UV-Based Approach for Accessing New Antibiotics

描述（由申请人提供）：本项目的目标是通过激活放线菌的沉默生物合成操纵子来发现新的抗生素。许多放线菌的全基因组测序表明，在给定物种中，编码次级代谢产物的操纵子比已知化合物多10-20倍。这为获得大量尚未开发的新抗生素提供了一个有吸引力的机会。基因工程已被用于开启放线菌中的沉默操纵子，导致次级代谢产物的产生。然而，工程操纵子被激活的速度非常低，基于这种方法，每年报告的代谢物不到10种。我们推断，在体外不产生抗菌剂的分离株的诱变将使沉默操纵子摆脱调控约束， 然后筛选将鉴定产生突变体。我们的初步数据表明，这种方法的效果令人惊讶，将一半以上的非活性生物转化为抗生素生产者。该方法是可扩展的，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2，000株无活性菌株用于抗生素生产，旨在获得新的，潜在有用的抗菌剂。生物和化学去复制将指示具有潜在新奇的化合物。我们将优先考虑对难以治疗的革兰氏阴性病原体具有活性的广谱化合物。将检测抗菌剂的效价、光谱、作用特异性和细胞毒性，并确定通过验证的化合物的结构。发现2-3种具有新化学性质的抗菌剂将作为这种方法的原理证明。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。 公共卫生相关性：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。由于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.