Biosynthesis and Synthetic Biology of Antibiotic Oligosaccharides

项目摘要 Orthosomycins是一个有效的抗生素寡糖家族，靶向广谱革兰氏阳性菌， 细菌，包括大多数耐药菌株。很少有人认识到，正霉素的子集也 革兰氏阴性耐药菌，包括肠杆菌科成员， 被疾病控制和预防中心列为人类健康的最大威胁。 正霉素在动物和哺乳动物体内均表现出高效力、良好的生物利用度和低毒性。 人类这个类的承诺是探索临床前，并通过随后的发展之一， orthosomycin、everninomicin A（Ziracin）。尽管Ziracin进入了III期临床试验，但未声明 药理学并发症导致战略决定停止这种支架的临床开发， 2000.在此期间，没有尝试改进正霉素。我们推测 由于退出原因不明，解决药理学责任是复杂的， 对everninomicin分子靶点的不完整理解，以及正骨霉素固有的挑战 化学合成，至少需要130个步骤。 最近，结合在细菌核糖体中的正霉素的结构已经被解决， 我们对它们的分子靶点和作用机制的理解，并创造机会， 核糖体相互作用和药理学性质通过有针对性的结构变化。同时，我们 开发了一套用于编辑生产生物体基因组的遗传工具，并推进了 了解正骨霉素组装的生化机制和途径。我们已经开始了，但是 未完成，探索了糖苷间原酸酯键的形成， 二氯异艾弗尼酸的附着，以及正霉素特有的尤里卡酸糖的生物合成 抗生素这种融合的进展，了解正骨霉素的生物合成和目标 识别提供了前所未有的机会，以解决限制临床应用的并发症， 这些分子通过提高它们的效力和药理学性质。为了实现这一目标，我们的具体 目的是（1）表征和调节正糖霉素与rProtein uL16的相互作用，（2）研究h89 和h91跨越正霉素的相互作用，以及（3）开发获得非天然正霉素类似物的途径， 影响rRNA h91口袋的靶向结构变化。 在这个项目中，我们概述了药理学责任的可能起源和解决方案。利用 生物合成的见解，我们将产生有针对性的变化，在支架的正霉素使用组合 基因、化学和生物化学方法。通过这些设计的变体，我们将确定 其结构变化可以改善核糖体结合，以及改变药理学性质， 解除负担，提高治疗指数。

PROJECT SUMMARY Orthosomycins are a family of potent antibiotic oligosaccharides that target a wide spectrum of gram-positive bacteria, including most antibiotic-resistant strains. It is less appreciated that subsets of orthosomycins also target gram-negative drug-resistant bacteria, including members of the Enterobacteriaceae family, which are ranked as some of the highest threats to human health by the Centers for Disease Control and Prevention. Orthosomycins demonstrate high potency, good bioavailability, and low toxicity in vivo in both animals and humans. The promise of this class was explored preclinically, and through subsequent development of one orthosomycin, everninomicin A (Ziracin). Despite Ziracin’s advancement to phase III clinical trials, unstated pharmacological complications led to a strategic decision to discontinue clinical development of this scaffold in 2000. In the intervening time, no attempts have been made to improve the orthosomycins. We speculate that addressing pharmacological liabilities was complicated due to the unknown reasons for withdrawal, the fragmentary understanding of the everninomicin molecular target, and the challenges inherent in orthosomycin chemical synthesis, which requires at least 130 steps. Recently, the structures of orthosomycins bound in the bacterial ribosome have been solved, revolutionizing our understanding of their molecular target and mechanism of action and creating opportunities to improve ribosome interactions and pharmacological properties via targeted structural changes. Contemporaneously, we developed a set of genetic tools for editing the genome of the producing organisms, as well as advanced the understanding of the biochemical mechanisms and pathways of orthosomycin assembly. We have initiated, but not completed, an exploration of the formation of the interglycosidic orthoester linkages, the formation and attachment of dichloroisoeverninic acid, and the biosynthesis of the eurekanate sugar, unique to orthosomycin antibiotics. This convergence of progress in the understanding of orthosomycin biosynthesis and target identification provides an unprecedented opportunity to address the complications limiting the clinical utility of these molecules by improving their potency and pharmacological properties. To further this goal, our specific aims are to (1) characterize and tune the interactions of orthosomycins with rProtein uL16, (2) investigate h89 and h91 spanning interactions of orthosomycins, and (3) develop access to unnatural orthosomycin analogs with targeted structural changes impacting the rRNA h91 pocket. Premise: In this project, we outline probable origins and solutions to pharmacological liabilities. Leveraging biosynthetic insights, we will generate targeted changes in the scaffolds of orthosomycins using a combined genetic, chemical, and biochemical approach. With these designed variants, we will determine the extent to which structural variations can improve ribosome binding, as well as modify pharmacological properties to remove liabilities and improve the therapeutic index.