Biosynthetic Analysis of Marine Cyanobacterial Pathways

描述(由申请人提供)：海洋蓝藻非常丰富，它们生产出具有生物活性和结构独特的天然产品。其中许多次级代谢物或其衍生物是药物开发项目中的先导化合物，旨在提供治疗癌症、细菌感染、炎症反应和作物保护的新疗法，以杀死有害的微生物病原体和昆虫。海洋和陆地蓝藻天然产物的分离和结构分析为获得大量非核糖体多肽合成酶/聚酮合成酶(NRPS/PKS)混合系统提供了途径。相应的代谢系统由一组耐人寻味的复杂多功能蛋白质组成，这些蛋白质与相关酶一起通过模块化的多步骤过程产生结构复杂的分子。在过去的几年里，Sherman、Gerwick和Smith实验室开发了一个互补的计划，以克隆和表征具有重大生物技术应用潜力的新型蓝藻次生代谢物的生物合成途径。尽管取得了长足的进展，但对蓝藻生物合成途径中的分子机制、催化活性、动力学特性和底物特性的全面了解才刚刚开始。这项拟议的研究将建立在我们在柯拉酸、甲胺咪胺和隐孢霉素/阿司他丁代谢系统方面取得的成就的基础上，这三条强大的途径一直是新信息的丰富来源。预期的代谢、生化和结构理解将有助于设计新的生物合成系统，以利用蓝藻天然产物途径日益增长的潜力。蓝藻天然产物产生新的先导化合物作为有用的药物的充分承诺，只有通过弥合知识和技术方面的一系列关键差距才能实现。解决这些挑战将需要开发和优化遗传和生化方法，使我们能够1)操纵蓝藻天然产物代谢系统来产生类似结构，2)利用独特的次生代谢物酶来创造新的生物活性分子，3)筛选新的化合物和类似物以确定有前景的新的抗癌化合物用于进一步的开发。具体目标是：1。利用蓝藻天然产物系统固有的多功能性来创造新的抗癌先导化合物。次级目标包括：a.利用独特的实验室培养和突变合成方法，调查蓝藻生物合成途径产生新类似物的能力。B.研究海洋蓝藻途径的独特酶能力，以设计新的代谢系统和定制过程，以产生新的生物活性化合物。使用结构生物学和定点突变方法来了解独特生物合成酶的精确生化机制。D.开发新的化学酶、体内和体外途径，以创造具有增强药用性能的新的抗癌药物2.对特定目标产生的新化合物进行生物测试1.从拟议研究中获得的新化合物将被转移到卫材研究所和密歇根大学化学基因组中心，使用与癌症相关的一系列生化和基于细胞的分析来分析生物活性。

DESCRIPTION (provided by applicant): Marine cyanobacteria are extraordinarily rich in their production of biologically active and structurally unique natural products. A number of these secondary metabolites or their derivatives are lead compounds in drug development programs aimed at providing new therapies to treat cancer, bacterial infections, inflammatory responses and in crop protection to kill harmful microbial pathogens and insects. Isolation and structural analysis of marine and terrestrial cyanobacterial natural products has provided access to an unusually large number of mixed non-ribosomal peptide synthetase/polyketide synthase (NRPS/PKS) systems. The corresponding metabolic systems are comprised of an intriguing set of complex multifunctional proteins that along with allied enzymes generate structurally complex molecules via a modular multi-step process. Over the past several years the Sherman, Gerwick and Smith laboratories have developed a complementary program to clone and characterize the biosynthetic pathways of novel cyanobacterial secondary metabolites that possess significant potential for biotechnological applications. Despite considerable progress, a full understanding of the molecular mechanisms, catalytic activities, kinetic properties, and substrate specificities within cyanobacterial biosynthetic pathways is just beginning to unfold. The proposed research will build upon our accomplishments on the curacin, jamaicamide and cryptophycin/ arenastatin metabolic systems, three robust pathways that have been a rich source of new information. The expected metabolic, biochemical and structural understanding will facilitate the design of new biosynthetic systems that harness the growing potential of cyanobacterial natural product pathways. The full promise of cyanobacterial natural products to yield new lead compounds for development as useful pharmaceuticals will only be realized by closing a series of key gaps in knowledge and technology. Solving these challenges will require development and optimization of genetic and biochemical methods that allow us to 1) manipulate cyanobacterial natural product metabolic systems to produce analog structures, 2) utilize unique secondary metabolite enzymes for creation of novel bioactive molecules and, 3) screen new compounds and analogs to identify promising new anticancer compounds for further development. The specific aims are: 1. To harness the inherent versatility of cyanobacterial natural product systems to create new anticancer lead compounds. Sub-aims include: a. Investigate ability of cyanobacterial biosynthetic pathways to generate novel analogs using unique laboratory culture and mutasynthesis methodologies. b. Investigate the unique enzymatic capabilities of marine cyanobacterial pathways to engineer new metabolic systems and tailoring processes to generate new bioactive compounds. c. Employ structural biology and site-directed mutagenesis approaches to understand the precise biochemical mechanisms of unique biosynthetic enzymes. d. Develop new chemoenzymatic, in vivo, and in vitro pathways to create new anticancer agents with enhanced medicinal properties 2. Perform bioassays on new compounds resulting from Specific Aim 1. a. New compounds derived from the proposed research will be transferred to Eisai Research Institute and University of Michigan Center for Chemical Genomics for analysis of biological activity using a series of biochemical and cell based assays relevant to cancer.