Expanding the genetic code of streptomycetes: a platform for new antibiotics

描述(由申请人提供)：用于治疗细菌感染的新型抗生素的发现和开发已经显著放缓。由于多重耐药细菌菌株的出现，这是一个严重威胁人类健康的问题，对医院和整个社区都构成了威胁。长期目标是创造新的方法来研究导致疗效或耐药性的机制，同时开发能够克服耐药性的抗生素。这项应用的目标是将非天然氨基酸定点结合到由土壤细菌链霉菌产生的核糖体合成的硫肽抗生素中。核心假设是，核糖体掺入这些非天然氨基酸将创造一种快速而通用的方法来研究现有和未来的硫肽作用机制，并极大地改善人类临床使用的硫肽的药理性质。这与以前的合成策略不同，以前的合成策略受到硫肽的高分子复杂性的阻碍。中心假设是基于非天然氨基酸被证明用于创建生物物理和细胞生物探针、生物活性多肽和抗体支架以及改进的药物生物偶联。提出这一建议的理由是，了解新的抗生素硫肽机制和创造新的硫肽抗生素支架有可能转化为治疗每年报告的大约200万例耐药细菌感染的新药。这一中心假设将以以下具体目标进行检验：1)阐明贝尼那霉素的抗生素作用机制；2)确定硫肽GE37468的改进类似物。将非天然氨基酸插入到硫肽中，将使标记研究能够检验它具有独特的双重作用模式的假设，而对GE37468‘S核心支架的修饰将产生改进的硫肽，以对抗多重耐药细菌菌株。非天然氨基酸的掺入将使用已建立的正交氨酰-tRNA合成酶(AARS)/tRNA对和已知的产生硫肽的基因簇进入链霉菌。这种方法是创新的，因为它将现有的生物合成核糖体机制与化学合成(非天然氨基酸)相结合，以产生具有精确工程结构和反应活性的硫肽。值得注意的是，这项研究将增加我们对新的抗生素机制的理解，并为硫肽抗生素类似物的创建创造一个新的模型。

DESCRIPTION (provided by applicant): The discovery and development of fundamentally new classes of antibiotics to treat bacterial infections has slowed dramatically. This is a critica problem to human health due to the emergence multi-drug resistant bacterial strains, which present a threat to both hospitals and communities at large. The long-term goal is to create new methods for studying the mechanisms leading to efficacy or resistance, while developing antibiotics that can overcome resistance. This application's objective is to site-specifically incorporate unnatural amino acids into ribosomally synthesized thiopeptide antibiotics produced by the soil bacteria, Streptomyces. The central hypothesis is that ribosomal incorporation of these unnatural amino acids will create a rapid and general method to study existing and future thiopeptide mechanisms of action, as well as drastically improve the pharmacological properties of thiopeptides for human clinical use. This contrasts with previous synthetic strategies, which have been hindered by the high molecular complexity of thiopeptides. The central hypothesis is based on the demonstrated utility of unnatural amino acids for the creation of biophysical and cell biological probes, biologically active peptide and antibody scaffolds, and improved medicinal bioconjugates. The rationale for this proposal is that understanding novel antibiotic thiopeptide mechanisms and creation of new thiopeptide antibiotics scaffolds has the potential to translate into new medicines to treat the roughly 2 million cases of drug-resistant bacterial infections reported each year. The central hypothesis will be tested with the following specific aims: 1) Elucidate berninamycin's antibiotic mechanism of action and 2) Identify improved analogues of thiopeptide GE37468. Insertion of unnatural amino acids into thiopeptides berninamycin will allow labeling studies to test the hypothesis that it possess a unique dual mode of action, and modification of GE37468's core scaffold will generate improved thiopeptides to test against multi-drug resistant bacterial strains. Incorporation of unnatural amino acids will be accomplished using established orthogonal aminoacyl- tRNA synthetase (aaRS)/tRNA pairs and known thiopeptide producing gene clusters into Streptomycetes. This approach is innovative because it combines both existing biosynthetic ribosomal machinery with chemical synthesis (unnatural amino acids) to produce thiopeptides with precisely engineered structure and reactivity. Significantly, this research stands to increase our understanding of novel antibiotic mechanisms, and create a new model for the creation of thiopeptide antibiotic analogues.