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.

革兰氏阴性细菌利用称为伴侣糖途径（杯）pili的细胞外纤维介导粘附 宿主和环境表面，促进入侵宿主组织，并促进与其他细菌的相互作用 形成生物膜。肝病大肠杆菌（UPEC）在1型钢琴上使用称为FIMH的杯子粘合剂蛋白（凝集素） 与膀胱上皮细胞上的甘露糖基化糖蛋白结合以介导尿的发作和进展 道感染（UTI）。这种结合事件引发细菌侵袭和细胞内细菌的形成 真核细胞中的社区（IBC）。使用合理的药物设计，FIMH的甘露糖苷拮抗剂已作为口服生物利用疗法开发，用于治疗和预防UTI。急性感染可以 自我溶解或发展为慢性膀胱炎，其特征是：i）持续的高滴度细菌和 感染后牺牲> 4周时，细菌膀胱负担; ii）慢性炎症和尿路上皮坏死； iii）膀胱粘膜下淋巴结增生和； iv）缺乏尿蓝蛋白表达的尿路上皮增生，这是表面细胞末端分化的标志物。类似的组织学发现 在患有持续的细菌和复发性尿路的人类中观察到。 FMLH，尖端相关 FML/F9/YDE PILI（以前表示FMLD）在UPEC发病机理中功能的粘附蛋白，提供适应性 慢性膀胱炎期间的优势。 FMLH特异性结合了GAL（β1-3）Galnac表位，该表位是作为一部分 在慢性膀胱炎期间，小鼠膀胱的重塑聚糖/半乳糖谱。 FMLH也已更新 与在培养基或正常情况下体外生长期间的表达相比，从UTI患者直接分离的尿液直接分离 尿液，提示特定于宿主的诱导。在此提案中，创新策略将合理地采用 将βGAL和βGalNAC配体作为FMLH的拮抗剂，单独或与FIMH抑制剂结合起来，为 用于治疗急性和慢性膀胱炎的新临床前治疗。使用FMLH的X射线结构和 鉴定了虚拟筛选，包括O-亚硝基苯基-β-半乳糖苷（ONPG）在内的新FMLH配体。 随后，使用与FMLH结合的Galβ-1-3-GALNAC（TF）和ONPG的X射线结构用于设计 改进的FMLH配体，结合亲和力比ONPG高几倍，由ELISA确定 结合测定。这种基于结构的设计策略将用于优化具有较高亲和力的新配体 和良好的药物样特性。该项目的目标将通过集成：i）基于X射线结构的药物设计与医学化学（AIM 1）； ii）生化筛选，功能细胞和组织结合测定 目标2）and;严格的药代动力学（PK）评估和药效学（PD）效率最多 有希望的FMLH配体，在慢性膀胱炎的鼠动物模型中 殖民化（目标3）。总体目标是将临床前候选FMLH拮抗剂作为独立 抗病毒疗法和/或作为与现有FIMH拮抗剂进行治疗的组合疗法和/或 预防UTI。