Dithiolopyrrolone Antibiotics: Biosynthesis, Mode of Action and Cellular Function

项目摘要 二硫代吡咯烷酮抗生素具有独特的二硫键桥连杂双环核心， 对细菌、真菌和哺乳动物癌细胞系的有效活性。虽然 已知二硫代吡咯烷酮已有60多年，它们的治疗作用模式， 生物合成和生理功能还不清楚。基因组挖掘方法 用于鉴定特定二硫代吡咯烷酮化合物的生物合成基因簇， 全霉素，其生产菌株，棒状链霉菌。这项初步工作建立了 二硫代吡咯烷酮支架的更深入的研究的基础，描述 本文所该提案包括三个具体目标：1）阐明 二硫代吡咯烷酮，包括全霉素。假设全霉素通过以下途径发挥其活性： 氧化还原循环和/或蛋白质修饰。将采用系统生物学方法， 将全霉素与具有已知作用机制的抗生素分组。同时， 将进行全霉素处理的细菌的转录谱研究，以进一步提供 关于行动方式的线索。还将在细菌中进行下拉实验 培养以鉴定分子靶标和全霉素的化学反应性; 2）研究 二硫代吡咯烷酮的生物合成途径。深入描述了秩序和 将进行关于全霉素的单独酶促转化的机制 生物合成途径，特别是氧化步骤中涉及的氧化还原化学， 双环形成。此外，将利用基因组挖掘方法来发现未知的 二硫代吡咯烷酮基因簇和新的二硫代吡咯烷酮化合物; 3）审查 全霉素在链霉菌中的作用及调控机制。虽然被认定为 抗生素和抗癌分子，二硫代吡咯烷酮被假设作为信号传导， 生产生物体的分子。将进行转录谱研究， 检测全霉素在S. clavuligerus和模式链霉菌S. 天蓝色。通过以下方式探索全霉素产生的调节机制 转录分析和遗传操作的调控基因中存在的集群。 这项提案中描述的研究将大大促进我们对自然界的理解。 组装二硫代吡咯烷酮及其作用机制的逻辑，提供了新的方法， 将其转化为癌症和传染病的可行疗法，并阐明 链霉菌中次级代谢产物的复杂调控网络， 占目前使用的大量药物。

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.