UV-Based Approach for Accessing New Antibiotics

描述(申请人提供)：这个项目的目标是通过激活放线菌沉默的生物合成操纵子来发现新的抗生素。许多放线杆菌的全基因组测序显示，在给定物种中，编码次生代谢物的操纵子比已知化合物多10-20倍。这打开了一个诱人的机会，可以获得大量未开发的新抗生素来源。基因工程已经被用来启动放线菌中沉默的操纵子，导致产生次生代谢物。然而，工程操纵子被激活的速度非常慢，基于这种方法每年报道的代谢物不到10种。我们推测，对在体外不产生抗菌剂的菌株进行突变将使沉默的操纵子从调控限制中解脱出来，并且 然后将进行筛选，以确定产生突变的菌株。我们的初步数据显示，这种方法效果出奇地好，将一半不活跃的生物体转化为抗生素生产商。这种方法是可扩展的，我们最近使用这种方法确定了两种潜在的新型抗菌剂。在第一阶段，我们将诱变/筛选2000株用于抗生素生产的非活性菌株，旨在获得新的、潜在有用的抗菌剂。生物和化学去重复将表明化合物具有潜在的新颖性。优先选择对难治革兰氏阴性菌具有抗菌活性的广谱化合物。这些抗菌剂将进行效力、光谱、作用特异性和细胞毒性测试，并将确定通过验证的化合物的结构。用新的化学方法寻找2-3种抗菌剂将成为这一方法的原则证明。这些发现将为第二阶段的大规模药物发现工作提供坚实的基础。 与公共卫生相关：该项目的总体目标是开发一种简单有效的方法来发现新的抗生素，以对抗在美国和世界各地构成严重健康威胁的耐药病原体。随着治疗这些危险的人类感染的药物管道继续减少，迫切需要发现抗生素的新方法。

DESCRIPTION (provided by applicant): The goal of this project is to discover novel antibiotics by activating the silent biosynthetic operons of Actinobacteria. Whole genome sequencing of many Actinobacteria showed that there are 10-20 times more operons coding for secondary metabolites than known compounds in a given species. This opens an attractive opportunity to access a large untapped source of new antibiotics. Genetic engineering has been used to turn on silent operons in Actinobacteria, leading to production of secondary metabolites. However, the pace at which engineered operons are activated is very low, less than ten metabolites a year are being reported based on this approach. We reasoned that mutagenesis of isolates that do not produce antimicrobials in vitro will relieve silent operons from regulatory constraints, and screening will then identify the producing mutants. Our preliminary data showed that the approach works surprisingly well, turning over half of the inactive organisms into antibiotic producers. The method is scalable, and we recently identified two potentially novel antimicrobials using this approach. In Phase I, we will mutagenize/screen 2,000 inactive strains for antibiotic production, aiming to obtain new, potentially useful antimicrobials. Biological and chemical dereplication will indicate compounds with potential novelty. We will give priority to broad spectrum compounds with activity against difficult to treat gram-negative pathogens. The antimicrobials will be tested for potency, spectrum, specificity of action and cytotoxicity, and th structure of compounds that pass validation will be determined. Finding 2-3 antimicrobials with novel chemistry will serve as proof-of-principle for this approach. These findings will provide a solid basis for a large-scale drug discovery effort in Phase II. PUBLIC HEALTH RELEVANCE: The overall goal of the project is to develop a simple and efficient method to discover novel antibiotics to combat drug-resistant pathogens that pose a serious health threat in the US and around the world. Novel approaches for discovering antibiotics are desperately needed as the drug pipeline for treating these dangerous human infections continues to diminish.