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）：为了回应 NIH NOT-OD-09-058 标题为“NIH 宣布恢复法案资金用于竞争性修订申请”的可用性，我们希望扩大在 NIH 拨款 2R01 CA78747 资助期间牵头的研究，题为“烯二炔生物合成与工程。”在美国，每四人死亡中就有一人死于癌症。因此，开发临床上有用的全新抗癌药物构成了国家健康和研究的当务之急。烯二炔是当今存在的最有效、高活性的抗癌剂，并且它们作为抗癌药物的用途已得到临床证实。一个巨大的挑战是开发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理研究和临床开发。我们在此竞争性修订申请中建议 (1) 从结构上（通过 X 射线晶体学方法）表征烯二炔聚酮合酶 (PKSE) 及其用于烯二炔核心生物合成的相关酶以及来自选定的 9 元和 10 元烯二炔的其他酶生物合成途径； (2) 生产并进一步分析具有独特可利用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)从基于基因组挖掘鉴定的微生物来源中分离和表征新的烯二炔。我们的假设是：(1) 参与烯二炔生物合成的选定新型酶的表征，特别是 C-1027、新制癌菌素 (NCS)、马杜肽 (MDP)、加利车霉素 (CAL)、埃斯拉霉素 (ESP) 和动力霉素 (DYN)）生物合成将对机械酶学和天然产物化学做出基础性贡献； (2) 通过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所表现出的生物活性；此类化合物需要进一步研究，只有通过增加产量才能实现； (3)基于基因组挖掘鉴定出的新微生物产生新颖且具有潜在医学重要性的烯二炔。此竞争性修订申请的具体目标是：(1) 所选烯二炔 PKS 和相关酶的体内和体外表征及其在 9 元（C-1027、NCS 和 MDP）和 10 元（CAL）中的作用、ESP 和 DYN) 烯二炔核心生物合成； (2) 通过 X 射线晶体学对烯二炔（C-1027、NCS、MDP、CAL 和 DYN）生物合成机器中选定的酶进行结构表征； (3) 分离工程化的 C-1027 类似物，以评估它们作为体内抗癌药物的作用； (4) 来自 S. ghanaensis NRRL B-12104、A. orientalis ATCC43491 和 S. citricolor IFO13005 的新型 9 元或 10 元烯二炔天然产物的分离和结构阐明。这些研究的结果将大大加快我们的烯二炔生物合成、工程和药物发现计划的节奏，通过（1）定义将新生线性多烯中间体从烯二炔 PKSE 转化为特征性烯二炔核心结构所需的最少酶，（2 ）通过解析选定途径中关键酶的结构，在中试规模上证明“结构基因组学”方法进行烯二炔生物合成的可行性，（3）推进将 C-1027 及其工程类似物进行体内测试，以实际将其开发为临床有用的新型抗癌药物，以及 (4) 通过分离新的烯二炔天然产物来扩大烯二炔抗癌药物和先导药物的组合。 公共健康相关性：在美国，每 4 人死亡中就有 1 人死于癌症，预计 2008 年将有 565,650 名美国人死于癌症。因此，优化现有药物并开发临床上有用的全新抗癌药物是一个重要的研究目标。烯二炔是当今存在的最有效、高活性的抗癌剂。尽管天然烯二炔作为临床药物的用途有限，但基于聚合物的递送系统和烯二炔-抗体缀合物在抗癌化疗中已显示出巨大的临床成功或前景，这表明当利用其极强的细胞毒性时，烯二炔可以开发成强大的药物并递送至特定的癌细胞。一个巨大的挑战是开发制造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理研究和临床开发。这项研究将研究烯二炔生物合成和工程化新型烯二炔类似物。结果包括将烯二炔及其类似物开发成潜在的抗癌药物。