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

• 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
• 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

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.

革兰氏阴性菌利用称为伴侣-引导者途径(CUP)的胞外纤维来调节黏附 宿主和环境表面，促进对宿主组织的入侵，并促进与其他细菌的相互作用 形成生物膜。尿路致病性大肠杆菌(UPEC)在1型菌毛上使用一种名为FimH的杯状黏附蛋白(凝集素) 与膀胱上皮细胞甘露糖化糖蛋白结合介导尿路疾病的发生发展 呼吸道感染(UTIs)。这种结合事件启动细菌入侵和细胞内细菌的形成 真核细胞中的群落(IBCs)。通过合理的药物设计，FimH的甘露糖苷拮抗剂已被开发为治疗和预防尿路感染的口服生物利用疗法。急性感染可以是 自愈或发展为慢性膀胱炎，其特征为：i)持续性、高滴度菌尿和 感染后4周的细菌膀胱负荷；ii)慢性炎症和尿路上皮坏死； 3)膀胱粘膜下层淋巴结节增生；4)尿路上皮增生，缺乏尿路上皮蛋白的表达，这是浅表小关节细胞终末分化的标志。相似的组织学发现 已经在患有持续性菌尿和复发性尿路感染的人类中观察到。FmlH，与TIP关联的 FML/F9/Yde菌毛粘附素(FmlD)通过提供适合性在UPEC发病机制中的作用 在慢性膀胱炎期间的优势。FmlH与Gal(β1-3)GalNAc表位特异性结合，后者是 慢性膀胱炎期间小鼠膀胱的糖链/半乳糖图谱。FmlH也在 尿路感染患者直接分离的尿液与体外培养或正常培养期间表达的比较 尿液，表明是宿主特异性诱导。在这一建议中，将理性地采取创新战略 发展β半乳糖和β半乳糖作为Fm1H的拮抗剂，单独或与FimH抑制剂联合应用，AS 治疗急慢性膀胱炎的新的临床前治疗方法。使用FmlH的X射线结构和 通过虚拟筛选，确定了新的FmlH配体，包括邻硝基苯基β半乳糖苷(ONPG)。 随后，利用Galβ-1-3-GalNAc(Tf)和与FmlH结合的ONPG的X射线结构进行了设计 改进的FmlH配体，结合亲和力比ONPG高几倍，通过ELISA测定 结合试验。这种基于结构的设计策略将被用来优化具有更高亲和力的新配体 和良好的类似药物的特性。该项目的目标将通过以下方式实现：i)基于X射线结构的药物设计与药物化学(目标1)相结合；ii)生化筛选、功能细胞和组织结合分析 目的2)和；严格的药代动力学(PK)评价和药效学(PD)药效试验 在慢性膀胱炎、尿毒症/肾脏感染和GIT小鼠动物模型中有前景的FmlH配体 殖民(目标3)。总体目标是开发临床前候选FmlH拮抗剂作为独立药物 抗毒力疗法和/或作为与现有FimH拮抗剂的联合治疗 预防尿路感染。