Development of WecA-targeting immune potentiators to treat carbapenem-resistant Enterobacteriaceae (CRE) infections

开发 WecA 靶向免疫增强剂来治疗碳青霉烯类耐药肠杆菌 (CRE) 感染

• 批准号: 10470327
• 负责人: Terry Roemer
• 金额: $ 100万
• 依托单位: PROKARYOTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10470327
• 关键词: Address; Anabolism; Antibiotics; Bacteremia; Bacteria; Bacterial Infections; Bacterial Pneumonia; Binding; Cancerous; Carbapenems; Cell Survival; Cells; Centers for Disease Control and Prevention (U.S.); Chemicals; Clinical; Colistin; Communities; Complement; Data; Development; Dose; Enterobacter; Enterobacteriaceae; Enterobacteriaceae Infections; Epidemic; Escherichia coli; Essential Genes; Exposure to; Formulation; Fosfomycin; Genes; Genetic; Goals; Gram-Negative Bacteria; Growth; HepG2; Hospitals; Imipenem; Immune; Immune Targeting; Immune system; Immunooncology; In Vitro; Infection; Innate Immune System; Ion Channel; Klebsiella pneumoniae; Lead; Libraries; Life; Maps; Mediating; Meropenem; Modeling; Multi-Drug Resistance; Mutation; New Agents; O Antigens; Oral; Organism; Orthologous Gene; Pathogenesis; Pathway interactions; Pattern; Permeability; Pharmaceutical Preparations; Pharmacology; Phase; Plasmids; Polymyxins; Production; Property; Pseudomonas aeruginosa; Rattus; Reporter; Reporting; Resistance; Risk; Rodent; Rodent Model; Route; Safety; Septicemia; Serum; Siderophores; Source; Superbug; Taro Vegetable; Testing; analog; antimicrobial; bactericide; base; beta-Lactam Resistance; beta-Lactamase; beta-Lactams; carbapenem resistance; carbapenem-resistant Enterobacteriaceae; commensal bacteria; doripenem; drug repurposing; extracellular; gene product; gut microbiota; in vivo; inhibitor; innovation; member; mortality; mutant; new therapeutic target; novel; novel therapeutics; overexpression; pathogen; periplasm; programmed cell death protein 1; public health relevance; resistance mechanism; scale up; screening; success; tigecycline

Abstract In 2013, the CDC designated carbapenem-resistant Enterobacteriaceae (CRE) an Urgent Threat, and in 2017, the WHO designated it a Priority 1 “critical superbug”. As few therapies remain to treat CRE, the risk of “pan- resistant” CRE, untreatable by any currently available antibiotic, increases. Entirely new agents with novel mechanisms of action (MOA) not cross-resistant to SOC agents languish. Our proposal aims to develop an O- antigen (O-a) biosynthetic inhibitory agent that potentiates serum-mediated killing (SMK) and is efficacious in a CRE rodent model 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 (Phase 1; Ph1). Screening, MOA studies, and Proof-of-Concept in vivo Studies. (1) Complete lux reporter screening of the tarocin focused library for SMK against WT and ΔtolC E. coli (Ec), (2) directly confirm tarocins inhibit EcWecA as their MOA for eliciting SMK, (3) demonstrate that tarocin-induced SMK extends to Kp, and (4) demonstrate proof-of-concept in vivo efficacy. Milestone 1. Screen ~600 additional tarocin analogs for SMK activity and identify up to 4 chemically distinct tarocin subseries demonstrating i) > 4-fold EC50 shift in SMK by EcWecA overexpression, ii) dose-dependent depletion of O-a in a whole-cell context, iii) causal drugR mutations mapping to EcwecA, iv) SMK activity against Kp DtolC, v) >90% HepG2 cell viability at 25X MIC, and vi) favorable 50% protective dose for survival in a rat septicemia model using an efflux-deficient Ec strain. Aim 2 (Ph2). Lead ID/Opt. Identify tarocins with potent WT Ec and Kp SMK by (1) empirically testing analogs for SMK against panel of Ec/Kp permeability/efflux deficient mutants, (2) employing recent physicochemical rules of GN entry, and (3) exploring siderophore conjugation to drive SAR efforts. We will also optimize PK and drug- like properties. Milestone 2. Identify up to 3 analogs demonstrating i) Ec/Kp WT activity (MIC in serum [MICs] <1 ug/ml), ii) PK exposure to cover MICs for 4 hrs, iii) Ec/Kp target/pathway engagement selectivity including tarocinR wecA mutation in Kp, v) Ec/Kp FOR <1x10-9 at 8X MICs, vi) MICs90 <2 ug/ml (100 isolates), and vii) > 90% HepG2/HEK293 viability at 50X MICs, clean vs CYP/ion channels (>10 uM), and PanLabs IC50 >10 uM. Aim 3 (Ph2). In vivo efficacy demonstration. (1) Optimize synthetic routes to efficiently prepare 3 analogs for formulation and dose-ranging rat PK studies, (2) identify formulation vehicles for the 3 analogs to enable oral and IV PK dosing in in vivo studies, and (3) demonstrate in vivo efficacy for our top compound in a rodent septicemia model using Ec and Kp strains. Milestone 3. Synthesize 500 mg (>95% purity) of up to 3 optimal analogs that best satisfy Aim 2 milestones, identify formulation in a safety approved vehicle that achieves 10X MICS90 target exposure, and demonstrate > 3 log reduction of Ec and Kp bacterial burden after 24h treatment.

摘要 2013年，CDC将耐碳青霉烯类肠杆菌科（CRE）指定为紧急威胁，2017年，WHO将其指定为优先级1的“关键超级细菌”。由于几乎没有治疗CRE的疗法，因此任何现有抗生素都无法治疗的“泛耐药”CRE的风险增加。具有新作用机制（MOA）的新药物对SOC药物没有交叉耐药性。我们的提议旨在开发一种O-抗原（O-a）生物合成抑制剂，其增强血清介导的杀伤（SMK）并且在CRE啮齿动物感染模型中有效。我们已经表明，O-a生物合成基因wecA，在标准生长条件下的非必需基因，是必不可少的生长和发病机制中存在的哺乳动物血清。我们的提案概述了开发我们先前发现并优化以抑制革兰氏阳性WecA直系同源物TarO的WecA合成抑制剂的计划。我们的目标是：目标1（第1阶段; Ph 1）。筛选、MOA研究和体内概念验证研究。(1)针对针对WT和ΔtolC E的SMK的塔罗辛聚焦文库的完全lux报告子筛选。大肠杆菌（Ec）中，（2）直接证实tarocin抑制EcWecA作为其引发SMK的MOA，（3）证明tarocin诱导的SMK延伸至Kp，和（4）证明体内功效的概念验证。里程碑1.筛选约600种额外的塔罗辛类似物的SMK活性，并鉴定多达4种化学上不同的塔罗辛亚系列，证明i）通过EcWecA过表达导致SMK的> 4倍EC 50偏移，ii）在全细胞背景下0-α的剂量依赖性消耗，iii）定位至EcwecA的致病性drugR突变，iv）SMK对Kp DtolC的活性，v）在25 X MIC下>90% HepG 2细胞活力，和vi）在使用流出缺陷型Ec菌株的大鼠败血症模型中，对于存活有利的50%保护剂量。目标2（Ph 2）。领导ID/Opt.通过（1）针对Ec/Kp渗透性/外排缺陷突变体组经验性测试SMK类似物，（2）采用GN进入的最新物理化学规则，以及（3）探索铁载体结合以推动SAR工作，鉴定具有有效WT Ec和Kp SMK的塔罗辛。我们还将优化药代动力学和药物性质。里程碑2.鉴定多达3种类似物，证明i）Ec/Kp WT活性（血清中的MIC [MIC] <1 μ g/ml），ii）PK暴露以覆盖MIC 4小时，iii）Ec/Kp靶标/途径接合选择性，包括Kp中的tarocinR wecA突变，v）Ec/Kp FOR <1x 10 -9，在8X MIC下，vi）MIC 90 <2 μ g/ml（100个分离株），和vii）在50 X MIC下> 90%的HepG 2/HEK 293活力，清洁相对于β 2/离子通道（>10 μ M），和PanLabs IC 50>10 μ M。目标3（Ph 2）。体内功效证明。(1)优化合成路线以有效制备用于制剂和剂量范围大鼠PK研究的3种类似物，（2）鉴定3种类似物的制剂媒介物以在体内研究中实现口服和IV PK给药，以及（3）使用Ec和Kp菌株在啮齿动物败血症模型中证明我们的顶级化合物的体内功效。里程碑3.合成500 mg（>95%纯度）最佳满足目标2里程碑的最多3种最佳类似物，在安全批准的载体中鉴定达到10 X MICS 90目标暴露的制剂，并在24小时处理后证明Ec和Kp细菌负荷的> 3个对数减少。