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.

抽象的 2013 年，CDC 将碳青霉烯类耐药肠杆菌科细菌 (CRE) 指定为紧急威胁，2017 年，世界卫生组织将其指定为 1 号优先级“严重超级细菌”，因为治疗 CRE 的疗法很少，因此存在“泛耐药”CRE 的风险。 ，目前任何可用的抗生素都无法治疗，具有新颖作用机制（MOA）的全新药物不会与 SOC 药物产生交叉耐药性。开发一种 O- 抗原 (O-a) 生物合成抑制剂，可增强血清介导的杀伤作用 (SMK)，并且在 CRE 啮齿动物感染模型中有效。对于哺乳动物血清存在下的生长和发病机制至关重要，我们的提案概述了开发 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菌。 WecA 直系同源物，TarO。我们的目标是：目标 1（第一阶段；Ph1）。 ΔtolC 大肠杆菌 (Ec)，(2) 直接证实 tarocins 抑制 EcWecA 作为其引发 SMK 的 MOA，(3) 证明tarocin 诱导的 SMK 延伸至 Kp，并且 (4) 证明了体内功效的概念验证。里程碑 1. 筛选约 600 个其他 tarocin 类似物的 SMK 活性，并鉴定多达 4 个化学上不同的 tarocin 子系列，证明 i) > 4 倍。 EcWecA 过表达导致 SMK 中的 EC50 变化，ii) 全细胞环境中 O-a 的剂量依赖性消耗，iii) 映射到的因果药物 R 突变EcwecA，iv) 针对 Kp DtolC 的 SMK 活性，v) 在 25X MIC 下 >90% HepG2 细胞活力，以及 vi) 在使用外排缺陷的 Ec Aim 2 (Ph2) 的大鼠败血症模型中，50% 的生存保护剂量有利。先导 ID/选项通过 (1) 根据一组实验测试 SMK 类似物来鉴定具有有效 WT Ec 和 Kp SMK 的 tarocins。 Ec/Kp 渗透性/外排缺陷突变体，(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。我们还将优化 PK 和类药物特性。 里程碑 2。类似物证明 i) Ec/Kp WT 活性（血清中的 MIC [MIC] <1 ug/ml），ii) PK 暴露覆盖 4 次的 MIC小时，iii) Ec/Kp 靶点/通路结合选择性，包​​括 Kp 中的 tarocinR wecA 突变，v) Ec/Kp FOR <1x10-9 at 8X MIC，vi) MIC90 <2 ug/ml（100 个分离株），以及 vii) > 50X MIC 下的 HepG2/HEK293 活力为 90%，干净通道与 CYP/离子通道相比 (>10 uM)，PanLabs IC50 >10 uM (Ph2) 体内功效验证 (1) 优化合成路线以有效制备 3 种类似物用于制剂和剂量范围的大鼠 PK 研究，(2) 确定制剂载体。 3 种类似物可在体内研究中实现口服和静脉注射 PK 给药，并且 (3) 在使用 Ec 和 Kp 菌株的啮​​齿动物败血症模型中证明我们的顶级化合物的体内功效。 3. 合成 500 mg（>95% 纯度）的最多 3 种最能满足 Aim 2 里程碑的最佳类似物，确定可实现 10X MICS90 目标暴露量的安全批准载体中的配方，并证明 Ec 和 Kp 细菌负荷减少 > 3 个对数治疗24小时后。