Discovery and Characterization of Novel Nitroaromatic Antibiotics

新型硝基芳香族抗生素的发现和表征

• 批准号: 8397213
• 负责人: Kou-San Ju
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8397213
• 关键词: Actinobacteria class; Agriculture; Anabolism; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Antifungal Agents; Antineoplastic Agents; Aromatic Amines; Bacteria; Bioinformatics; Biological; Biological Assay; Biological Factors; Biological Products; Biomedical Research; Breathing; Cell Line; Characteristics; Chemicals; Chromatography; Collection; DNA; Dioxygenases; Enzymes; Gene Cluster; Gene Deletion; Genes; Genome; Genomics; Goals; Health; Human; Incidence; Individual; Infection; Ions; Knock-out; Knowledge; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Medicine; Methods; Mining; Molecular Genetics; Multi-Drug Resistance; Nitric Oxide Synthase; Pathway interactions; Pattern; Pharmaceutical Preparations; Phylogenetic Analysis; Plasmids; Production; Property; Recording of previous events; Reporting; Research; Role; Sampling; Screening procedure; Sequence Analysis; Services; Source; Spottings; Structure; Techniques; Testing; anticancer research; base; combat; functional group; genome sequencing; high throughput screening; insight; knockout gene; microbial; microorganism; milligram; next generation; novel; pathogen; programs; research study; resistant strain

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.

描述（由申请人提供）：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的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，由于传统筛选方法的有前途的候选人持续下降，预计利润水平较低。本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及其生物合成的表征来应对新抗生素的挑战。这些化合物是一类未被充分研究的天然产物，也是新药和药物的有前途的来源。虽然迄今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作为药物的丰富历史为具有该官能团的新药的合成提供了灵感，即使在今天也在继续。“基因组挖掘”方法将用于筛选整个美国农业部农业研究服务（ARS）放线菌菌株收集（约7,500），以发现新型硝基芳香族抗生素。整个USDA-ARS放线菌集合中每个菌株的基因组DNA将被提取并排列在96孔格式中，为天然产物发现创建统一的高通量平台。具有合成硝基芳香族化合物能力的菌株将使用PCR筛选来鉴定，所述PCR筛选使用芳香胺双加氧酶的简并引物，所述芳香胺双加氧酶是负责将硝基添加到芳香环上的一组独特的酶，并且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。候选菌株的培养基提取物将通过各种色谱法、衍生化和质谱分析进行筛选，以鉴定新型和生物活性硝基芳烃。将对选定的化合物进行纯化（根据其丰度和初步生物活性进行优先排序），以表征其化学和生物学特性。同时，下一代测序和分子遗传学的最新进展将被应用于揭示其生物合成的基础。全基因组测序，基因敲除和异源表达实验将用于识别和阐明途径基因在硝基芳烃生物合成中的作用。这些研究的结果不仅有望产生可能用作药物和抗生素的新化合物，而且还将扩大我们对这类相对未开发的天然产物的了解。 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的发病率在世界范围内持续增加，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。该项目旨在通过发现从微生物中生产的新药和抗生素并研究它们是如何制造的来应对这一挑战。