Combinatorial biosynthesis and metabolism studies of novel tetracenomycins

项目摘要/摘要 新型四环素的组合生物合成及代谢研究 蒽环类药物是天然产物抗生素，是最有效的抗癌药物之一。 诊所。蒽环类化合物的生物合成修饰是一种很有前途的合成新的化学类似物的策略 具有不同的抗癌活性和扩大的治疗窗口。埃洛霉素(ELM，1)是一种 橄榄链霉菌Tü2353产生的以四环己烯为特征的蒽环类抗生素 苷元和添加的8-O-2，3，4-三-O-甲基-a-L鼠李糖。埃洛阿霉素和四环素 (中药)由于抑制DNA拓扑异构酶II而表现出温和的抗增殖活性。此外， 脱氧糖部分的优化对于提高细胞穿透性、效力、清除性和 类似物的新陈代谢。然而，我们仍然没有完全了解结构-活性-关系 中药脱氧糖对细胞增殖活性和药物代谢的影响。此外，尽管准备了&gt；20 不同的ELM类似物，这些类似物的SAR尚未在癌症小组中进行严格评估 细胞系。我们的长期目标是生产具有更好的抗增殖活性的蒽环类类似物。至 为了促进类似物的生产，我们将利用变铅青链霉菌(Cos16F4)表达系统来改变 通过组合生物合成的四环素类药物的脱氧糖部分。这一综合平台将 促进用于人类癌症的新药先导的开发。拟议方案的总体目标 研究包括三个方面：(1)合成新的中药衍生物；(2)验证其抗癌活性 以及(3)建立一种S9组分分析方法，以评估1，4，4，6，6，6，6，6，6，6，6，6，6，6，6，6，6，5，5，6，6，6，5，6，6，5，5，6，6，6，6，6，5，6，5，6，5，5，5，5，5，6，5，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，我们的 假设(1)ElmGT具有足够的“底物弹性”来转移预定的TDP-脱氧糖 8-去甲基四环素C的供体，(2)添加的脱氧糖将通过以下途径改变抗癌活性 与拓扑异构酶II和DNA结合；(3)通过与肝脏的不同结合改变药物代谢 细胞色素P450s。我们的具体目标将检验这些假设：(目标1)我们将异源表达“糖” 变铅青链球菌(COS16F4)中的“质粒”改变中药脱氧糖的部分，(目标2)我们将评估 新的中药类似物的抗增殖活性，(目标3)我们将建立一种S9组分的分析方法来确定 中药类似物的代谢谱。其基本原理是“底物-灵活的”糖基转移酶ElmGT， 应该接受新颖的TDP-脱氧糖供体。这一贡献意义重大，因为预期 生产平台将更加充分地探索中药类似物的化学空间。此外，这项研究是 创新是因为我们正在研究脱氧糖修饰对抗肿瘤活性和 四环素的代谢，药物的性质，目前还不太清楚。

Project Summary/Abstract Combinatorial biosynthesis and metabolism studies of novel tetracenomycins Anthracyclines are natural product antibiotics that are among the most effective anticancer drugs used in the clinic. Biosynthetic modification of anthracyclines is a promising strategy to generate new chemical analogs with differentiated anticancer activities and expanded therapeutic windows. Elloramycin (ELM, 1) is an anthracycline antibiotic produced by Streptomyces olivaceus Tü 2353 that features a tetracyclic elloramycinone aglycone and an appended 8-O-2,3,4-tri-O-methyl-a-L-rhamnose sugar. Elloramycins and tetracenomycins (TCMs) exhibit mild antiproliferative activity due to inhibition of DNA topoisomerase II. Furthermore, optimization of the deoxysugar moiety is essential for refining the cellular penetration, potency, clearance, and metabolism of the analogs. However, we still do not fully understand the structure-activity-relationships of the TCM deoxysugar on antiproliferative activity and drug metabolism. Furthermore, despite preparation of >20 different analogs of ELM, the SAR of these analogs has not yet been assessed rigorously in a panel of cancer cell lines. Our long-term goal is to produce anthracycline analogs with improved antiproliferative activity. To facilitate production of analogs, we will exploit the S. lividans (cos16F4) expression system to alter the deoxysugar moiety of tetracenomycins via combinatorial biosynthesis. This comprehensive platform will facilitate development of new drug leads for use in human cancer. The overall objectives of the proposed research are three-fold: (1) to synthesize novel TCM derivatives, (2) to validate the anticancer activity of the TCMs, and (3) to develop a S9 fraction assay to evaluate metabolism of 1 and the most active analogs. Our hypotheses are that (1) ElmGT will be “substrate-flexible” enough to transfer the intended TDP-deoxysugar donors to 8-demethyl-tetracenomycin C, (2) the appended deoxysugar will alter the anticancer activity via binding to topoisomerase II and DNA, and (3) it will alter drug metabolism via differential binding to hepatic cytochrome P450s. Our specific aims will test these hypotheses: (Aim 1) we will heterologously express “sugar plasmids” in S. lividans (cos16F4) to alter the TCM deoxysugar moiety, (Aim 2) we will evaluate the antiproliferative activity of the new TCM analogs, (Aim 3) we will develop a S9 fraction assay to determine the metabolic profile of TCM analogs. The rationale is that the “substrate-flexible” glycosyltransferase, ElmGT, should accept the novel TDP-deoxysugar donors. This contribution is significant because the expected production platform will more fully explore the chemical space of TCM analogs. Furthermore, this research is innovative because we are investigating the impact of deoxysugar modifications on antitumoral activity and metabolism of tetracenomycins, drug properties which are currently not well-understood.