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，抗生素可以说是唯一的医疗手段 比任何其他干预措施都拯救了更多的生命。然而，这种拯救生命的干预现在正在进行 受到抗生素耐药性问题的威胁，这一问题的速度超过了新抗生素的发现，导致 世界卫生组织和疾控中心宣布抗生素耐药性是对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#年的全球经济产生巨大影响 目前的抗生素领域没有发生重大变化 我们已经开发了新的策略来连接基因组学和高通量化学筛选 改变抗生素发现的技术，这里的重点是主要的革兰氏阴性病原体 铜绿假单胞菌(PSA)，特异性靶向其必需外膜蛋白(OMP)和外膜 膜相关蛋白(OMAP)，以规避对异种细胞质的需要 积累。我们以多重方式对条形码的池进行了化学筛选， 其中每个必要的OMP/OMAP靶基因都已被 被启动子替换所击倒。下一代测序用于枚举放大的条形码 以测量池中每个突变菌株对小分子的反应。控制低电平 这些菌株中每一种目的蛋白的表达都使它们对抑制物过敏 相应的目标。重要的是，这一策略使我们(1)扩大了小分子的数量 通过识别如果单纯针对野生型进行筛查就不会被发现的小分子来候选 PSA，(2)结合全细胞筛选和基于靶点的发现，以及(3)靶向PSA的基本蛋白 功能未知或缺乏高通量检测。使用这种方法瞄准9个基本的OMP/OMAP目标 在一次筛选中，我们已经确定了针对外膜蛋白LptD和OprL的候选蛋白 分别是内毒素转运和细胞膜完整性所必需的。因为这些目标缺乏强有力的 在功能分析方面，我们开发了一种高通量的转录驱动流水线和机器 学会识别和优先考虑有很大可能特别抑制这些靶点的分子。我们现在 建议开发具有全新作用机制的HITS，以引导优化和演示 活体概念验证。

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.