SMALL MOLECULE BACTERIAL LECTIN ANTAGONISTS FOR UTI TREATMENT AND PREVENTION

用于治疗和预防尿路感染的小分子细菌凝集素拮抗剂

基本信息

批准号：
9234333
负责人：
SCOTT J. HULTGREN
金额：
$ 48.57万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-01 至 2021-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9234333
关键词：
Acute Acute Cystitis Adhesions Affinity Animal Model Antibiotic Resistance Antibiotic Therapy Bacteria Bacterial Adhesins Bacteriuria Binding Binding Proteins Bioavailable Biochemical Biological Biological Assay Biological Availability Biophysics Bladder Bladder Tissue Carbon Cells Cellular Assay Child Chronic Chronic Cystitis Collaborations Combined Modality Therapy Communities Cost of Illness Crystallization Cystitis Development Docking Drug Design Drug Kinetics Elderly Environment Enzyme-Linked Immunosorbent Assay Epithelial Cells Epithelium Epitopes Escherichia Eukaryotic Cell Evaluation Event Female Fiber Gal-GalNAc Galactosamine Galactose Galactosides Gene Cluster Genome Glycoproteins Glycosides Goals Gram-Negative Bacteria Growth Hemophilus Histologic Human Hyperplasia In Vitro Infection Infective cystitis Inflammation Interferometry Intestines Klebsiella Knowledge Lectin Libraries Ligands Liquid substance Liver Microsomes Mannose Mannosides Mediating Metabolic Microbial Biofilms Modeling Molecular Chaperones Molecular Models Molecular Weight Mus Mutation Necrosis Nitrogen Oral Pathogenesis Pathogenicity Pathway interactions Patients Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacodynamics Pilum Plasma Plasma Proteins Polysaccharides Positioning Attribute Pre-Clinical Model Prevention Property Proteins Pseudomonas Recurrence Roentgen Rays Salmonella Scanning Solubility Structure Submucosa Surface System Testing Therapeutic Time Tissues Treatment Efficacy Urinary tract infection Urine Uropathogenic E. coli Urothelial Hyperplasia Virulence Virulence Factors Woman Yersinia analog aqueous base combat design efficacy testing extracellular fitness improved in vivo inhibitor/antagonist innovation kidney infection male molecular modeling mouse model novel therapeutics pre-clinical preclinical evaluation prevent renal abscess screening small molecule small molecule libraries sugar tissue/cell culture virtual

项目摘要

Gram-negative bacteria utilize extracellular fibers called chaperone-usher pathway (CUP) pili to mediate adhesion to host and environmental surfaces, facilitate invasion into host tissues, and promote interaction with other bacteria to form biofilms. Uropathogenic E. coli (UPEC) use a CUP adhesion protein (lectin) called FimH on the type 1 pilus to bind to mannosylated glycoproteins on bladder epithelial cells to mediate the onset and progression of urinary tract infections (UTIs). This binding event initiates bacterial invasion and formation of intracellular bacterial communities (IBCs) in the eukaryotic cell. Using rational drug design, mannoside antagonists of FimH have been developed as orally bioavailable therapeutics for the treatment and prevention of UTIs. Acute infection can either self-resolve or develop into chronic cystitis, which is characterized by: i) persistent, high titer bacteriuria and bacterial bladder burdens at sacrifice >4 weeks post- infection; ii) chronic inflammation and urothelial necrosis; iii) lymphonodular hyperplasia in the bladder submucosa and; iv) urothelial hyperplasia with a lack of uroplakin expression, which is a marker for terminal differentiation in superficial facet cells. Similar histological findings have been observed in humans suffering persistent bacteriuria and recurrent UTI. FmlH, the tip-associated adhesin of Fml/F9/Yde pili (previously denoted FmlD) functions in UPEC pathogenesis by providing a fitness advantage during chronic cystitis. FmlH specifically binds to Gal(β1-3)GalNac epitopes which appear as part of a remodeled glycan/galactose profile of the mouse bladder during chronic cystitis. FmlH is also upregulated in urines directly isolated from patients with UTI compared to expression during in vitro growth in media or normal urine, suggesting a host-specific induction. In this proposal, innovative strategies will be taken to rationally develop βGal and βGalNAc ligands as antagonists of FmlH, alone or in combination with FimH inhibitors, as new preclinical therapeutics for treatment of acute and chronic cystitis. Using an X-ray structure of FmlH and virtual screening, new FmlH ligands, including O-nitrophenyl-β-galactoside (ONPG) were identified. Subsequently, the X-ray structures of Galβ-1-3-GalNAc (TF) and ONPG bound to FmlH were used to design improved FmlH ligands, with binding affinities several times higher than ONPG, as determined by an ELISA binding assay. This structure-based design strategy will be used to optimize new ligands with higher affinity and good drug-like properties. This project's aims will be accomplished by integrating: i) X-ray structure-based drug design with medicinal chemistry (Aim 1); ii) biochemical screening, functional cell and tissue binding assays Aim 2) and; rigorous pharmacokinetic (PK) evaluation and pharmacodynamic (PD) efficacy testing of the most promising FmlH ligands, in murine animal models of chronic cystitis, urosepsis/kidney infection, and GIT colonization (Aim 3). The overall goal is to develop preclinical candidate FmlH antagonists as standalone anti-virulence therapeutics and/or as combination therapy with existing FimH antagonists for the treatment and prevention of UTIs.

革兰氏阴性细菌利用称为伴侣引导通路 (CUP) 菌毛的细胞外纤维来介导粘附附着在宿主和环境表面，促进侵入宿主组织，并促进与其他细菌的相互作用尿路致病性大肠杆菌 (UPEC) 在 1 型菌毛上使用一种称为 FimH 的 CUP 粘附蛋白（凝集素）来形成生物膜。与膀胱上皮细胞上的甘露糖化糖蛋白结合，介导泌尿系疾病的发生和进展这种结合事件引发细菌入侵和细胞内细菌的形成。通过合理的药物设计，FimH 的甘露糖苷拮抗剂已被开发为口服生物可利用的治疗剂，可用于治疗和预防急性感染。自愈或发展为慢性膀胱炎，其特征为：i) 持续、高滴度菌尿和感染后>4周处死时的细菌性膀胱负担；ii)慢性炎症和尿路上皮坏死； iii) 膀胱粘膜下层淋巴结节增生；iv) 尿路上皮增生，缺乏尿斑蛋白表达，尿斑蛋白表达是浅表小面细胞终末分化的标志物。已在患有持续性菌尿和复发性 FmlH（与尖端相关）的人类中观察到。 Fml/F9/Yde pili 的粘附素（以前称为 FmlD）通过提供适应性在 UPEC 发病机制中发挥作用 FmlH 在慢性膀胱炎期间具有特异性结合 Gal(β1-3)GalNac 表位，该表位作为慢性膀胱炎期间小鼠膀胱的重构聚糖/半乳糖谱也在 FmlH 中上调。直接从 UTI 患者分离的尿液与在培养基或正常体外生长期间的表达进行比较尿液，表明宿主特异性诱导在该提案中，将采取合理的创新策略。开发 βGal 和 βGalNAc 配体作为 FmlH 的拮抗剂，单独或与 FimH 抑制剂组合，如使用 FmlH 的 X 射线结构治疗急性和慢性膀胱炎的新临床前疗法。通过虚拟筛选，鉴定出了新的 FmlH 配体，包括 O-硝基苯基-β-半乳糖苷 (ONPG)。随后，利用与 FmlH 结合的 Galβ-1-3-GalNAc (TF) 和 ONPG 的 X 射线结构来设计改进的 FmlH 配体，经 ELISA 测定，其结合亲和力比 ONPG 高数倍这种基于结构的设计策略将用于优化具有更高亲和力的新配体。该项目的目标将通过整合以下方式来实现：i) 基于 X 射线结构的药物设计与药物化学（目标 1）；生化筛选、功能细胞和组织结合测定。目标 2) 和；严格的药代动力学 (PK) 评价和药效学 (PD) 功效测试在慢性膀胱炎、尿脓毒症/肾脏感染和胃肠道感染的小鼠动物模型中，有前景的 FmlH 配体定植（目标 3）是开发临床前候选 FmlH 拮抗剂作为独立的药物。抗毒力治疗和/或与现有 FimH 拮抗剂联合治疗，用于治疗和预防尿路感染。