Dithiolopyrrolone Antibiotics: Biosynthesis, Mode of Action and Cellular Function

二硫代吡咯酮抗生素：生物合成、作用方式和细胞功能

基本信息

批准号：
8720018
负责人：
Bo Li
金额：
$ 24.81万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8720018
关键词：
Accounting Affect Affinity Affinity Labels Anabolism Animal Model Antibiotics Antineoplastic Agents Bacteria Biochemical Pathway Biochemistry Bioinformatics Biological Factors Biological Process Cancer cell line Cell physiology Chemicals Chemistry Communicable Diseases Coupled DNA DNA-Directed RNA Polymerase Data Development Disulfides Engineering Environment Enzymatic Biochemistry Enzymes Escherichia coli Exhibits Family Flavins Foundations Future Gene Cluster Gene Expression Genes Genetic Genome Homologous Gene Human In Vitro Individual Investigation Label Learning Light Logic Malignant Neoplasms Mentors Methods Microbial Genetics Microbiology Mining Modeling Molecular Target Nature Organism Oxidation-Reduction Oxidative Stress Pathway interactions Pharmaceutical Preparations Pharmacologic Substance Phase Physiological Post-Translational Protein Processing Production Proteomics RNA chemical synthesis Radio Regulation Regulator Genes Research Role Signal Transduction Signaling Molecule Streptomyces Structure Structure-Activity Relationship Systems Biology Testing Therapeutic Thinking Training Work affinity labeling analog antimicrobial base biological adaptation to stress design enzyme mechanism experience follow-up fungus genetic manipulation improved in vivo insight medical schools member microorganism novel novel therapeutics oxidation promoter research study scaffold skills synthetic enzyme tool

项目摘要

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.

项目概要二硫代吡咯酮抗生素具有独特的二硫键桥杂双环核心并表现出对细菌、真菌和哺乳动物癌细胞系具有有效的活性。虽然二硫并吡咯酮类药物已为人所知六十多年，其治疗作用方式，生物合成和生理功能尚不清楚。基因组挖掘方法用于鉴定特定二硫代吡咯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。本次前期工作确定描述了对二硫代吡咯酮支架进行更深入研究的基础在此处。该提案包括三个具体目标： 1) 阐明二硫代吡咯酮类，包括全霉素。据推测，Holomycin 通过以下方式发挥其活性：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组合。结合起来，将进行全霉素处理细菌的转录谱研究，以进一步提供有关行动模式的线索。 Pull down 实验也将在细菌中进行培养以确定全霉素的分子靶标和化学反应性； 2）调查二硫代吡咯酮的生物合成途径。顺序的深入表征将针对全霉素进行个体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的形成。此外，基因组挖掘方法将用于发现未知的二硫代吡咯酮基因簇和新型二硫代吡咯酮化合物； 3) 审查全霉素在链霉菌中的功能和调控机制。虽然被认定为抗生素和抗癌分子中，二硫代吡咯酮被假设充当信号传导其生产生物体的分子。将进行转录谱研究检查全霉素对 S. clavuligerus 和模型链霉菌菌株 S. 天蓝色。将通过以下方式探索全霉素生产的调控机制对簇中存在的调节基因进行转录分析和遗传操作。本提案中描述的研究将显着增进我们对自然的理解组装二硫代吡咯酮的逻辑及其作用机制，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次级代谢产物的复杂调控网络占当前使用的药物的大量。