D-peptide Inhibitors of Uropathogenic E. coli Adhesion Proteins to Treat Urinary Tract Infections

项目摘要 尿致病性E.大肠杆菌（UPEC）进行附着 宿主细胞以促进尿路感染（UTI）中的定殖和疾病进展。主 与UPEC毒力相关的粘附蛋白是FimH，其结合尿上皮细胞上的D-甘露糖， FmlH，其结合肾脏和发炎膀胱细胞上的N-乙酰半乳糖胺。该提案旨在发展 防止UPEC在尿路上皮细胞中附着和随后内化的UTI治疗， 清除尿道中的细菌。阻断高度保守的结合口袋的治疗 FimH/FmlH阻止UPEC附着于宿主细胞并从尿路清除细菌6，9，11。然而，在这方面， 设计到达泌尿道的抑制剂以降低复发感染的风险（例如，通过长 半衰期和不破坏肠道细菌）仍然具有挑战性。我们的目标是利用镜像噬菌体 显示以鉴定UPEC粘附的D-肽抑制剂，从而从泌尿道中消除细菌。我们 假设阻止附着于宿主细胞的D-肽抑制剂可以作为新的 泌尿生殖道感染治疗。D-肽是这种应用的理想选择，因为它们可以通过肾脏清除 过滤（在尿液中积累），半衰期长，可以配制成长效注射剂。 为了筛选D肽抑制剂，我们将首先化学合成镜像D靶蛋白 (FimH/FmlH），使用具有D-氨基酸和天然化学连接（NCL）的固相肽合成（SPPS）。 这些D蛋白将通过与重组L蛋白的二级结构和结合进行比较来验证 活动利用镜像噬菌体展示，我们将筛选不同的噬菌体库中的L-肽结合剂， 合成D-靶蛋白。下一代测序（NGS）将用于鉴定高亲和力命中， 然后会在D-中合成以抑制天然的L-目标。 我们将通过直接结合和竞争研究确定我们的抑制剂的亲和力。晶体结构 我们的D-肽与FimH或FmlH的复合物将使用X射线晶体学获得，并将告知 抑制剂亲和力优化。将使用一组40种UPEC菌株进行体外试验，以验证D- 肽抑制宿主细胞附着、血凝和生物膜形成。康贝特人将以 图1显示了小鼠中最有希望的D-肽的药代动力学（PK）曲线。最后，我们将评估 在小鼠急性和慢性UTI模型中的治疗。该项目将为UPEC提供有价值的信息 皮利结合相互作用，推进我们的化学蛋白质合成知识，并产生D-肽抑制剂 用于精确UTI治疗的导线。

PROJECT SUMMARY Adhesion proteins on the pilus-covered surface of Uropathogenic E. coli (UPEC) are required for attachment to host cells to facilitate colonization and disease progression in urinary tract infections (UTIs). The primary adhesion proteins associated with UPEC virulence are FimH, which binds D-mannose on uroepithelial cells, and FmlH, which binds N-acetylgalactosamine on kidney and inflamed bladder cells. This proposal aims to develop UTI treatments that prevent the attachment and subsequent internalization of UPEC in uroepithelial cells, eliminating bacteria from the urinary tract. Treatments that block the highly conserved binding pockets of FimH/FmlH prevent UPEC attachment to host cells and clear bacteria from the urinary tract6,9,11. However, designing inhibitors that reach the urinary tract to reduce the risk for recurrent infections (e.g., through a long half-life and without disrupting commensal bacteria) remains challenging. Our goal is to use mirror-image phage display to identify D-peptide inhibitors of UPEC adhesion to eliminate bacteria from the urinary tract. We hypothesize that D-peptide inhibitors that prevent attachment to host cells could serve as novel antibiotic-sparing UTI treatments. D-peptides are ideal for this application because they are cleared via kidney filtration (accumulate in urine), have a long half-life, and can be formulated as a long-acting injection. To screen for D-peptide inhibitors, we will first chemically synthesize the mirror-image D-target proteins (FimH/FmlH) using solid-phase peptide synthesis (SPPS) with D-amino acids and native chemical ligation (NCL). These D-proteins will be validated by comparison to recombinant L-proteins for secondary structure and binding activity. Using mirror-image phage display, we will screen diverse phage libraries for L-peptide binders to the synthesized D-target proteins. Next-generation sequencing (NGS) will be used to identify high-affinity hits that will then be synthesized in D- to inhibit the natural L-target. We will determine the affinity of our inhibitors via direct binding and competition studies. The crystal structures of our D-peptides in complex with FimH or FmlH will be obtained using X-ray crystallography and will inform inhibitor affinity optimization. In vitro assays will be performed using a panel of 40 UPEC strains to validate D- peptide inhibition of host cell attachment, hemagglutination, and biofilm formation. We will determine the pharmacokinetic (PK) profile of the most promising D-peptides in mice. Finally, we will evaluate the efficacy of treatments in mouse acute and chronic UTI models. This project will contribute valuable information about UPEC pili binding interactions, advance our chemical protein synthesis knowledge, and generate D-peptide inhibitor leads for precision UTI treatment.