Dithiolopyrrolone Antibiotics: Biosynthesis, Mode of Action and Cellular Function

项目摘要 二硫代吡咯酮类抗生素共享一个独特的二硫键桥联杂环核心并展示 对细菌、真菌和哺乳动物癌细胞具有很强的活性。尽管 二硫代吡咯酮类药物已有60多年的历史，其治疗作用模式， 生物合成和生理功能还不是很清楚。一种基因组挖掘方法 用于鉴定一种特定的二硫代吡咯酮化合物的生物合成基因簇， 全息霉素，其产生菌株棒状链霉菌。这项初步工作确立了 描述了对二硫代吡咯酮支架进行更深入研究的基础 在这里。这项建议包括三个具体目标：1)阐明 二硫代吡咯酮类药物，包括全息霉素。霍洛霉素被认为是通过 氧化还原循环和/或蛋白质修饰。将采用系统生物学的方法来 将已知作用机制的抗生素与全霉素组。同时， 将进行全霉素处理的细菌的转录图谱研究，以进一步提供 关于行动模式的线索。下拉实验也将在细菌中进行。 培养鉴定全霉素的分子靶点(S)和化学反应活性；2)考察 二硫代吡咯酮的生物合成途径。订单和订单的深入表征 关于全息霉素，将进行个别酶转化的机制 生物合成途径，特别是氧化步骤中涉及的氧化还原化学和 形成双环。此外，将利用基因组挖掘的方法来发现未知的 二硫代吡咯酮基因簇和新的二硫代吡咯酮化合物；3)考察 全菌素在链霉菌中的作用及其调控机制。尽管被确认为 抗生素和抗癌分子，二硫代吡咯酮被假设为信号 它们生产有机体所需的分子。将进行转录特征分析研究以 在棒状链霉菌和链霉菌模型菌株中检测全霉素的作用。 天蓝色。将通过以下途径来探索全息霉素生产的调控机制 该簇中存在的调控基因的转录分析和遗传操作。 这项建议中描述的研究将极大地促进我们对自然 组装二硫代吡咯酮及其作用机理的逻辑，提供了新的方法 将它们转化为治疗癌症和传染病的可行疗法，并阐明 工业主力链霉菌中复杂的次生代谢物调控网络 占了当前使用的大量药品。

Project Summary Dithiolopyrrolone antibiotics share a unique disulfide-bridged heterobicyclic core and exhibit potent activities against bacteria, fungi, and mammalian cancer cell lines. Although the dithiolopyrrolones have been known for over sixty years, their therapeutic mode of action, biosynthesis, and physiological functions are not well understood. A genome-mining approach was used to identify the biosynthetic gene cluster of a particular dithiolopyrrolone compound, holomycin, in its producing strain, Streptomyces clavuligerus. This preliminary work established the foundation for more intensive investigations of the dithiolopyrrolone scaffold, described herein. This proposal includes three specific aims: 1) Elucidating the modes of action of dithiolopyrrolones including holomycin. Holomycin is hypothesized to exert its activity through redox cycling and/or protein modification. A systems biology approach will be undertaken to group holomycin with antibiotics with known mechanisms of action. In conjunction, transcriptional profiling studies of holomycin-treated bacteria will be carried out to further provide clues regarding the mode of action. Pull down experiments will also be performed in bacterial culture to identify the molecular target(s) and chemical reactivity of holomycin; 2) Investigating the biosynthetic pathway of dithiolopyrrolones. In-depth characterization of the order and mechanisms of individual enzymatic transformations will be carried out regarding the holomycin biosynthetic pathway, in particular the redox chemistry involved in the oxidation steps and bicyclic ring formation. Further, a genome-mining approach will be utilized to uncover unknown dithiolopyrrolone gene clusters and novel dithiolopyrrolone compounds; 3) Scrutinizing the functions and regulatory mechanisms of holomycin in Streptomyces. Though identified as antibiotics and anticancer molecules, dithiolopyrrolones are hypothesized to serve as signaling molecules for their producing organisms. Transcriptional profiling studies will be undertaken to examine the effects of holomycin in S. clavuligerus and model Streptomyces strain, S. coelicolor. The regulatory mechanism of holomycin production will be explored through transcriptional analysis and genetic manipulation of the regulatory genes present in the cluster. The studies described in this proposal will significantly advance our understanding of Nature's logic to assemble dithiolopyrrolones and their mechanisms of action, provide new ways to convert them into viable therapeutics for cancer and infectious diseases, and shed light on the intricate regulatory network of secondary metabolites in Streptomyces, the industrial workhorses accounting for a large number of drugs in current use.