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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（由申请人提供）：微生物天然产物，也称为次级代谢产物，传统上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，基于天然产物的抗感染药物的发现率有所下降。细菌病原体对我们现有药物库的耐药性越来越强，加上我们无法生产新的抗生素，在20世纪60年代和21世纪初之间，传染病已经成为21世纪世纪的主要健康挑战之一。在这个应用程序中，从微生物来源寻找新的生物活性分子的路障进行了讨论，并提出了创新的，多学科的方法，他们的快速发现。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的次级代谢产物的基因组-在正常生长条件下不表达。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，我们提出了几个新的和高通量的策略唤醒沉默基因簇。在一种方法中，我们建议使用多个遗传报告基因来提供4-5个沉默基因簇表达的同时读出。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活化剂。在第二种策略中，我们提出了基于质谱法（MS）的方法，作为激活隐藏簇的读出，而激发子筛选将提供必要的小分子。这两种方法，多基因报告和MS为基础的方法，使高通量发现的神秘代谢物。为了加速这些分子的鉴定和结构阐明，我们提出了基于HPLC-MS的差异代谢组学与MS/MS网络方案相结合的应用。最后，潜在的调节途径，打开沉默的集群响应外源性小分子也将被检查。总之，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的生物测定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.