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，CDC将耐碳青霉烯类肠杆菌科（CRE）指定为紧急威胁，2017年， 世界卫生组织将其列为优先级1的“关键超级细菌”。由于很少有治疗CRE的方法，“泛- 也存在任何现有抗生素都无法治疗的“耐药”CRE。具有新颖性的新药物 作用机制（MOA）不交叉耐药的SOC药剂衰退。开发这样的“一流” 该制剂还提供了急需的口服CRE治疗的潜力，从而提供了一种新的 逐步减少治疗，以减少住院时间和不断增长的医疗费用。我们的建议旨在发展一个 一种治疗多药耐药（MDR）导致的危及生命的细菌感染的全新治疗方法 肠杆菌科，包括CRE。使用创新的基于过表达的共培养筛选， 大肠杆菌（Ec），我们确定了四个结构不同的系列小分子抑制剂靶向MsbA， 一种负责脂多糖的基本且广泛保守的革兰氏阴性（GN）ABC转运蛋白 (LPS)革兰氏阴性菌外膜（OM）的生物发生和构建。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研究将扩大我们的初步MOA和微生物 MsbA抑制剂（MsbAi）的特征包括其他GN细菌，特别是克雷伯氏菌 肺炎克雷伯氏菌（Kp）。具体而言，我们将在（1）体外生物化学测定中证明Kp MsbA靶标抑制 和（2）通过测定我们的MsbAis对Kp β tolC菌株的MIC的全细胞背景， 在此Kp背景下选择MsbAisR突变体。 目标二。证明MsbAis的化学易处理性。目标2的目标是执行命中领先（H2 L） 每种MsbAi的药物化学结构活性关系（SAR）研究表明，>1个系列是 化学上易于处理，并且可以建立可量化的先导物前优化标准，包括i）Ec和 Kp MsbA体外效力（IC 50 < 0.25 µg/ml），ii）Ec和Kp WT活性（<16 µg/ml），以及iii）> 90% HepG 2细胞 在50 X MIC下对EcΔtolC的活力。MsbA实现这些里程碑是一个合理的起点， 利用目标2中获得的SAR和目标3中的SBDD信息进行未来的潜在客户优化。 目标3.与SSGCID合作获得Ec和Kp MsbAi-MsbA共晶体结构。的目标 目标3活动是发起原核生物、基因泰克公司和西雅图结构研究所之间的合作 由Peter Myler博士领导的传染病基因组学中心（SSGCID）优化了适合 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复合的蛋白质。