Enediyne Biosynthesis and Engineering

烯二炔生物合成与工程

• 批准号: 7811497
• 负责人: Ben Shen
• 金额: $ 17.84万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7811497
• 关键词: Active Sites; Acyl Carrier Protein; American; Anabolism; Antineoplastic Agents; Biochemical; Biological; Biological Factors; C 1027; Cancer Etiology; Cells; Cessation of life; Characteristics; Chemicals; Chemistry; Clinical; Collaborations; Complex; Coupling; Development; Dynemicin; Engineering; Enzymatic Biochemistry; Enzymes; Family; Funding; Genes; Genetic; Genome; Genome Scan; Goals; Grant; Health; Hypoxia; In Vitro; Malignant Neoplasms; Maps; Medical; Methodology; Methods; Mining; Natural Products Chemistry; Outcome; Outcome Study; Parents; Pathway interactions; Peptides; Peripheral; Pharmaceutical Preparations; Polyenes; Polymers; Production; Property; Reaction; Recombinants; Recovery; Research; Roentgen Rays; Role; Scanning; Source; Streptomyces; Structure; System; Testing; United States National Institutes of Health; X-Ray Crystallography; Zinostatin; analog; antibody conjugate; antitumor agent; base; cancer cell; chemotherapy; chromophore; clinical application; combinatorial; cytotoxicity; drug discovery; genetic manipulation; in vivo; microbial; microbial genome; microorganism; novel; parent grant; polyketide synthase; programs; public health relevance; response; structural genomics; success

DESCRIPTION (provided by applicant): In response to NIH NOT-OD-09-058 titled "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications," we wish to extend studies spearheaded during the funding of NIH grant 2R01 CA78747 titled "Enediyne Biosynthesis and Engineering." Cancer causes one of every four deaths in the US. The development of fundamentally new, clinically useful anticancer drugs therefore constitutes a national health and research imperative. The enediynes are the most potent, highly active anticancer agents in existence today, and their use as anticancer drugs has been demonstrated clinically. A great challenge is to develop ways to prepare enediynes and their structural analogs and to discover new enediyne natural products for mechanistic studies and clinical development. We propose in this Competitive Revision application to (1) structurally (by X-ray crystallographic means) characterize the enediyne polyketide synthases (PKSEs) and their associated enzymes for enediyne core biosynthesis as well as other enzymes from selected 9- and 10- membered enediyne biosynthetic pathways; (2) produce and further analyze engineered enediynes with distinct exploitable biophysical properties lending themselves to potential clinical applications; and (3) isolate and characterize new enediynes from microbial sources identified on the basis of genome mining. Our hypotheses are that: (1) characterization of selected novel enzymes involved in enediyne biosynthesis especially C-1027, neocarzinostatin (NCS), maduropeptin (MDP), calicheamicin (CAL), esperamicin (ESP), and dynemicin (DYN)) biosynthesis will make fundamental contributions to mechanistic enzymology and natural product chemistry; (2) enediynes produced by combinatorial biosynthetic methods can and do display biological activities superior to those displayed by the parent compound; such compounds warrant further study enabled only through increased production; and (3) new microorganisms identified on the basis of genome mining produce novel, and potentially medically important, enediynes. The specific aims for this Competitive Revision application are: (1) In vivo and in vitro characterization of the selected enediyne PKSs and associated enzymes and their roles in both 9- (C-1027, NCS, and MDP) and 10-membered (CAL, ESP, and DYN) enediyne core biosynthesis; (2) Structural characterization of selected enzymes from enediyne (C- 1027, NCS, MDP, CAL, and DYN) biosynthetic machineries by X-ray crystallography; (3) Isolation of engineered C-1027 analogs to evaluate them as anticancer agents in vivo; and (4) Isolation and structural elucidation of novel 9- or 10-membered enediyne natural products from S. ghanaensis NRRL B-12104, A. orientalis ATCC43491, and S. citricolor IFO13005. The outcomes from these studies will greatly accelerate the tempo of our enediyne biosynthesis, engineering, and drug discovery program by (1) defining the minimal enzymes necessary to convert a nascent linear polyene intermediate from the enediyne PKSE to the characteristic enediyne core structure, (2) demonstrating, on a pilot scale, the feasibility of a "structural genomics" approach to enediyne biosynthesis by solving the structures of key enzymes from selected pathways, (3) advancing C-1027 and its engineered analogs into in vivo testing to realistically develop them into clinically useful, new anticancer drugs, and (4) expanding the portfolio of enediyne anticancer drugs and drug leads by isolating new enediyne natural products. PUBLIC HEALTH RELEVANCE: Cancer causes 1 of every 4 deaths in the US, and 565,650 Americans are expected to die of cancer in 2008. It is therefore a critical research goal to optimize available drugs and to develop fundamentally new, clinically useful anticancer drugs. The enediynes are the most potent, highly active anticancer agents in existence today. Although the natural enediynes have seen limited use as clinical drugs, polymer-based delivery systems and enediyne-antibody conjugates have shown great clinical success or promise in anticancer chemotherapy, demonstrating that the enediynes can be developed into powerful drugs when their extremely potent cytotoxicity is harnessed and delivered to specific cancer cells. A great challenge is to develop methods to make enediynes and their structural analogs and to discover new enediyne natural products for mechanistic studies and clinical developments. This research will study enediyne biosynthesis and engineered novel enediyne analogs. The outcomes include development of enediynes and their analogs into potential anticancer drugs.

描述（由申请人提供）：作为对NIH no - od -09-058题为“NIH宣布竞争性修订申请恢复法案资金的可用性”的回应，我们希望扩展NIH拨款2R01 CA78747题为“Enediyne生物合成和工程”期间的研究。在美国，每四个人中就有一个死于癌症。因此，开发全新的、临床上有用的抗癌药物是国家卫生和研究的当务之急。烯二炔是目前存在的最有效、最高效的抗癌药物，它们作为抗癌药物的用途已被临床证明。一个巨大的挑战是找到制备烯二炔及其结构类似物的方法，并发现新的烯二炔天然产物用于机制研究和临床开发。在这篇竞争性修订申请中，我们建议(1)从结构上（通过x射线晶体学方法）表征烯二炔聚酮合成酶（pkse）及其相关的烯二炔核心生物合成酶，以及选定的9-和10-成员烯二炔生物合成途径中的其他酶；(2)生产并进一步分析具有独特可开发生物物理特性的工程烯二炔，使其具有潜在的临床应用价值；(3)在基因组挖掘的基础上从微生物源中分离和鉴定新的烯二炔。我们的假设是：（1)选定的参与内二炔生物合成的新酶的表征，特别是C-1027、neocarzinostatatin （NCS）、maduropeptin （MDP）、calicheamicin （CAL）、esperamicin （ESP）和dynemicin (DYN)）的生物合成将为机制酶学和天然产物化学做出基础性贡献；(2)组合生物合成方法产生的烯二炔能够并且确实表现出优于母体化合物的生物活性；这些化合物只有通过增加产量才能得到进一步的研究；(3)在基因组挖掘的基础上发现的新微生物产生新的、具有潜在医学重要性的烯二炔。该竞争性修订应用的具体目标是：(1)在体内和体外鉴定选定的内二炔PKSs和相关酶及其在9- （C-1027、NCS和MDP）和10- （CAL、ESP和DYN）内二炔核心生物合成中的作用；(2)利用x射线晶体学对烯二炔（C- 1027、NCS、MDP、CAL和DYN）生物合成机制中选定酶的结构进行表征；(3)分离C-1027工程类似物，在体内评价其抗癌作用；(4)从S. ghanaensis NRRL B-12104、A. orientalis ATCC43491和S. citricolor IFO13005中分离和鉴定新的9-或10元烯二炔天然产物。这些研究的结果将极大地加快我们的烯二因生物合成、工程和药物发现计划的速度，通过(1)定义将新兴线性多烯中间体从烯二因PKSE转化为特征烯二因核心结构所需的最小酶，(2)在中试规模上通过解决选定途径中关键酶的结构，证明“结构基因组学”方法在烯二因生物合成方面的可行性。(3)推进C-1027及其工程类似物的体内试验，使其真正成为临床有用的新型抗癌药物；(4)通过分离新的烯二炔天然产物，扩大烯二炔类抗癌药物和药物先导物的组合。