Novel inhibitors of bacterial cell envelope biogenesis for prophylaxis against carbapenem-resistant Enterobacteriaceae

摘要 2017年，世卫组织将耐碳青霉烯类肠杆菌科（CRE）指定为优先级1的“关键超级细菌”。 由于很少有治疗CRE的方法，目前可用的任何治疗方法都无法治疗“泛耐药”CRE的风险。 抗生素也存在，并且具有新作用机制（MOA）的全新药物不会对 特种作战部探员们都很憔悴我们认为，除了治疗CRE感染的新策略外， 还应考虑首先预防CRE感染的发生。我们的建议 旨在开发一种O-抗原（O-a）生物合成抑制剂，其可增强血清介导的杀伤（SMK） 并且在预防性CRE啮齿动物感染模型中有效。我们已经证明了O-α的生物合成 基因wecA，在标准生长条件下的非必需基因，是生长和发病所必需的， 哺乳动物血清的存在我们的提案概述了开发WecA合成抑制剂的计划， 我们先前发现并优化了抑制革兰氏阳性WecA直系同源物TarO。我们的目标是： 目标1. MOA验证和测定开发。证明tarocins直接靶向WecA以引发SMK 方面的影响.建立靶标结合（TE）和通路结合（PE）筛选试验。构建同基因集 E. coli（Ec）、克雷伯氏菌（K.肺炎假单胞菌（Kp）、铜绿假单胞菌（Pa）和A. 鲍曼不动杆菌（Ab）指导药物化学SAR。里程碑（M）1.鉴别多达4种化学性质不同的塔罗辛 WecA过表达导致SMK进展至导联ID且EC 50偏移> 4倍的亚系列，剂量- O-a的依赖性消耗，以及映射到wecA的致病性drugR突变。 目标2.电极导线识别。通过对SMK进行实证分析，推动SAR努力实现WT活动， 一组Ec/Kp/Pa/Ab渗透性和外排缺陷突变体，采用新兴的“Hergenrother GN条目的物理化学规则，并探索铁载体缀合的效用。M2.最多识别2个 用于进展为具有Ec和Kp WT活性的Lead Opt的不同tarocin亚系列（人血清（HS）-MIC < 16 μ g/ml），暴露于覆盖HS-MIC 4小时，Ec TE和PE选择性，Ec和Kp FOR < 1 x 10-9，在8X HS下。 MIC，10 X HS-MIC时哺乳动物细胞活力> 90%，且HS-MIC 90 < 64 ug/ml（n=50个分离株）。 目标3。电极导线优化。优化PK、药物样特性和体外活性。M3.鉴别3种塔罗辛 具有Ec/Kp WT活性的进展至Aim 4的类似物（HS-MIC < 1 μ g/ml），暴露以覆盖HS-MIC 8 小时，Ec TE和PE选择性，在4X HS-MIC下Ec/Kp FOR E1 x 10-9，在10 X HS-MIC下>90% HepG 2/HEK 293细胞活力 HS-MIC，干净的β-内酰胺/离子通道（>10 μ M），PanLabs IC 50> 10 μ M，HS-MIC 90> 2 μ g/ml（n=100个分离株）。 目标4。体内功效证明。最多使用三种先导优化化合物， CRE菌血症和大腿感染的啮齿动物预防模型。M4.将1种塔罗辛类似物鉴定为前 在24小时治疗后显示细菌负荷减少> 3 log的临床预防候选者， 预防性大鼠CRE菌血症和大腿感染模型。

Abstract In 2017, the WHO designated carbapenem-resistant Enterobacteriaceae (CRE) a Priority 1 “critical superbug”. As few therapies remain to treat CRE, the risk of “pan-resistant” CRE untreatable by any currently available antibiotic also exists, and entirely new agents with novel mechanisms of action (MOA) not cross-resistant to SOC agents languish. We believe that in addition to new strategies to treat CRE infections, greater consideration should also be given to preventing CRE infections from occurring in the first place. Our proposal aims to develop an O-antigen (O-a) biosynthetic inhibitory agent that potentiates serum-mediated killing (SMK) and is efficacious in prophylactic CRE rodent models of infection. We have shown that the O-a biosynthesis gene wecA, a nonessential gene under standard growth conditions, is essential for growth and pathogenesis in the presence of mammalian serum. Our proposal outlines a plan to develop synthetic inhibitors of WecA that we previously discovered and optimized to inhibit the Gram-positive WecA ortholog, TarO. Our Aims are: Aim 1. MOA validation and assay development. Demonstrate tarocins directly target WecA to elicit SMK effects. Establish target engagement (TE) and pathway engagement (PE) screening assays. Build isogenic set of permeability and efflux deficient mutants in E. coli (Ec), K. pneumoniae (Kp), P. aeruginosa (Pa), and A. baumannii (Ab) to guide med chem SAR. Milestone (M) 1. Identify up to 4 chemically distinct tarocin subseries for progression to Lead ID with > 4-fold EC50 shift in SMK by WecA overexpression, dose- dependent depletion of O-a, and causal drugR mutations mapping to wecA. Aim 2. Lead Identification. Drive SAR efforts to achieve WT activity by empirically assaying for SMK against a panel of Ec/Kp/Pa/Ab permeability and efflux deficient mutants, employing the emerging ‘Hergenrother physicochemical rules of GN entry’, and exploring the utility of siderophore conjugation. M2. Identify up to 2 distinct tarocin subseries for progression to Lead Opt with Ec and Kp WT activity (Human Serum (HS)-MIC < 16 ug/ml), exposure to cover HS-MIC for 4 hrs, Ec TE and PE selectivity, Ec and Kp FOR < 1 x 10-9 at 8X HS- MIC, > 90% mammalian cell viability at 10 X HS-MIC, and HS-MIC90 < 64 ug/ml (n=50 isolates). Aim 3. Lead Optimization. Optimize PK, drug-like properties, and in vitro activity. M3. Identify 3 tarocin analogs for progression to Aim 4 with Ec/Kp WT activity (HS-MIC < 1 ug/ml), exposure to cover HS-MIC 8 hrs, Ec TE and PE selectivity, Ec/Kp FOR £ 1x 10-9 at 4X HS-MIC, >90% HepG2/HEK293 cell viability at 10X HS-MIC, clean CYP/ion channels (>10 uM), PanLabs IC50 > 10 uM, and HS-MIC90 £ 2 ug/ml (n=100 isolates). Aim 4. In vivo efficacy demonstration. Demonstrate efficacy with up to three lead optimized compounds in rodent prophylaxis models of CRE bacteremia and thigh infections. M4. Identify 1 tarocin analog as a pre- clinical prophylaxis candidate that demonstrates > 3 log reduction of bacterial burden after 24h treatment in prophylactic rat CRE bacteremia and thigh infection models.