Mode of Action Core

摘要 这一行动模式(MOA)核心将支持我们CETR的所有3个项目。我们核心的目的是预测 项目确定的新型先导化合物的MOA，并通过体外和体内核心进行过滤。抗菌药物的开发需要了解药物靶标和MOA，以便能够进行下游改进和 改进销售线索。我们建议与尖端工具一起强调化学遗传策略。 以及识别毒品线索的MOA的方法。我们将利用健康测试的原理，在失去一个的地方 或者，基因的两个副本都会使突变的真菌或细菌对特定药物高度或低敏感。这个 这一分析的结果包括编码特定药物靶点的基因，或药物新陈代谢的效应物。 我们将利用现有的覆盖致病真菌和细菌基因组的突变文库，包括白色念珠菌和大肠杆菌。我们的战略将使我们能够在一组综合的药物目标上筛选线索，而不仅仅是一个目标。因此，对于抗真菌候选药物来说，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点和MOA； 所谓的单倍性不足图谱，其中菌株特定的条形码唯一地标记突变，而那些具有特殊适合性的突变通过Illumina测序来检测。对于抗菌铅，将使用高通量筛网 目的：检测革兰氏阴性菌突变文库的药物敏感性。对于大肠杆菌，这包括一个双等位基因 条形码缺失文库和CRISPRi文库，使必要基因的条件表达控制成为可能。 在一种平行的方法中，将选择耐药突变株，对基因组进行测序，确定SNPs并确定目标。药物靶点和MOA将通过下游的遗传和生化研究来确认。 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MOA的销售线索(总计最多10个)。 我们的核心是重要的，因为它将产生关于有前景的抗菌药物的至关重要的新知识 线索。需要对药物MOA的洞察来推动抗耐药传染病新药的开发 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 美国国立卫生研究院CETR计划实现了改善公众健康的目标。

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.