Biosynthesis of Maytansinoids and Analogs

描述(申请人提供)：由放线菌产生的阿萨米托星家族的阿萨霉素类微生物代谢物，以及结构几乎相同的植物来源的美丹辛及其同系物是非常有效的抗肿瘤药物。尽管有毒性，美坦辛一度被认为是临床开发的杰出候选者，但它在II期临床试验中未能显示出显著的疗效，可能是由于剂量限制毒性。然而，人们对这些化合物的兴趣仍在继续，例如，作为抗体靶向传递的“弹头”，它们的高效力要求进一步努力修改它们的结构，以确定具有较高抗肿瘤活性且毒性低于母体化合物的类似物。这些化合物的结构复杂性将修饰结构的化学方法限制在由天然产物起始材料半合成而成的方法；更深层次的主干结构修饰需要基于产生母体化合物的生物合成机制的遗传改变的生化方法。 考虑到这一原理，我们已经克隆并测序了拟青霉的抗丝分裂素(ASM)生物合成基因簇，并正在分析其单个基因的功能。在接下来的四年里，我们希望通过确定所有下游修饰基因/酶的功能，在异源宿主中表达组装阿米替丁主干的聚酮合成酶(PKS)并研究其结构和操作模式，以及澄清ASM PKS功能所需的罕见的聚酮链延伸单元的结构和形成方式来继续这项工作。基于从这项工作中获得的见解，我们将组装一个系统，该系统允许所有ASM生物合成基因在异源宿主天蓝色链霉菌(Streptomyces Coelicolor)的一系列基因盒中表达，在外部启动子的控制下产生转氨菌素。一旦这个体系建立，我们将通过在表达的基因簇中引入遗传修饰来证明生产阿司米托星类似物的可行性。此外，为了提高亲本和工程突变化合物的产量，我们计划研究阿司米托星在A.pretiosum中产生的遗传控制。因此，这项工作将为制备阿司匹林的结构类似物提供工具，这些结构类似物可以作为抗癌药物评估改善的治疗比率。

DESCRIPTION (provided by applicant): The ansamitocin family of ansamycin-type microbial metabolites produced by the Actinomycete Actinosynnema pretiosum and the structurally almost identical plant-derived maytansine and its congeners are extraordinarily potent antitumor agents. Despite its toxicity, maytansine at one time was considered an outstanding candidate for clinical development, but it failed to show significant efficacy in phase II clinical trials, probably due to dose-limiting toxicity. However, interest in these compounds continues, e.g., as "warheads" for antibody-targeted delivery, and their high potency calls for further efforts to modify their structures with the goal of identifying analogs which retain high antitumor activity coupled with lower toxicity than the parent compounds. The structural complexity of these compounds limits chemical approaches to modified structures to those accessible by semi-synthesis from the natural product starting materials; more deep-seated backbone structural modifications require biochemical approaches based on genetic alteration of the biosynthetic machinery generating the parent compounds. With this rationale in mind we have cloned and sequenced the ansmitocin (asm) biosynthetic gene cluster from A. pretiosum and are in the process of analyzing the functions of its individual genes. In the next 4-year period of this grant we wish to continue this work by determining the functions of all the downstream modification genes/enzymes, expressing the polyketide synthase (PKS) assembling the backbone of ansamitocin in a heterologous host and studying its structure and mode of operation, and clarifying the structure and mode of formation of a rare polyketide chain extension unit required for the function of the asm PKS. Based on the insights gained from this work, we will then assemble a system, which allows the expression of all the asm biosynthetic genes to produce ansamitocins in a heterologous host, Streptomyces coelicolor, from a series of gene cassettes under the control of an external promoter. Once this system is established, we will demonstrate the feasibility of producing ansamitocin analogs by introducing genetic modifications into the expressed gene cluster. In addition we plan to investigate the genetic control of ansamitocin production in A. pretiosum with the aim to increase yields of the parent and engineered mutant compounds. This work will thus provide the tools for the preparation of structural analogs of the ansamitocins, which can be evaluated for an improved therapeutic ratio as anticancer agents.