Enediyne Antitumor Antibiotic Synthesis - Streptomycetes

烯二炔抗肿瘤抗生素合成 - 链霉菌

基本信息

批准号：
6898468
负责人：
Ben Shen
金额：
$ 10.69万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-09-01 至 2007-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6898468
关键词：
SDS polyacrylamide gel electrophoresis Streptomyces aminosugar antineoplastic antibiotics drug design /synthesis /production gene expression genetic manipulation high performance liquid chromatography molecular cloning neocarzinostatin nucleic acid sequence protein purification southern blotting

项目摘要

DESCRIPTION (provided by applicant): Enediyne antibiotics are the most potent, highly active antitumor agents in existence today. Neocarzinostatin (NCS) and its conjugates have shown great clinical promise in anticancer chemotherapy, and several calicheamicin-antibody and C-1027-antibody conjugates have produced outstanding results in recent clinical trials or model studies. Therefore, it is a critical research goal to develop ways to make the enediynes and their analogs for further studies. This application proposes to clone and characterize the genes for NCS production in Streptomyces carzinostaticus, complementing our on going effort for C-1027 in Streptomyces globisporus, in an attempt to synthesize novel antitumor drugs by manipulating genes governing enediyne antibiotic biosynthesis. This proposal is based on the hypothesis that the NCS chromophore is derived from three biosynthetic building blocks an enediyne core, a deoxy aminosugar, and a polyketide naphthoic acid moiety. Three corollaries of this hypothesis are (1) that the extensive knowledge of deoxysugar and polyketide biosynthesis in actinomycetes should greatly aid the effort to clone the genes for deoxy aminosugar and naphthoic acid biosynthesis from S. carzinostaticus, (2) that the structural similarity between the enediyne cores of NCS and C-1027 provides an unique opportunity to decipher the genetic and biochemical basis of enediyne core assembly, and (3) that the genetic tools and technology developed in Streptomyces species for antibiotic biosynthesis should be directly applicable to manipulating NCS and C-1027 biosynthesis genes for yield improvement and analog or novel enediyne production. The specific aims for the five-year award period are (1) to clone and sequence the entire NCS biosynthetic gene cluster from S. carzinostaticus, (2) to develop a genetic system for S. carzinostaticus to manipulate NCS biosynthesis in viva, (3) to confirm the cloned S. carzinostaticus DNA encoding NCS biosynthesis and to localize the boundaries of the NCS gene cluster by gene disruption and replacement experiments, and (4) to identify genes essential for the enediyne core biosynthesis by comparing the NCS and C-1027 biosynthetic gene clusters and to demonstrate the synthesis of an enediyne core structure by expressing the corresponding genes in a heterologous host. The outcome of these studies will reveal new insights into the biosynthesis of the enediyne family of antibiotics, shedding light on rational engineering of enediyne biosynthesis in these as well as other enediyne-producing organisms, and could realistically lead to the making of fundamentally new, clinically useful antitumor agents

描述（由申请人提供）：Enediyne抗生素是最有效的，当今存在的高度活跃的抗肿瘤剂。 Neocarzinostatin（NCS）和它的缀合物在抗癌化学疗法中表现出巨大的临床承诺，还有几种Calicheamicin-抗体和C-1027-抗体结合物具有在最近的临床试验或模型研究中产生了出色的结果。因此，这是开发方法来制造方法的关键研究目标 Enediynes及其类似物进行进一步研究。此申请提出克隆并表征链霉菌中NCS产生的基因 Carzinostaticus，补充我们在链霉菌中为C-1027的努力全球孢子试图通过操纵新型抗肿瘤药物来综合新型抗肿瘤药物控制Enediyne抗生素生物合成的基因。该提议基于 NCS发色团源自三个生物合成的假设构建嵌段，一个deoxy氨基加糖和聚酮化合物萘酸部分。该假设的三个推论是（1）在放线菌中对脱氧和聚酮化合物生物合成的广泛了解应大力帮助克隆脱氧氨基加的基因和脑链链霉菌的萘酸生物合成，（2）结构 NCS和C-1027的Enediyne内核之间的相似性提供了独特的破译Enediyne Core的遗传和生化基础的机会组装和（3）遗传工具和技术在抗生素生物合成的链霉菌物种应直接适用操纵NCS和C-1027生物合成基因，以提高产量和模拟或新型Enediyne生产。五年奖励的具体目标周期为（1）以克隆并对整个NCS生物合成基因簇进行测序从脑链球链球菌开始，（2）开发用于S.的遗传系统 carzinostaticus在Viva中操纵NCS生物合成，（3）确认克隆的Carzinostaticus DNA编码NCS生物合成并定位 NCS基因簇的边界通过基因破坏和替代实验和（4）识别endiyne核心必不可少的基因通过比较NCS和C-1027生物合成基因簇和到生物合成通过表达来证明endiyne核心结构的合成异源宿主中的相应基因。这些研究的结果将揭示有关Enediyne家族的生物合成的新见解抗生素，阐明了Enediyne生物合成合理工程在这些以及其他产生的生物中，可以实际上导致制造根本新的，临床上有用的抗肿瘤剂