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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

基本信息

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
项目类别：
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

项目摘要

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(申请人提供)：微生物天然产物，也被称为次生代谢物，传统上一直是药物先导的重要来源，特别是在抗生素的开发中。然而，近年来，以天然产品为基础的抗感染药物的发现率有所下降。细菌病原体对现有药物的抗药性不断增强，加上我们无法产生新的抗生素在20世纪60年代至21世纪初，传染病已成为21世纪的关键卫生挑战之一。在这一应用中，讨论了从微生物来源中寻找新的生物活性分子的障碍，并提出了快速发现它们的创新的、多学科的方法。寻找新的天然产物的主要挑战是，大多数微生物生物合成基因簇--从简单的构建块合成复杂次级代谢物的基因集--在正常生长条件下不表达。这些基因簇被称为“沉默的”或“隐秘的”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结果产物的结构鉴定是现有方法的低通量过程。为了应对这些挑战，我们提出了几种新的高通量策略来唤醒沉默的基因簇。在一种方法中，我们建议使用多个遗传报告器来同时读出4-5个沉默基因簇的表达。然后使用小分子文库的高通量筛选来鉴定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学(MS)的方法作为激活神秘簇的读出，而激发子筛选将提供必要的小分子。这两种方法，多个遗传记者和基于MS的方法，都能够高通量地发现隐秘的代谢物。为了加速这些分子的鉴定和结构鉴定，我们建议将基于高效液相-质谱仪的差异代谢组学与MS/MS联网方案相结合。最后，还将研究响应外源小分子而开启沉默簇的潜在调控途径。综上所述，上述方法旨在快速发现隐蔽基因簇的激发子和产物，同时允许理解调节这些过程的内源性调控途径。随后在内部和提交给NIH和NCI的生物测试将评估细菌隐蔽代谢物的潜在治疗效用。

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（4）

Thailandenes, Cryptic Polyene Natural Products Isolated from Burkholderia thailandensis Using Phenotype-Guided Transposon Mutagenesis.

DOI：
10.1021/acschembio.9b00883
发表时间：
2020-05-15
期刊：
ACS CHEMICAL BIOLOGY
影响因子：
4
作者：
Park, Jong-Duk;Moon, Kyuho;Miller, Cheryl;Rose, Jessica;Xu, Fei;Ebmeier, Christopher C.;Jacobsen, Eremy R.;Mao, Dainan;Old, William M.;DeShazer, David;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.