Dithiolopyrrolone Antibiotics: Biosynthesis, Mode of Action and Cellular Function

描述(申请人提供)：二硫代吡咯酮抗生素共享一个独特的二硫键桥联杂双环核心，并显示出对细菌、真菌和哺乳动物癌细胞株的强大活性。尽管二硫代吡咯酮类化合物已有60多年的研究历史，但其治疗作用模式、生物合成和生理功能尚不清楚。利用基因组挖掘的方法，在棒状链霉菌中鉴定了一种特殊的二硫代吡咯酮化合物--全息霉素的生物合成基因簇。这项初步工作为更深入地研究本文所述的二硫代吡咯酮支架奠定了基础。这项建议包括三个具体目的：1)阐明包括全霉素在内的二硫代吡咯酮类化合物的作用模式。霍诺霉素被认为是通过氧化还原循环和/或蛋白质修饰来发挥其活性。将采用系统生物学的方法将全息霉素与已知作用机制的抗生素进行分组。同时，将对全息霉素处理的细菌进行转录图谱研究，以进一步提供有关作用模式的线索。还将在细菌培养中进行下拉实验，以确定全息霉素的分子靶点(S)和化学反应活性；2)研究二硫代吡咯酮的生物合成途径。关于全息霉素的生物合成途径，特别是氧化步骤和双环形成过程中涉及的氧化还原化学，将深入描述各个酶转化的顺序和机制。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)仔细研究全霉素在链霉菌中的功能和调节机制。尽管二硫代吡咯酮被鉴定为抗生素和抗癌分子，但它们被假设为它们的产生生物体的信号分子。将进行转录图谱研究，以检查棒状链霉菌和模式链霉菌天蓝色链霉菌中全霉素的作用。将通过转录分析和对簇中存在的调控基因的遗传操作来探索全息霉素生产的调控机制。这项建议中描述的研究将极大地促进我们对自然界组装二硫代吡咯酮的逻辑及其作用机制的理解，为将它们转化为癌症和传染病的可行疗法提供新的方法，并揭示链霉菌中复杂的次生代谢物调控网络，链霉菌是目前使用的大量药物的工业主力。 与公共卫生相关：对天然产品的研究已证明为发现和设计新型药物提供了肥沃的土壤。二硫代吡咯酮类抗生素是一类独特的天然产物，其研究将为这些抗生素的产生提供新的途径，并为未来提高其在治疗传染病和癌症方面的药理作用提供基础。此外，产生二硫代吡咯酮的微生物产生大量的天然产物，对这些微生物的深入了解将为发现新的治疗方法奠定基础。

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.