SMALL MOLECULE BACTERIAL LECTIN ANTAGONISTS FOR UTI TREATMENT AND PREVENTION

革兰氏阴性菌利用细胞外纤维称为伴侣引导途径（CUP）皮利介导粘附 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。肾盂肾炎大肠大肠杆菌（UPEC）在1型菌毛上使用称为FimH的CUP粘附蛋白（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激酶的发生和进展， 尿路感染（UTI）。这种结合事件引发细菌侵入和细胞内细菌粘附的形成。 真核细胞中的IBC。使用合理的药物设计，FimH的甘露糖苷拮抗剂已被开发为用于治疗和预防UTI的口服生物可利用的治疗剂。急性感染可以是 自行消退或发展成慢性膀胱炎，其特征在于：i）持续性、高滴度菌尿， 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）膀胱粘膜下层中的淋巴结增生和; iv）缺乏尿斑蛋白表达的尿路上皮增生，尿斑蛋白是浅表小面细胞中终末分化的标志物。相似的组织学结果 已经在患有持续性菌尿和复发性UTI的人中观察到。FmlH，提示相关 Fml/F9/Yde皮利的粘附素（之前表示为FmlD）通过提供适应性在UPEC发病机制中发挥作用 在慢性膀胱炎中的优势。FmlH特异性结合Gal（β1-3）GalNac表位，其作为FmlH的一部分出现。 慢性膀胱炎期间小鼠膀胱的重塑聚糖/半乳糖谱。FmlH也上调， 直接从UTI患者分离的尿液与在培养基或正常培养基中体外生长期间的表达相比 尿液，表明宿主特异性诱导。在这一建议中，将采取创新策略， 开发βGal和βGalNAc配体作为FmlH拮抗剂，单独或与FimH抑制剂组合， 用于治疗急性和慢性膀胱炎的新的临床前疗法。使用FmlH的X射线结构， 通过虚拟筛选，鉴定了新的FmlH配体，包括O-硝基苯基-β-半乳糖苷（ONPG）。 随后，使用与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，其结合亲和力比ONPG高几倍，如通过ELISA测定的 结合分析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 和良好的药物样性质。本项目的目标将通过整合：i）基于X射线结构的药物设计与药物化学（目标1）; ii）生物化学筛选，功能细胞和组织结合试验 目的2）和;严格的药代动力学（PK）评价和药效学（PD）有效性测试， 在慢性膀胱炎、尿脓毒症/肾感染和GIT的鼠动物模型中， 殖民化（目标3）。总体目标是开发临床前候选FmlH拮抗剂作为独立的药物。 抗毒性治疗剂和/或与现有FimH拮抗剂的联合治疗， 预防UTI。

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.