Salinosporamide Biosynthesis and Engineering

盐孢酰胺生物合成与工程

• 批准号: 8370821
• 负责人: BRADLEY S MOORE
• 金额: $ 28.44万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8370821
• 关键词: Acids; Address; Affinity; Amino Acids; Anabolism; Antineoplastic Agents; Bacteria; Biochemical Reaction; Biochemistry; Biological; Biological Factors; Biomedical Engineering; Bortezomib; Chlorine; Clinical; Clinical Treatment; Clinical Trials; Coenzyme A; Complex; Development; Dose-Limiting; Educational process of instructing; Engineering; Enzymes; Evaluation; FDA approved; Family; Fluorine; Foundations; Genetic Engineering; Genome; Goals; Human; Knowledge; Lactams; Lactones; Libraries; Malignant Neoplasms; Marines; Microbe; Mining; Molecular; Multiple Myeloma; Names; Natural Resistance; Oxidoreductase; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Productivity; Proteasome Binding; Proteasome Inhibitor; Reaction; Recombinants; Regulation; Research; Resistance; Resistance development; Role; Side; Structure-Activity Relationship; Surface; System; Toxic effect; Translations; Velcade; Work; analog; base; design; drug candidate; halogenation; inhibitor/antagonist; innovation; multicatalytic endopeptidase complex; multidisciplinary; novel; pharmacophore; polyketide synthase; prephenate; programs; response; salinosporamide A; synthetic biology; thioester

DESCRIPTION (provided by applicant): Salinosporamide A is a potent irreversible proteasome inhibitor presently in phase Ib human clinical trials for the treatment of multiple myeloma and other cancers. This marine bacterial natural product has a distinctive mechanism of action based on its ?-lactam-?-lactone pharmacophore that differs from the only FDA-approved proteasome inhibitor, the peptide boronate bortezomib. During the last period of support, we established the biosynthetic foundation of salinosporamide assembly and discovered a number of novel enzymatic reactions in halogenation, prephenate biochemistry, and polyketide precursor supply. Translation of this basic knowledge allowed us to rationally design through genetic engineering new salinosporamide analogues for biological evaluation. This work helped determine the structure-activity relationships within the salinosporamide family of anticancer agents. Despite our significant progress to date, we still only have a cursory understanding of how salinosporamide is biosynthesized due to its unprecedented assembly from novel molecular building blocks. Numerous questions remain, while new opportunities have surfaced in response to discoveries made in this ongoing research program. Opportunities in enzyme discovery, synthetic biology, chemoenzymatic synthesis, genome mining, and proteasome biochemistry are uniquely suited for this natural product biosynthetic program. To accomplish the broad goals outlined in this application, we propose a multidisciplinary project involving five specific aims. First, we plan to functionally and structurally characterize the SalC ketosynthase and its key biosynthetic role in the formation of the ?-lactam-?-lactone core of salinosporamide. Second, we will apply the function of SalC to develop a streamlined chemoenzymatic synthesis of salinosporamide derivatives based on a focused library of ?-lactam-?-lactones from synthetic acylamino acid thioesters with recombinant salinosporamide biosynthetic enzymes. Third, we aim to functionally characterize the biosynthetic enzymes responsible for the synthesis of salinosporamide's novel amino acid residue, cyclohexenylalanine, which is paramount to its potent proteasome binding affinity. Fourth, we will functionally characterize the dedicated proteasome ?-subunit SalI and its hypothesized role in S. tropica self-resistance against salinosporamide. And fifth, we plan to develop new crotonyl-CoA reductase-based expression systems for the engineered biosynthesis of new polyketide synthase extender units with halogenated (fluorine and chlorine) and branched side chains for the design of new polyketide molecules. PUBLIC HEALTH RELEVANCE: The recent FDA approval of the peptide boronate bortezomib (Velcade) as a first-in-class inhibitor of the 20S proteasome to treat multiple myeloma and other cancers has fueled the discovery and development of new drugs to address the long-term utility of bortezomib due to dose-limiting toxicities and the development of resistance. One of the most promising drug candidates currently in human clinical trials is salinosporamide A (USAN name marizomib), a natural product possessing a complex, densely functionalized ?-lactam-?-lactone pharmacophore that is chemically distinct from bortezomib and other peptide-based proteasome inhibitors. Because of the clinical promise and mechanistic novelty of salinosporamide A, we aim to characterize the fundamental mechanism of salinosporamide biosynthesis in order to develop bioengineering approaches to enhance the productivity of the natural product and rationally design analogs for biological evaluation.

描述（由申请人提供）：盐孢酰胺A是目前在IB期临床试验中用于治疗多发性骨髓瘤和其他癌症的有效不可逆蛋白酶体抑制剂。这种海洋细菌天然产物具有基于其乳酰胺 - ？ - 赖乳酮药效团具有独特的作用机理，与唯一由FDA批准的蛋白酶体抑制剂，肽硼酸硼酸硼酸硼酸硼酸硼酸苯甲酸酯。在支持的最后一个时期，我们建立了盐氧化盐组装的生物合成基础，并发现了许多新型的酶促反应，前卤代生物化学和Polyketide前体供应。这种基本知识的翻译使我们能够通过基因工程进行合理设计，以进行生物学评估。这项工作有助于确定抗癌剂盐孢子菌家族中的结构活性关系。尽管我们迄今为止取得了重大进展，但由于其前所未有的分子构建块，我们仍然对盐孢胺如何被生物合成有粗略的了解。仍然存在许多问题，而新的机会浮出水面，以应对正在进行的研究计划中的发现。酶发现，合成生物学，化学酶合成，基因组挖掘和蛋白酶体生物化学的机会非常适合这种天然产物生物合成计划。为了实现本应用程序中概述的广泛目标，我们提出了一个涉及五个特定目标的多学科项目。首先，我们计划在功能和结构上表征SALC 酮合酶及其在盐酸盐的 - 乳酰胺核心核心的形成中的关键生物合成作用。其次，我们将利用SALC的功能来开发基于盐氧化盐衍生物的精简化学酶合成，该合成基于聚焦 - lactam-？？ - 从合成酰基氨基氨基硫酸硫代酯与重组盐氧化盐生物合成生物合成酶的集合的库。第三，我们的目的是在功能上表征负责盐孢酶合成的新型氨基酸残基环己基丙氨酸的生物合成酶，这对于其有效的蛋白酶体结合亲和力至关重要。第四，我们将在功能上表征专用的蛋白酶体？ - 亚基sali及其在对盐孢菌胺的热带自我抗性中的假设作用。第五，我们计划开发新的基于CROTONYL-COA还原酶的表达系统，以用于新的Polyketide合酶扩展单元的工程生物合成，并具有卤代（氟和氯）和分支侧链，以设计新的Polyketide分子。 公共卫生相关性：FDA最近批准肽硼酸硼酸硼替佐米（Velcade）是20S蛋白酶体的第一类抑制剂，用于治疗多发性骨髓瘤和其他癌症，这推动了由于有毒剂的长期实用性的发现和开发，该药物由于有毒的毒性而导致的毒性和发展毒性和发展毒性而产生了抵抗力和这种抵抗力，这是不适的。目前在人类临床试验中的最有前途的候选药物之一是盐氧甲酰胺A（乌斯曼名称Marizomib），它是一种具有复杂且功能齐密化的天然产物？ - 乳酸盐 - ？ - 乳乳酮药丸与乳酸化学上不同于bortezomib和其他基于肽的蛋白酶体的化学差异。由于盐酸盐A的临床希望和机械新颖性，我们旨在表征盐氧化盐生物合成的基本机制，以开发生物工程方法，以增强自然产物的生产力和合理设计类似物的生产力以进行生物学评估。