Innovative technologies to transform antibiotic discovery. Project 1 Genomic applications to transform Gram-negative Antibiotic discovery

改变抗生素发现的创新技术。

• 批准号: 10242002
• 负责人: DEBORAH T HUNG
• 金额: $ 187.02万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-07 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242002
• 关键词: Antibiotic Resistance; Antibiotics; Bacterial Infections; Bar Codes; Binding; Biological; Biological Assay; Biological Availability; Biophysics; Cell membrane; Cells; Censuses; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chemicals; Clinical; Collection; Complex; Coupled; Crystallization; Data Analyses; Dependence; Development; Drug Industry; Expression Profiling; Failure; Gene Expression Profiling; Genes; Genetic; Genetic Engineering; Genome; Genomics; Gram-Negative Bacteria; Health; Hospitals; Human; In Vitro; Infection; Intervention; Kinetics; Lead; Life; Machine Learning; Measures; Medical; Membrane Proteins; Modeling; Monitor; Mus; Natural Products; New Agents; Pathway interactions; Penicillins; Permeability; Pharmaceutical Preparations; Polysaccharides; Process; Property; Proteins; Pseudomonas aeruginosa; Resistance; Safety; Savings; Specificity; Structure; Structure-Activity Relationship; Technology; Testing; Therapeutic Index; Time; Triage; Xenobiotics; Zebrafish; bacterial resistance; base; biophysical properties; clinical candidate; combinatorial; cost; cytotoxicity; efflux pump; experience; genetic approach; genome sequencing; genome-wide; high throughput screening; improved; in vivo; inhibitor/antagonist; innovative technologies; interest; knock-down; lead candidate; lead optimization; lead series; machine learning algorithm; mutant; next generation sequencing; novel; novel strategies; pathogen; peptidoglycan-associated lipoprotein; potency testing; promoter; protein expression; response; scaffold; screening; small molecule; small molecule libraries; success; targeted agent; transcriptomics; transposon sequencing; uptake; whole genome

Since Alexander Fleming's discovery of penicillin, antibiotics have been arguably the single medical intervention that has saved more lives than any other. However, this life saving intervention is now being threatened by the problem of antibiotic resistance that is outpacing the discovery of new antibiotics, resulting in the WHO and CDC declaring antibiotic resistance as one of the greatest threats to human health. Projections include the possibility of 10 million deaths per year by 2050 with tremendous impact on the global economy in the absence of a significant shift in the current antibiotic landscape We have developed novel strategies to interface genomics and high-throughput chemical screening technologies to transform antibiotic discovery, with a focus here on the major Gram-negative pathogen Pseudomonas aeruginosa (PsA), specifically targeting its essential outer membrane proteins (OMPs) and outer membrane associated proteins (OMAPs) in order to circumvent the need for xenobiotic cytoplasmic accumulation. We have performed chemical screening in a multiplexed fashion against a pool of bar-coded, genetically engineered target-specific strains in which each of the essential OMP/OMAP target genes has been knocked-down by promoter replacement. Next generation sequencing is used to enumerate amplified barcodes to measure the census of each mutant strain in the pool in response to a small molecule. Controlled low expression of the protein of interest in each of these strains hypersensitizes them to inhibitors of the corresponding target. Importantly, this strategy has allowed us (1) to expand the numbers of small molecule candidates by identifying small molecules that would have eluded discovery if screening simply against wild-type PsA, (2) to couple whole cell screening with target-based discovery, and (3) to target essential proteins of unknown function or lack a high-throughput assay. Using this approach to target 9 essential OMP/OMAP targets in a single screen, we have identified candidates targeting the outer membrane proteins LptD and OprL, proteins required for LPS transport and cell membrane integrity, respectively. Because these targets lack robust functional assays, we have developed a high-throughput transcriptomics-driven pipeline coupled with machine learning to identify and prioritize molecules with a high likelihood of specifically inhibiting these targets. We now propose to develop the hits with completely novel mechanisms of action to lead optimization and demonstrate in vivo proof of concept.

自从亚历山大·弗莱明（Alexander Fleming）发现青霉素以来，抗生素可以说是单一的医学 干预措施比其他任何人都挽救了更多的生命。但是，这种救生干预现在正在 受到超过发现新抗生素的抗生素耐药性问题的威胁，导致 世卫组织和疾病预防控制中心宣布抗生素抗性为对人类健康的最大威胁之一。预测 包括到2050年每年1000万人死亡的可能性，对全球经济的影响很大 当前的抗生素景观没有显着转移 我们已经开发了新颖的策略来接口基因组学和高通量化学筛选 转化抗生素发现的技术，重点是主要的革兰氏阴性病原体 铜绿假单胞菌（PSA），专门针对其必需的外膜蛋白（OMP）和外部 膜相关蛋白（OMAP），以规避对异生物生物细胞质的需求 积累。我们以多重方式进行了化学筛选， 基因工程靶标特异性菌株，其中每个必需的OMP/OMAP靶基因一直是 被发起人替换击倒。下一代测序用于枚举放大条形码 响应小分子，测量池中每个突变菌株的普查。控制低 这些菌株中感兴趣的蛋白质的表达使它们超敏，以使其抑制剂 相应的目标。重要的是，这种策略使我们（1）能够扩大小分子的数量 候选人通过识别仅针对野生型筛查的小分子，这些小分子会发现发现发现 PSA，（2）将整个细胞筛选与基于目标的发现以及（3）靶向靶向的必需蛋白质 功能未知或缺乏高通量测定法。使用这种方法来靶向9个基本OMP/OMAP目标 在单个屏幕中，我们确定了针对外膜蛋白LPTD和OPRL的候选物 LPS运输和细胞膜完整性所需。因为这些目标缺乏强大的 功能分析，我们开发了一个高通量转录组驱动的管道，并与机器相结合 学会识别和优先级分子，并具有很高的特异性抑制这些目标的可能性。我们现在 提议通过完全新颖的作用机理来开发击球，以领导优化并证明 体内概念证明。