Biosynthetic Analysis of Marine Cyanobacterial Pathways

描述（由申请人提供）：海洋蓝细菌在其生物活性和结构独特的天然产物的生产方面非常丰富。许多这些次级代谢物或其衍生物是药物开发计划中的主要化合物，旨在提供治疗癌症，细菌感染，炎症反应的新疗法，以及作物保护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提供了一个非常大的混合非核糖体肽合成酶/聚酮合酶（NRPS/PKS）系统的数量。相应的代谢系统由一组复杂的多功能蛋白质组成，这些蛋白质沿着相关酶通过模块化的多步骤过程产生结构复杂的分子。在过去的几年中，谢尔曼，格威克和史密斯实验室已经开发了一个互补的计划，克隆和表征新的蓝藻次生代谢产物的生物合成途径，具有显着的生物技术应用的潜力。尽管取得了相当大的进展，充分了解的分子机制，催化活性，动力学特性，和底物特异性内蓝藻生物合成途径才刚刚开始展开。拟议的研究将建立在我们对curacin，jamaicamide和cryptophycin/ arenastatin代谢系统的成就之上，这三个强大的途径已经成为新信息的丰富来源。预期的代谢，生物化学和结构的理解将有助于设计新的生物合成系统，利用蓝藻天然产物途径的潜力不断增长。蓝藻天然产物产生新的先导化合物作为有用的药物开发的全部前景只有通过缩小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物化学方法，使我们能够1）操纵蓝藻天然产物代谢系统以产生类似物结构，2）利用独特的次级代谢产物酶来产生新的生物活性分子，3）筛选新的化合物和类似物，以确定有前途的新抗癌化合物，以进一步开发。具体目标是：1.利用蓝藻天然产物系统的固有多功能性来创造新的抗癌先导化合物。次级目标包括：调查能力的蓝藻生物合成途径，以产生新的类似物使用独特的实验室培养和突变合成方法。B.研究海洋蓝藻途径的独特酶促能力，以设计新的代谢系统和定制过程，产生新的生物活性化合物。C.采用结构生物学和定点诱变方法来了解独特的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，以创建具有增强药用特性的新抗癌药物2。对特定目标1产生的新化合物进行生物测定。a.来自拟议研究的新化合物将被转移到Riskai研究所和密歇根大学化学基因组学中心，使用一系列与癌症相关的生物化学和细胞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.