Enediyne Biosynthesis and Engineering

描述(由申请人提供)：为了回应NIH NOT-OD-09-058题为“NIH宣布恢复法案资金可用于竞争性修订申请”的问题，我们希望延长在NIH资助2R01 CA78747期间带头进行的名为“烯二炔生物合成和工程”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、临床有用的抗癌药物构成了国家健康和研究的当务之急。烯二炔类化合物是当今存在的最有效、活性最高的抗癌药物，它们作为抗癌药物的用途已在临床上得到证明。一个巨大的挑战是开发制备烯二炔类化合物及其结构类似物的方法，并为机制研究和临床开发发现新的烯二炔类天然产物。在这一竞争性修订中，我们建议(1)从结构上(通过X射线结晶学手段)表征烯二炔聚酮合成酶(PKSE)及其用于烯二炔核心生物合成的相关酶，以及从选定的9元和10元烯二炔生物合成途径中选择的其他酶；(2)生产和进一步分析具有独特可开发生物物理性质的工程烯二炔，使其具有潜在的临床应用；以及(3)从基于基因组挖掘的微生物来源中分离和表征新的烯二炔。我们的假设是：(1)与烯二炔生物合成有关的一些新酶的特性，特别是C-1027、新卡西诺抑素(NCS)、马多肽素(MDP)、石灰霉素(CAL)、埃斯帕米星(ESP)和动力霉素(Dyn))的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能够而且确实显示出比母体化合物更好的生物活性；此类化合物只有通过提高产量才能进一步研究；以及(3)在基因组挖掘的基础上鉴定的新微生物产生新的并且具有潜在医学意义的二炔类化合物。本竞争性修订应用的具体目的是：(1)所选择的烯二炔PKS及其相关酶的体内和体外表征及其在9-(C-1027、NCS和MDP)和10-元(CAL、ESP和DYN)烯二炔核心生物合成中的作用；(2)通过X射线结晶学表征从烯二炔(C-1027、NCS、MDP、CAL和DYN)生物合成机制中选择的酶的结构特征；(3)分离工程C-1027类似物以评估它们作为体内抗癌药物的作用；(4)新的9元或10元烯二炔天然产物的分离和结构鉴定。这些天然产物分别来自加那氏链霉菌NRRL B-12104、东方链球菌ATCC43491和橙色链球菌IFO13005。这些研究的结果将极大地加快我们的烯二炔生物合成、工程和药物发现计划的步伐，方法是：(1)定义将新生的线性多烯中间体从endiyne PKSE转化为特征的endiyne核心结构所需的最低限度的酶；(2)在中试规模上证明通过从选定的途径解决关键酶的结构来进行endiyne生物合成的“结构基因组学”方法的可行性；(3)将C-1027及其工程类似物推进体内测试，以便现实地将它们开发成临床上有用的新型抗癌药物；以及(4)通过分离新的endiyne天然产品来扩大endiyne抗癌药物和药物先导化合物的产品组合。 与公共卫生相关：在美国，每4人中就有1人死于癌症，预计2008年美国将有565,650人死于癌症。因此，优化现有药物并从根本上开发临床上有用的抗癌药物是一个重要的研究目标。烯二炔类化合物是目前存在的最有效、活性最高的抗癌药物。尽管天然烯二炔类药物的临床应用有限，但基于聚合物的给药系统和烯二炔抗体结合物在抗癌化疗中显示出巨大的临床成功或前景，表明当利用其极强的细胞毒性并将其传递给特定的癌细胞时，烯二炔类化合物可以开发成强大的药物。一个巨大的挑战是开发方法来制造烯二炔及其结构类似物，并发现用于机制研究和临床开发的新的烯二炔天然产物。这项研究将研究烯二炔的生物合成和工程设计的新型烯二炔类似物。其成果包括将烯二炔类及其类似物开发为潜在的抗癌药物。

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.