Dithiolopyrrolone Antibiotics: Biosynthesis, Mode of Action and Cellular Function

描述（由申请人提供）：二硫代吡洛酮抗生素具有独特的二硫桥接杂双环核心，对细菌、真菌和哺乳动物癌细胞系具有有效的活性。虽然二硫代吡洛酮类药物已被发现60多年，但其治疗方式、生物合成和生理功能尚不清楚。采用基因组挖掘方法鉴定了一种特殊的二硫代吡洛酮化合物——全息霉素在其生产菌株——链霉菌clavuligerus中的生物合成基因簇。这项初步工作为本文所述的二硫代吡洛酮支架的更深入研究奠定了基础。本课题有三个具体目的：1)阐明包括霍霉素在内的二硫代吡洛酮类药物的作用方式。据推测，Holomycin通过氧化还原循环和/或蛋白质修饰发挥其活性。将采用系统生物学方法将holomycin与已知作用机制的抗生素进行分组。同时，将开展全霉素处理细菌的转录谱研究，以进一步提供有关作用方式的线索。下拉实验也将在细菌培养中进行，以确定holomycin的分子靶点和化学反应性；2)二硫代吡喃酮生物合成途径的研究。深入表征单个酶转化的顺序和机制将进行关于全息霉素生物合成途径，特别是氧化步骤和双环形成所涉及的氧化还原化学。此外，基因组挖掘方法将用于发现未知的二硫代吡洛酮基因簇和新的二硫代吡洛酮化合物；3)探究全霉素在链霉菌中的作用及调控机制。二硫代吡洛酮虽然被确定为抗生素和抗癌分子，但被假设为其产生生物的信号分子。转录谱研究将进行，以检查全息霉素在S. clavuligerus和模型链霉菌菌株，S. coelicolor的影响。通过转录分析和对集群中存在的调控基因的遗传操作，探索全息霉素生产的调控机制。本提案中描述的研究将大大促进我们对自然逻辑的理解，以组装二硫代吡罗酮及其作用机制，提供新的途径，将其转化为可行的治疗癌症和传染病的方法，并揭示链霉菌次生代谢物的复杂调控网络，这是目前使用的大量药物的工业支柱。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Studies in natural products have proven fertile ground for discovery and design of novel pharmaceuticals. The proposed research on the dithiolopyrrolone antibiotics, a unique group of natural products, will afford new ways to generate these antibiotics, and provide basis for future efforts to improve their pharmacological effects in treating infectious diseases and cancer. Further, the dithiolopyrrolone-producing microorganisms generate a large wealth of natural products, and a deeper understanding of these microorganisms will build foundation for the discovery of novel therapeutics.