Mode of Action Core

行动模式核心

• 批准号: 10592385
• 负责人: BRUCE Steven KLEIN
• 金额: $ 76.39万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592385
• 关键词: Mode; Action; Core

ABSTRACT This mode-of-action (MOA) core will support all 3 projects in our CETR. Our core’s purpose is to divine the MOA of novel lead compounds identified by the projects and filtered through the in vitro and in vivo cores. An-timicrobial development requires knowledge of a drug target and MOA to enable downstream refinements and improvements of leads. We propose to emphasize chemical-genetic strategies together with cutting edge tools and approaches to discern MOA of drug leads. We will exploit the principle of fitness testing where loss of one or both copies of a gene renders a mutant fungus or bacterium hyper- or hypo-sensitive to a specific drug. The hits from this analysis include genes encoding the specific drug target, or effectors of the drug's metabolism. We will capitalize on available mutant libraries that span the genomes of pathogenic fungi and bacteria, in-cluding Candida albicans and Escherichia coli. Our strategy will allow us to screen leads across a comprehen-sive set of drug targets rather than just a single target. Thus, for antifungal candidates, drug-induced growth alteration of strains in a library of heterozygous deletion mutants will reveal a potential drug target and MOA; a so-called haploinsufficiency profile where strain-specific barcodes uniquely mark mutants, and those with al-tered-fitness are detected by Illumina sequencing. For antibacterial leads, high throughput screens will be used to assay mutant libraries of Gram-negative species for drug sensitivity. For E. coli, this includes a two-allele barcoded deletion library and a CRISPRi library that enables conditional expression control of essential genes. In a parallel approach, drug-resistant mutants will be selected, genome sequenced, SNPs identified and targets implicated. Drug targets and MOAs will be confirmed with downstream genetic and biochemical studies. We have several leads in hand to begin our studies. From ongoing work by our group, we expect to study two leads annually for MOA (up to 10 total). Our core is significant because it will generate vital new knowledge about promising antimicrobial drug leads. The insight on drug MOA will be needed to fuel development of new drugs against resistant infectious disease. Our research findings will exert a sustained and powerful influence on the field because it will improve the care of patients infected with resistant fungal and bacterial pathogens. These achievements will help the NIH CETR program realize its goals of improving the public’s health.

摘要 该行动模式（MOA）核心将支持我们CETR中的所有3个项目。我们核心的目的是 通过项目鉴定并通过体外和体内核心筛选的新型先导化合物的MOA。抗菌药物的开发需要了解药物靶标和MOA，以实现下游改进， 改善领导。我们建议强调化学遗传策略与尖端工具 以及药物线索MOA的识别方法。我们将利用适应性测试的原则，其中损失一个 或者基因的两个拷贝使突变的真菌或细菌对特定药物高度敏感或低度敏感。的 这项分析的结果包括编码特定药物靶点或药物代谢效应物的基因。 我们将利用现有的突变库，跨越致病真菌和细菌的基因组，包括白色念珠菌和大肠杆菌。我们的策略将使我们能够在一组全面的药物靶点中筛选线索，而不仅仅是单一的靶点。因此，对于抗真菌候选物，药物诱导的生长 杂合缺失突变体文库中菌株的改变将揭示潜在的药物靶标和MOA; 所谓的单倍不足谱，其中菌株特异性条形码独特地标记突变体，并且通过Illumina测序检测具有改变的适合度的突变体。对于抗菌电极导线，将使用高通量筛选 以测定革兰氏阴性物种的突变体文库的药物敏感性。大肠在大肠杆菌中，这包括一个双等位基因 条形码化缺失文库和CRISPRi文库，其能够实现必需基因的条件表达控制。 在一个平行的方法中，将选择耐药突变体，进行基因组测序，识别SNPs，并将其与基因组测序相结合。受到牵连。药物靶标和MOA将通过下游遗传和生化研究进行确认。 我们手头有几条线索可以开始我们的研究。从我们小组正在进行的工作中，我们希望研究两个 每年为MOA提供领导（总计10个）。 我们的核心是重要的，因为它将产生关于有前途的抗菌药物的重要新知识 线索.需要对药物MOA的深入了解，以促进抗耐药感染性疾病新药的开发。 疾病我们的研究成果将对该领域产生持续而强大的影响，因为它将改善 对感染耐药真菌和细菌病原体的患者的护理。这些成就将有助于 NIH CETR计划实现了改善公众健康的目标。