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

自从亚历山大·弗莱明发现青霉素以来，抗生素一直是可以说是唯一的医疗手段。 比其他任何干预措施都挽救了更多的生命。然而，这种拯救生命的干预措施现在正在 抗生素耐药性问题的威胁超过了新抗生素的发现，导致 世界卫生组织和疾病预防控制中心宣布抗生素耐药性是人类健康的最大威胁之一。预测 包括到2050年每年有1000万人死亡的可能性，这将对全球经济产生巨大影响， 当前抗生素格局没有发生重大变化 我们已经开发了新的策略，接口基因组学和高通量化学筛选 改变抗生素发现的技术，重点关注主要的革兰氏阴性病原体 铜绿假单胞菌（PsA），特异性靶向其必需的外膜蛋白（OMP）和外膜蛋白（OMP）， 膜相关蛋白（OMAPs），以避免需要异生质细胞质 积累我们已经以多重方式对条形码库进行了化学筛选， 基因工程化的靶特异性菌株，其中每种必需的OMP/OMAP靶基因已被 通过启动子置换敲低。下一代测序用于计数扩增的条形码 以测量池中响应于小分子的每个突变菌株的普查。控制性低 在这些菌株中的每一种中感兴趣的蛋白质的表达使它们对免疫抑制剂超敏。 对应的目标。重要的是，这种策略使我们（1）扩大了小分子的数量， 通过鉴定小分子来筛选候选人，如果简单地针对野生型进行筛选， PsA，（2）将全细胞筛选与基于靶点的发现相结合，以及（3）靶向 功能未知或缺乏高通量测定。使用这种方法瞄准9个基本OMP/OMAP目标 在一次筛选中，我们已经鉴定了靶向外膜蛋白LptD和OprL的候选蛋白， LPS转运和细胞膜完整性所需的。因为这些目标缺乏强大的 功能测定，我们已经开发了一个高通量转录组学驱动的管道， 学习识别和优先考虑具有特异性抑制这些靶点的高可能性的分子。我们现在 建议开发具有全新作用机制的命中物，以引导优化和演示 体内概念验证。

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.