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.

抽象的 这种行动方式（MOA）核心将支持我们CERT的所有3个项目。我们的核心目的是神圣 新型铅化合物的MOA由项目鉴定并通过体外和体内核过滤。一阵发生物的开发需要了解药物靶标和MOA以实现下游改进，并且 铅的改进。我们建议强调化学遗传策略以及尖端工具 并辨别毒品铅的MOA的方法。我们将利用健身测试的原则，其中损失一个 或两种基因的副本都使突变的真菌或细菌对特定药物过度敏感。这 该分析中的命中包括编码特定药物靶标的基因或药物代谢的作用。 我们将利用跨越致病真菌和细菌基因组的可用突变库，覆盖的白色念珠菌和大肠杆菌。我们的策略将使我们能够筛选一系列全面的药物目标，而不仅仅是一个目标。对于抗真菌候选者，药物诱导的生长 杂合缺失突变体库中菌株的改变将揭示潜在的药物靶标和MOA。 所谓的单倍体不足型曲线，其中菌株特异性条形码独特地标记突变体，以及通过Illumina序列检测到具有拟合度的突变体。对于抗菌导线，将使用高吞吐量屏幕 主张革兰氏阴性物种的突变库，以提高药物敏感性。对于大肠杆菌 条形码删除库和CRISPRI库，可实现基本基因的条件表达控制。 在平行的方法中，将选择耐药突变体，测序基因组，鉴定为SNP和焦油。药物靶标和MOA将通过下游遗传和生化研究确认。 我们手头有几条线索开始学习。由于我们小组正在进行的工作，我们希望研究两个 每年用于MOA（最多10个）。 我们的核心很重要，因为它将产生有关有希望抗菌药物的重要新知识 铅。需要对毒品MOA的见解，以推动新药的开发，以防止抗性感染力 疾病。我们的研究发现将对该领域产生持续和强大的影响，因为它将改善 感染了耐药菌和细菌病原体的患者的护理。这些成就将有助于 NIH CTR计划实现了改善公众健康的目标。