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: 10242174
• 负责人: Terry Roemer
• 金额: $ 14万
• 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/10242174
• 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 ΔtolC 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，CDC 将耐碳青霉烯类肠杆菌科 (CRE) 列为紧急威胁，并于 2017 年， 世界卫生组织将其指定为 1 级“严重超级细菌”。由于治疗 CRE 的疗法所剩无几，“泛- 目前任何可用抗生素都无法治疗的“耐药性”CRE 也存在。具有新颖性的全新药物 作用机制（MOA）不与 SOC 药物交叉耐药。开发这样的“一流” 该药物还为急需的口服 CRE 治疗提供了潜力，从而提供了一种新的 降压治疗以减少住院时间和不断增加的医疗费用。我们的建议旨在开发一个 治疗因多重耐药性 (MDR) 引起的危及生命的细菌感染的全新疗法 肠杆菌科，包括 CRE。使用创新的基于过度表达的共培养筛选 大肠杆菌 (Ec)，我们鉴定了四种结构不同的系列针对 MsbA 的小分子抑制剂， 一种重要且广泛保守的革兰氏阴性 (GN) ABC 转运蛋白，负责脂多糖 （LPS）生物发生和革兰氏阴性外膜（OM）的构建。建立在坚实的基础上 在初步数据的基础上，我们的目标是： 目标 (A) 1. 扩大 MOA 研究。目标 1 研究将扩展我们初步的 MOA 和微生物学 MsbA 抑制剂 (MsbAi) 的特征包括其他 GN 细菌，特别是克雷伯氏菌 肺炎链球菌（Kp）。具体来说，我们将在 (1) 体外生化测定中证明 Kp MsbA 靶标抑制作用 (2) 通过确定我们的 MsbAis 对 Kp ΔtolC 菌株的 MIC，然后确定全细胞环境 在此 Kp 背景中选择 MsbAisR 突变体。 目标 2. 证明 MsbAis 的化学可处理性。目标 2 的目标是实现“Hit to Lead”（H2L） 每个 MsbAi 的药物化学结构活性关系 (SAR) 研究并证明 >1 系列是 化学上易于处理，并且可以建立可量化的先导优化标准，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)，以及 iii) > 90% HepG2 细胞 在 50X MIC 下针对 EcΔtolC 的活力。 MsbAis 实现这些里程碑是一个合理的起点 未来的先导优化利用目标 2 中获得的 SAR 和目标 3 中获得的 SBDD 信息。 目标 3. 与 SSGCID 合作获得 Ec 和 Kp MsbAi-MsbA 共晶结构。目标是 目标 3 活动是发起 Prokaryotics、Genentech 和西雅图结构研究所之间的合作 Peter Myler 博士领导的传染病基因组学中心 (SSGCID) 优化了适合的条件 获得 apo 形式的 Kp MsbA 以及 Ec 和/或 Kp MsbA 的高分辨率 X 射线晶体结构 与 MsbAis 形成复合物的蛋白质。