Discovery and Characterization of Novel Nitroaromatic Antibiotics

描述(申请人提供)：随着病原体中的抗生素耐药性水平、多药耐药菌株的感染以及癌症发病率在全球范围内持续增加，迫切需要新的抗生素来对抗这些对人类健康日益增长的威胁。不幸的是，只有少数生物制药公司保留了积极的天然产品发现计划，这是因为传统筛选方法的有前途的候选公司持续下降，而且预计利润水平较低。该项目旨在通过从培养的放线菌中发现新的硝基芳香族天然产物并对其生物合成进行表征来应对新抗生素的挑战。这些化合物是一类未被研究的天然产物，也是新药和药物的有前途的来源。虽然到目前为止只有少量的硝基芳香族天然产物被报道，但这些化合物广泛的生物活性和丰富的药用历史激发了合成具有这个官能团的新药的灵感，这种做法至今仍在继续。“基因组挖掘”方法将被用于筛选整个美国农业部-农业研究服务(ARS)放线菌菌株集合(约7500株)，以发现新的硝基芳香族抗生素。整个USDA-ARS放线菌库中每个菌株的基因组DNA将被提取并以96孔格式排列，为天然产品发现创建一个统一的高通量平台。能够合成硝基芳香化合物的菌株将通过使用芳香胺双加氧酶(负责将硝基添加到芳环上的一组独特的酶)的简并引物进行PCR筛选，以及对含有一氧化氮合酶的天然产物基因簇的测序基因组进行生物信息学分析来鉴定。候选菌株的介质提取物将通过各种层析、衍生化和质谱分析进行筛选，以鉴定新的和具有生物活性的硝基芳烃。将对选定的化合物进行提纯(根据其丰度和初步生物活性进行优先排序)，以确定其化学和生物学特性。同时，将应用下一代测序和分子遗传学的最新进展来揭示它们的生物合成基础。将使用全基因组测序、基因敲除和异源表达实验来鉴定和阐明途径基因在硝基芳烃生物合成中的作用。这些研究的结果预计不仅会产生可能用作药物和抗生素的新化合物，而且还会扩大我们对这类相对未被探索的天然产品的了解。 公共卫生相关性：随着病原体的抗生素耐药性水平、多药耐药菌株的感染以及癌症发病率在全球范围内继续增加，迫切需要新的药物来应对这些对人类健康日益增长的威胁。该项目试图通过发现从微生物中产生的新药和抗生素并调查它们是如何制造的来应对这一挑战。

DESCRIPTION (provided by applicant): As levels of antibiotic resistance in pathogens, infections by multi-drug resistant strains, and incidence of cancer continues to increase worldwide, new antibiotics are urgently needed to combat these growing threats to human health. Unfortunately, only few biopharmaceutical companies have retained aggressive natural product discovery programs due to the continued decline in promising candidates from traditional screening methods and low levels of projected profits. This project seeks to answer the challenge for new antibiotics through the discovery of novel nitroaromatic natural products from cultivated actinomycetes and the characterization of their biosynthesis. These compounds are an understudied class of natural products and a promising source of new drugs and medicines. Although only a small number of nitroaromatic natural products have been reported to date, the wide-ranging bioactivities and a rich history as medicines of these compounds has served as inspiration for the synthesis of new drugs with this functional group, which continues even today. A "genome-mining" approach will be used to screen the entire USDA-Agricultural Research Service (ARS) actinomycete strain collection (ca 7,500) for the discovery of novel nitroaromatic antibiotics. Genomic DNA from each strain in the entire USDA-ARS actinomycete collection will be extracted and arrayed in 96-well format, creating a unified high-throughput platform for natural product discovery. Strains with the ability to synthesize nitroaromatic compounds will be identified using PCR screening using degenerate primers to aromatic amine dioxygenases, a unique group of enzymes responsible for the addition of the nitro group onto aromatic rings, and bioinformatic analyses of sequenced genomes for natural product gene clusters containing nitric oxide synthase. Media extracts from candidate strains will be screened by a variety of chromatography, derivatization, and mass spectrometry analyses to identify novel and bioactive nitroarenes. Select compounds will be purified (prioritized based on their abundance and preliminary bioactivities) for characterization of their chemical and biological properties. Simultaneously, recent advances in next-generation sequencing and molecular genetics will be applied to uncover the basis for their biosynthesis. Whole genome sequencing, gene-knockouts, and heterologous expression experiments will be used identify and elucidate the role of pathway genes in nitroarene biosynthesis. Results from these studies are expected to not only yield new compounds that may useful as drugs and antibiotics, but also expand our knowledge about this relatively unexplored group of natural products. PUBLIC HEALTH RELEVANCE: As levels of antibiotic resistance in pathogens, infections by multi-drug resistant strains, and the incidence of cancer continues to increase worldwide, new medicines are urgently needed to combat these growing threats to human health. This project seeks to answer this challenge by discovering new drugs and antibiotics produced from microorganisms and investigating how they are made.