SMALL MOLECULE BACTERIAL LECTIN ANTAGONISTS FOR UTI TREATMENT AND PREVENTION

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 以形成生物膜。尿路致病性大肠杆菌 (UPEC) 在 1 型菌毛上使用一种称为 FimH 的 CUP 粘附蛋白（凝集素） 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 泌尿道感染（UTI）。这种结合事件引发细菌入侵并形成细胞内细菌 真核细胞中的群落（IBC）。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，用于治疗和预防尿路感染。急性感染可以 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周处死时的细菌膀胱负荷； 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，尿斑蛋白是浅表小面细胞终末分化的标志物。相似的组织学发现 已在患有持续性菌尿和复发性尿路感染的人类中观察到。 FmlH，尖端相关 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 慢性膀胱炎时的优势。 FmlH 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聚糖/半乳糖谱的重塑。 FmlH 也上调 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。本提案将采取创新策略，合理 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 用于治疗急性和慢性膀胱炎的新临床前疗法。使用 FmlH 的 X 射线结构和 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 结合测定。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 和良好的药物样特性。该项目的目标将通过整合来实现： i) 基于 X 射线结构的药物设计与药物化学（目标 1）； ii) 生化筛选、功能细胞和组织结合测定 目标 2) 和；最严格的药代动力学（PK）评估和药效（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 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。

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.