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: 10415522
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10415522
• 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/antagonist; 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，疾控中心将耐碳青霉烯类肠杆菌科(CRE)列为紧急威胁，2017年，世界卫生组织将其列为优先事项1-“关键超级细菌”。由于治疗CRE的疗法寥寥无几，目前任何现有抗生素都无法治愈的“泛耐药”CRE的风险增加。具有新的作用机制(MOA)且对SOC药剂没有交叉耐药性的全新药剂正在衰败。我们的建议旨在开发一种O-抗原(O-a)生物合成抑制剂，以增强血清介导的杀伤(SMK)，并在CRE啮齿动物感染模型中有效。我们已经证明，O-a生物合成基因WECA是标准生长条件下的一个非必需基因，在哺乳动物血清存在的情况下对生长和发病是必不可少的。我们的提案概述了一项开发WECA合成抑制剂的计划，我们之前发现并优化了该抑制剂，以抑制革兰氏阳性WECA同源基因Taro。我们的目标是：目标1(阶段1；PH1)。筛选、MOA研究和体内概念验证研究。(1)完成针对针对WT和Δ的SMK靶向文库的Lux报道筛选，(2)直接证实Tarocin抑制EcWecA作为其诱导SMK的MOA，(3)证明Tarocin诱导的SMK延伸到KP，(4)在体内证明概念验证的有效性。里程碑1.筛选另外600个用于SMK活性的Tarocin类似物，并确定多达4个化学上不同的Tarocin亚系列，证明i)&Gt；4通过EcWecA过度表达使SMK的EC50增加4倍，ii)全细胞环境中O-a的剂量依赖耗竭，iii)映射到EcwecA的原因药物R突变，iv)SMK对KP DtolC，v)&Gt；90%的HepG2细胞活性在25X MIC下，以及vi)在使用外排缺陷EC菌株的大鼠败血症模型中存活的有利的50%保护剂量。目标2(PH2)。销售线索ID/选项。通过(1)针对EC/KP渗透性/外排缺陷突变体小组进行SMK类似物的经验性测试，(2)采用最新的GN进入的物理化学规则，以及(3)探索铁载体结合以驱动SAR努力，确定具有有效WT EC和KP SMK的Tarocin。我们还将优化PK和类药物属性。里程碑2.确定最多3个类似物，证明i)EC/KP WT活性(血清[MICs]和lt；1 ug/ml)，ii)PK暴露于MIC 4小时，iii)EC/KP靶/通路参与选择性，包括Kp中的tarocinR WECA突变，v)EC/KP在8倍MICs时为1x10-9，vi)MICs90&lt；2 ug/ml(100个分离株)，以及vii)&gt；90%的HepG2/HEK293在50倍MICs，清洁的CYP/离子通道(&gt；10 um)，以及PanLabs 50&GT；10 um。目标3(PH2)。体内药效演示。(1)优化合成路线，以高效地制备3种类似物，用于配方和剂量范围的大鼠PK研究；(2)确定3种类似物的配方载体，以便在体内研究中口服和静脉注射PK；以及(3)在使用EC和KP菌株的啮齿动物败血症模型中展示我们顶级化合物的体内疗效。里程碑3.合成500毫克(95%纯度)最符合目标2里程碑的3种最佳类似物，在安全批准的车辆中确定配方，达到10倍的MICS90目标暴露，并在24小时治疗后证明EC和KP的细菌负荷对数减少。