Characterization of MsbA inhibitors as potential antibiotic leads to treat carbapenem-resistant Enterobacteriaceae (CRE)

MsbA 抑制剂作为潜在抗生素的特性可用于治疗耐碳青霉烯类肠杆菌 (CRE)

• 批准号: 9978345
• 负责人: Terry Roemer
• 金额: $ 24.5万
• 依托单位: PROKARYOTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-19 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978345
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; American; Aminoglycosides; Anti-Bacterial Agents; Antibiotics; Bacterial Drug Resistance; Bacterial Infections; Binding Sites; Biochemical; Biogenesis; Biological Assay; CDC2 gene; Carbapenems; Cell Survival; Cells; Cellular Structures; Centers for Disease Control and Prevention (U.S.); Cephalosporins; Chemicals; Clinical; Coculture Techniques; Colistin; Collaborations; Communicable Diseases; Communities; Complex; Cryoelectron Microscopy; Crystallization; Data; Deacetylase; Development; Dose; Drug Design; Enterobacter; Enterobacteriaceae; Enterobacteriaceae Infections; Enzymes; Escherichia coli; Escherichia coli drug resistance; European; Fosfomycin; Foundations; Fright; Future; Genomic Centers for Infectious Diseases; Goals; Gram-Negative Bacteria; Health Care Costs; HepG2; Hospitals; In Vitro; Klebsiella pneumoniae; Lactamase; Lactams; Lead; Left; Length of Stay; Letters; Life; Ligand Binding; Lipopolysaccharide Biosynthesis Pathway; Lipopolysaccharides; Mediating; Membrane; Meropenem; Microbiology; Molecular Conformation; Multi-Drug Resistance; Mutation; New Agents; Oral; Organism; Pathway interactions; Pattern; Permeability; Pharmaceutical Chemistry; Pharmaceutical Preparations; Polymyxins; Proteins; Reporting; Resistance; Resolution; Risk; Roentgen Rays; Series; Siderophores; Solid; Structure; Structure-Activity Relationship; Superbug; Tetracyclines; Therapeutic; Time; Zinc; antimicrobial; bactericide; base; beta-Lactam Resistance; carbapenem resistance; carbapenem-resistant Enterobacteriaceae; clinically relevant; commensal bacteria; doripenem; genome sequencing; gut microbiota; improved; in vitro activity; inhibitor/antagonist; innovation; lead optimization; membrane biogenesis; molecular site; mortality; mutant; novel; novel therapeutics; overexpression; pathogen; periplasm; resistance frequency; small molecule inhibitor; structural genomics; tigecycline; whole genome

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 also exists. Entirely new agents with novel mechanisms of action (MOA) not cross-resistant to SOC agents languish. Development of such a ‘first-in-class’ agent also offers the potential for much-needed orally-administered CRE therapeutic, thus providing a new step-down therapy to reduce hospital stay and growing healthcare costs. Our proposal aims to develop an entirely novel therapeutic to treat life-threatening bacterial infections due to multidrug resistant (MDR) Enterobacteriaceae, including CRE. Using an innovative overexpression-based co-culture screen in Escherichia coli (Ec), we identified four structurally distinct series of small molecule inhibitors targeting MsbA, an essential and broadly conserved Gram-negative (GN) ABC transporter responsible for lipopolysaccharide (LPS) biogenesis and construction of the Gram-negative outer membrane (OM). Building upon a solid foundation of preliminary data, our Aims are: Aim (A) 1. Expanded MOA studies. Aim 1 studies will expand upon our preliminary MOA and microbiological characterization of the MsbA inhibitor (MsbAi) hits to include other GN bacteria, particularly Klebsiella pneumoniae (Kp). Specifically, we will demonstrate Kp MsbA target inhibition in (1) an in vitro biochemical assay and (2) a whole-cell context by determining MICs of our MsbAis against a Kp tolC strain and subsequently selecting for MsbAisR mutants in this Kp background. Aim 2. Demonstrate chemical tractability of MsbAis. The goal of Aim 2 is to perform Hit to Lead (H2L) medicinal chemistry structure activity relationship (SAR) studies for each MsbAi and demonstrate >1 series is chemically tractable and that quantifiable pre-lead optimization criteria can be established, including i) Ec and Kp MsbA in vitro potency (IC50 < 0.25 µg/ml), ii) Ec and Kp WT activity (<16 µg/ml), and iii) > 90% HepG2 cell viability at 50X MIC against EcΔtolC. MsbAis achieving these milestones serve as a justifiable starting point for a future Lead Optimization leveraging the resulting SAR gained in Aim 2 and SBDD information from Aim 3. Aim 3. Obtain Ec and Kp MsbAi-MsbA co-crystal structures in collaboration with SSGCID. The goal of Aim 3 activities is to initiate a collaboration between Prokaryotics, Genentech, and the Seattle Structural Genomics Center for Infectious Disease (SSGCID) led by Dr. Peter Myler to optimize conditions suitable for obtaining high resolution x-ray crystal structures of Kp MsbA in the apo form as well as Ec and/or Kp MsbA proteins in complex with MsbAis.

2013年，疾病预防控制中心设计的耐碳青霉烯肠杆菌科（CRE）是紧急威胁，2017年，2017年 世卫组织将其指定为优先级1“关键超级细菌”。由于几乎没有治疗CRE的疗法，因此“泛滥的风险 当前任何可用的抗生素都无法治疗耐药性。 行动机制（MOA）对SOC代理人的抗性不足。开发这样的“一流” 代理商还提供了急需的口服ADMINESTER CRE疗法的潜力，从而提供了新的 降低治疗，以减少住院和增加医疗费用。我们的建议旨在发展 完全新颖的热热以治疗由于多药耐药性（MDR）引起的威胁生命的细菌感染 肠杆菌科，包括Cre。使用创新的基于过表达的共培养屏幕 大肠杆菌（EC），我们确定了针对MSBA的四个结构上不同的小分子抑制剂， 负责脂多糖的基本且宽阔的革兰氏阴性（GN）ABC转运蛋白 （LPS）革兰氏阴性外膜的生物发生和构建（OM）。基于固体 初步数据的基础，我们的目标是： 目的（a）1。扩展的MOA研究。 AIM 1研究将扩大我们的初步MOA和微生物学 MSBA抑制剂（MSBAI）命中的表征包括其他GN细菌，尤其是Klebsiella 肺炎（KP）。特别是，我们将在（1）体外生化测定中证明KP MSBA目标抑制 （2）通过确定我们的MSBAI的麦克风对kp托菌株的麦克风，然后 在此KP背景中选择MSBAISR突变体。 AIM 2。证明MSBAIS的化学障碍性。 AIM 2的目标是执行铅（H2L）的命中率（H2L） 每个MSBAI的药物化学结构活动关系（SAR）研究> 1系列是 可以建立化学处理和可量化的铅优化标准，包括i）EC和 KP MSBA体外效力（IC50 <0.25 µg/ml），II）EC和KP WT活性（<16 µg/ml），III）> 90％HEPG2细胞 50倍MIC的生存能力针对ECΔTOLC。 MSBAI实现这些里程碑是一个合理的起点 利用AIM 3的AIM 2和SBDD信息获得的未来潜在客户优化。 AIM 3。与SSGCID合作获取EC和KP MSBAI-MSBA共晶体结构。目标 AIM 3活动是在原核生物，Genentech和Seattle结构性之间进行合作 彼得·迈勒（Peter Myler）博士领导的基因组学传染病中心（SSGCID）优化适合适合的条件 在APO形式以及EC和/或KP MSBA中获得KP MSBA的高分辨率X射线晶体结构 蛋白质与MSBAIS复杂化。