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）：Salinosporamide A 是一种有效的不可逆蛋白酶体抑制剂，目前正在进行 Ib 期人体临床试验，用于治疗多发性骨髓瘤和其他癌症。这种海洋细菌天然产物具有基于其 β-内酰胺-β-内酯药效团的独特作用机制，与 FDA 批准的唯一蛋白酶体抑制剂肽硼酸硼替佐米不同。在最后的支持期间，我们建立了盐孢酰胺组装的生物合成基础，并在卤化、预苯酯生物化学和聚酮化合物前体供应方面发现了许多新颖的酶促反应。这些基础知识的转化使我们能够通过基因工程合理设计新的盐孢菌酰胺类似物用于生物学评价。这项工作有助于确定抗癌药物盐孢酰胺家族内的结构-活性关系。尽管迄今为止我们取得了重大进展，但由于盐孢菌素是由新型分子构件前所未有地组装而成，因此我们对盐孢菌素的生物合成方式仍然只有粗略的了解。仍然存在许多问题，而针对这项正在进行的研究计划中的发现，新的机遇已经出现。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领域的机会特别适合这种天然产物生物合成计划。为了实现本申请中概述的广泛目标，我们提出了一个涉及五个具体目标的多学科项目。首先，我们计划从功能和结构上表征 SalC 酮合酶及其在盐孢菌酰胺β-内酰胺-β-内酯核心形成中的关键生物合成作用。其次，我们将应用 SalC 的功能来开发盐孢酰胺衍生物的简化化学酶合成方法，该方法基于使用重组盐孢酰胺生物合成酶从合成酰基氨基酸硫酯中合成的 β-内酰胺-β-内酯的集中库。第三，我们的目标是对负责合成盐孢菌酰胺的新型氨基酸残基环己基丙氨酸的生物合成酶进行功能表征，这对于其有效的蛋白酶体结合亲和力至关重要。第四，我们将从功能上表征专用蛋白酶体β-亚基 SalI 及其在 S. tropica 对盐孢菌素自我抗性中的假设作用。第五，我们计划开发新的基于巴豆酰辅酶A还原酶的表达系统，用于工程生物合成具有卤化（氟和氯）和支化侧链的新型聚酮化合物合酶延伸单元，以设计新的聚酮化合物分子。 公共健康相关性：最近 FDA 批准肽硼酸硼替佐米 (Velcade) 作为 20S 蛋白酶体的一流抑制剂来治疗多发性骨髓瘤和其他癌症，这推动了新药的发现和开发，以解决长期问题由于剂量限制性毒性和耐药性的发展，硼替佐米的用途。目前在人体临床试验中最有前途的候选药物之一是 salinosporamide A（USAN 名称 marizomib），这是一种天然产物，具有复杂的、密集功能化的 β-内酰胺-β-内酯药效团，其化学性质不同于硼替佐米和其他基于肽的蛋白酶体抑制剂。由于盐孢酰胺 A 的临床前景和机制新颖性，我们的目标是表征盐孢酰胺生物合成的基本机制，以开发生物工程方法来提高天然产物的生产力，并合理设计用于生物学评价的类似物。