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）：为了响应NIH NOT-OD-09-058标题为“NIH宣布恢复法案资金的竞争性修订申请的可用性”，我们希望扩展在NIH资助2 R 01 CA 78747标题为“烯二炔生物合成和工程”的资助期间率先开展的研究。“癌症导致美国每四例死亡中就有一例。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。烯二炔类化合物是目前存在的最有效的、高活性的抗癌剂，并且它们作为抗癌药物的用途已经在临床上得到证实。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。我们在此竞争修订申请中建议（1）在结构上（通过X-射线晶体学方法）表征烯二炔聚酮化合物脱氢酶（PKSE）及其用于烯二炔核心生物合成的相关酶以及来自所选9元和10元烯二炔生物合成途径的其它酶;（2）产生并进一步分析具有独特的可利用的生物物理性质的工程化烯二炔，使其本身具有潜在的临床应用;和（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所选新酶的表征，特别是C-1027、新抑癌素（NCS）、马度抑肽酶（MDP）、加利车霉素（CAL）、埃斯波霉素（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本贡献;（2）通过组合生物合成方法产生的烯二炔可以并且确实显示出比母体化合物所显示的生物活性上级的生物活性;只有通过增加产量才能对这些化合物进行进一步的研究;和（3）基于基因组挖掘鉴定的新微生物产生新的和潜在的医学上重要的烯二炔。该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- 10和10 - 11两种酶中的作用。（C-1027、NCS和MDP）和10元（CAL、ESP和DYN）烯二炔核心生物合成;（2）来自烯二炔的所选酶的结构表征（C- 1027、NCS、MDP、CAL和DYN）生物合成机器的X射线晶体学（3）分离工程化的C-1027类似物以评价它们作为体内抗癌剂;和（4）从S. ghanaensis NRRL B-12104，A. orientalis ATCC 43491和S. citricolor IFO13005.这些研究的结果将大大加快我们的烯二炔生物合成、工程化和药物发现计划的克里思，方法是：（1）确定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小酶，（2）在中试规模上证明，“结构基因组学”方法通过解决来自所选途径的关键酶的结构来进行烯二炔生物合成的可行性，（3）推进C-1027及其工程类似物进入体内测试，以将它们实际开发成临床上有用的新抗癌药物，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药物先导物的组合。 公共卫生相关性：在美国，癌症导致每4例死亡中就有1例，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全新的、临床上有用的抗癌药物是一个关键的研究目标。烯二炔类化合物是目前存在的最有效的、高活性的抗癌剂。尽管天然烯二炔作为临床药物的应用有限，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显示出巨大的临床成功或前景，表明当利用烯二炔的极强的细胞毒性并将其递送至特定癌细胞时，烯二炔可以开发成强效药物。一个巨大的挑战是开发方法来制备烯二炔及其结构类似物，并发现新的烯二炔天然产物用于机理研究和临床开发。本研究将研究烯二炔的生物合成和工程化的新型烯二炔类似物。研究成果包括将烯二炔及其类似物开发成潜在的抗癌药物。