with MS/MS networking schemes. Finally, the underlying regulatory pathways that turn on silent clusters in response to exogenous small molecules will also be examined. Together, the approaches above aim to rapidly discover both elicitors and products of cryptic gene clusters, while allowing an understanding of the endogenous regulatory pathways that mediate these processes. Subsequent bioassays carried out in house and by submission to the NIH and the NCI will assess the potential therapeutic utility of bacterial cryptic metabolites.

 描述（由申请人提供）：微生物天然产物，也称为次生代谢物，传统上一直是药物先导物的重要来源，特别是在抗生素的开发中，然而，近年来发现率有所下降。细菌病原体对我们现有的药物库的耐药性不断增强，加上我们无法生产新型抗生素。 20 世纪 60 年代至 2000 年代初，传染病已成为 21 世纪的主要健康挑战之一。在本应用中，讨论了从微生物来源寻找新生物活性分子的障碍，以及快速发现这些分子的创新、多学科方法。提出寻找新天然产物的主要挑战是大多数微生物生物合成基因簇（从简单构件合成复杂次生代谢物的基因组）不表达。这些被称为“沉默”或“神秘”基因簇，激活它们的方法将对药物发现产生重大影响。第二个挑战是沉默基因簇的激活和所得产物的结构阐明。为了解决这些挑战，我们提出了几种新的高通量策略来唤醒沉默基因簇，在一种方法中，我们建议使用多个基因生产者来同时读出表达。 4-5 人然后使用小分子文库的高通量筛选来识别这些簇的激发子或激活子在第二种策略中，我们提出了基于质谱（MS）的方法作为神秘簇的激活读数。而激发子筛选将提供必需的小分子。为了加速这些分子的识别和结构阐明，我们建议应用多种基因产生器和基于 MS 的方法。最后，还将研究基于 HPLC-MS 的差异代谢组学与 MS/MS 网络方案相结合，以响应外源小分子而开启沉默簇的潜在调控途径。神秘基因簇的产物，同时允许了解介导这些过程的内源性调控途径，随后在内部进行的生物测定以及提交给 NIH 和 NCI 将评估细菌神秘代谢物的潜在治疗效用。

项目成果

A genetics-free method for high-throughput discovery of cryptic microbial metabolites.

一种用于高通量发现神秘微生物代谢物的无遗传学方法。

• 发表时间: 2019
• 影响因子: 14.8
• 作者: Xu, Fei;Wu, Yihan;Zhang, Chen;Davis, Katherine M;Moon, Kyuho;Bushin, Leah B;Seyedsayamdost, Mohammad R
• 通讯作者: Seyedsayamdost, Mohammad R

Mapping the Trimethoprim-Induced Secondary Metabolome of Burkholderia thailandensis.

绘制泰国伯克霍尔德杆菌甲氧苄啶诱导的次级代谢组图。

• 发表时间: 2016
• 影响因子: 4
• 作者: Okada, Bethany K;Wu, Yihan;Mao, Dainan;Bushin, Leah B;Seyedsayamdost, Mohammad R
• 通讯作者: Seyedsayamdost, Mohammad R

Reporter-Guided Transposon Mutant Selection for Activation of Silent Gene Clusters in Burkholderia thailandensis.

记者引导的转座子突变体选择，用于激活泰国伯克霍尔德氏菌沉默基因簇。

• 发表时间: 2020
• 作者: Mao, Dainan;Yoshimura, Aya;Wang, Rurun;Seyedsayamdost, Mohammad R
• 通讯作者: Seyedsayamdost, Mohammad R

The Polyene Natural Product Thailandamide A Inhibits Fatty Acid Biosynthesis in Gram-Positive and Gram-Negative Bacteria.

多烯天然产物 Thaiamide A 抑制革兰氏阳性和革兰氏阴性细菌的脂肪酸生物合成。

• DOI: 10.1021/acs.biochem.8b00678
• 发表时间: 2018-07-05
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Yihan Wu;M. Seyedsayamdost
• 通讯作者: M. Seyedsayamdost

Antibiotic dialogues: induction of silent biosynthetic gene clusters by exogenous small molecules.

抗生素对话：通过外源小分子诱导沉默的生物合成基因簇。

• 发表时间: 2017-01
• 影响因子: 11.3
• 作者: Okada, Bethany K;Seyedsayamdost, Mohammad R
• 通讯作者: Seyedsayamdost, Mohammad R