Analytical Metagenomics Paradigm for Structure Based Screening

基于结构的筛选的分析宏基因组学范式

• 批准号: 8310684
• 负责人: DAVID Alan MEAD
• 金额: $ 16.38万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-15 至 2013-11-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8310684
• 关键词: Academia; Address; American; Analytical Chemistry; Anti-Infective Agents; Antibiotic Resistance; Antibiotics; Area; Bacteria; Bacterial Infections; Biological Factors; Businesses; Cell Extracts; Chemicals; Clinical; Cloning; Collaborations; Communicable Diseases; Complex; DNA; Data; Databases; Development; Environment; Event; Fermentation; Genbank; Generations; Genes; Genetic Variation; Genomics; Goals; Incidence; Lead; Libraries; Life; Marriage; Metagenomics; Methicillin Resistance; Methodology; Methods; Molecular Weight; Multi-Drug Resistance; Noise; Pathway interactions; Pharmaceutical Preparations; Phase; Proteomics; Public Health; Publications; Recombinants; Research; Research Proposals; Resistance; Resources; Science; Scientist; Screening procedure; Sequence Analysis; Services; Shuttle Vectors; Signal Transduction; Small Business Innovation Research Grant; Soil; Source; Structure; Technology; Therapeutic; Time; Triage; analytical method; base; blind; cost; drug discovery; expression cloning; fight against; improved; innovation; man; metabolomics; methicillin resistant Staphylococcus aureus; microbial; microbial community; microbiome; microorganism; microorganism culture; new technology; next generation; novel; pathogen; professor; scaffold; small molecule; tool

DESCRIPTION (provided by applicant): Each year, 100,000 Americans perish due to untreatable bacterial infections. The societal benefits of new antibiotic compounds that are effective against numerous multiple drug resistant pathogens would be significant. The best possible source for new antibiotic scaffolds with potentially novel mechanisms of action is within natural environments, particularly soils, which have the greatest diversity of microbial life. Our recent research advances the science of metagenomics, the cloning of DNA from entire microbial communities, to discover novel antibiotics and identify the best lead candidates for clinical development. A team of scientists from academia and Lucigen Corporation have united four key technological breakthroughs that together resulted in the next generation metagenomic library. This library combined 1) an improved methodology for the isolation and purification of high molecular weight genomic DNA from soil microorganisms; 2) a new broad host range shuttle vector for enhanced expression of cloned DNAs; 3) a random shear cloning method to produce very large insert sizes (>100 kb); and 4) a rapid and improved screening method to identify antibiotic-producing clones within a metagenomic library. The library was screened against a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), resulting in the identification of 28 metagenomic clones that produce anti-MRSA compounds, a hit rate of 1 in 685. Twelve of these anti-MRSA clones were analyzed by sequencing and found to have very large insert sizes (average 113.5 kb) and novel genetic diversity not encountered before. Moreover, one of the clones was found to produce a novel metabolite. These results are 10-100 fold more efficient than previous efforts. However, the primary bottleneck now is elucidating the structure of these compounds. Conventional technologies often require many man-years of effort to elucidate a single structure. This Phase I research proposal endeavors to improve that level by approximately ten-fold. In fact we propose to find new small molecules from this next generation metagenomics library by screening for structures. If successful the rate of natural product discovery could be accelerated many fold compared to functional based screens. PUBLIC HEALTH RELEVANCE: In the fight against microbial infectious disease we are losing ground due to the development of antibiotic resistance and our inability to find replacement drugs. The loss of life and the burden of treatment is a significant public health threat to American citizens. The proposed research unleashes a new set of tools for drug discovery that is 10-100 times more efficient than conventional technologies.

描述（由申请人提供）：每年，由于细菌感染不可治疗，有100,000名美国人灭亡。针对多种多种耐药病原体有效的新抗生素化合物的社会益处将是显着的。具有潜在新型作用机制的新抗生素支架的最佳来源是在自然环境中，尤其是土壤，这些土壤具有微生物寿命最大的多样性。我们最近的研究推进了宏基因组学的科学，即从整个微生物群落的DNA克隆，以发现新颖的抗生素并确定临床开发的最佳铅候选者。来自学术界和Lucigen Corporation的科学家团队已经团结了四个关键的技术突破，从而导致了下一代元基因组图书馆。该文库结合了1）改进的方法，用于从土壤微生物中分离和纯化高分子量基因组DNA； 2）一种新的广泛的寄主范围航天飞机向量，以增强克隆DNA的表达； 3）一种随机的剪切克隆方法，可产生非常大的插入尺寸（> 100 kb）； 4）一种快速而改进的筛选方法，以鉴定宏基因组文库中产生抗生素的克隆。对图书馆进行了筛查，筛选了金黄色葡萄球菌（MRSA）的临床分离株，从而鉴定出28种产生抗MRSA化合物的宏基因组克隆的鉴定，685中的命中率为1，而在685中的命中率为1个，并通过序列分析了这些抗MRSA Clone，并通过序列分析了，并发现了这些抗MRSSA的平均水平（均具有多样性（分析）（均已分析）（均具有多样性）。以前没有遇到过。此外，发现其中一个克隆会产生一种新型的代谢产物。这些结果比以前的工作效率更高10-100。但是，现在的主要瓶颈正在阐明这些化合物的结构。传统技术通常需要多种努力来阐明单一结构。这阶段I研究建议努力将该水平提高约十倍。实际上，我们建议通过筛选结构来从这个下一代宏基因组学库中找到新的小分子。如果成功，与基于功能的屏幕相比，自然产品发现的速率可能会加速许多折叠。 公共卫生相关性：在与微生物传染病作斗争中，由于抗生素耐药性的发展和无法找到替代药物的发展，我们正在失去立场。丧生和治疗负担是对美国公民的重大公共卫生威胁。拟议的研究释放了一套新的药物发现工具，其效率比传统技术高10​​-100倍。