Biosynthesis and Synthetic Biology of Antibiotic Oligosaccharides

项目总结 正霉菌素是一类有效的抗生素低聚糖家族，以广谱的革兰氏阳性菌为靶标 细菌，包括大多数抗药性菌株。不太了解的是，直霉菌素的亚群也 针对革兰氏阴性耐药细菌，包括肠杆菌科家族的成员，它们是 被疾病控制和预防中心列为对人类健康的最大威胁之一。 正霉素在动物和动物体内均显示出高效、良好的生物利用度和低毒 人类。这门课的前景是在临床前探索的，并通过随后开发的一个 正霉素、伊维诺米星A(齐拉星)。尽管齐拉星进入了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年。在这段时间里，没有人试图改进正霉素类。我们推测 由于停药的原因未知，解决药物责任问题很复杂， 对伊维诺米星分子靶点的片段性了解，以及正霉素固有的挑战 化学合成，这至少需要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，这是一场革命性的 我们对它们的分子靶点和作用机制的了解，并创造机会来改进 通过靶向结构变化的核糖体相互作用和药理学特性。与此同时，我们 开发了一套用于编辑生产有机体基因组的遗传工具，以及先进的 了解正霉素组装的生化机制和途径。我们已经发起了，但是 未完成，探索糖苷间邻酯键的形成、形成和 正霉素所特有的二氯异乙烯基酸的结合和优尿酸糖的生物合成 抗生素。正霉素生物合成和靶标研究进展的趋同 鉴定为解决限制其临床应用的并发症提供了前所未有的机会。 这些分子通过改善它们的效力和药理特性。为了推进这一目标，我们的具体目标 目的是(1)表征和调整原霉菌素与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 有针对性的结构变化影响rRNA h91口袋。 前提：在这个项目中，我们概述了药物责任的可能来源和解决方案。利用 生物合成的洞察，我们将产生有针对性的改变，在支架上使用联合 遗传、化学和生化方法。有了这些设计的变体，我们将确定 哪些结构变化可以改善核糖体结合，以及改变药理性质 解除债务，提高治疗指标。

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.