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

尿路致病性大肠杆菌粘附蛋白 D 肽抑制剂治疗尿路感染

• 批准号: 10932838
• 负责人: Marcy Robins
• 金额: $ 4.12万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10932838
• 关键词: Acetylgalactosamine; Acute; Adhesions; Affinity; Amino Acids; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Avidity; Bacteria; Bacterial Adhesins; Bacteriophages; Binding; Biological Assay; Bladder; Bypass; Catheterization; Cell-Matrix Junction; Cells; Chemicals; Chronic; Circular Dichroism; Circular Dichroism Spectroscopy; Collaborations; Complex; Consensus Sequence; Cytoplasm; Disease Progression; Drug Delivery Systems; Drug Kinetics; Drug resistance; Filtration; Fluorescence Polarization; Galactose; Goals; Growth; Half-Life; Hemagglutination; Image; Immune response; Infection; Inflammation; Injections; Kidney; Knowledge; Laws; Lead; Lectin; Libraries; Ligation; Lower urinary tract; Mammalian Cell; Mannose; Measures; Mediating; Methods; Microbial Biofilms; Modeling; Molecular Conformation; Monitor; Mus; Output; Pathogenesis; Peptide Hydrolases; Peptide Phage Display Library; Peptide Synthesis; Peptides; Periodicity; Phage Display; Phase; Pilum; Population; Proteins; Randomized; Recombinant Proteins; Recombinants; Recurrence; Reporting; Research; Risk; Risk Reduction; Secondary Protein Structure; Solid; Specificity; Structure; Surface; Transitional Epithelium; Treatment Efficacy; Urinary tract; Urinary tract infection; Urination; Urine; Uropathogenic E. coli; Validation; Virulence; Woman; X-Ray Crystallography; alternative treatment; antimicrobial; bacterial community; chemical synthesis; commensal bacteria; cost; cytotoxicity; deep sequencing; design; efficacy evaluation; efficacy study; enantiomer; fitness; glycomimetics; gut microbiome; gut microbiota; immune clearance; immunogenicity; improved; in vitro Assay; in vivo; inhibitor; kidney cell; mouse model; next generation sequencing; novel; peptide L; peptide chemical synthesis; prevent; recurrent infection; resistant strain; screening; vaginal microbiota

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.

项目摘要 附着的尿道病大肠杆菌（UPEC）的粘附蛋白需要 宿主细胞以促进尿路感染（UTI）的定殖和疾病进展。主要 与UPEC毒力相关的粘附蛋白是FIMH，它结合了d-甘露糖在叶状细胞上，并且 FMLH结合肾脏和发炎的膀胱细胞上的N-乙酰基半乳糖胺。该建议旨在发展 防止UPEC在近皮细胞中的附着和随后内在化的UTI处理， 从尿路中消除细菌。阻止高度保守的结合口袋的治疗 FIMH/FMLH防止UPEC附着在宿主细胞上，并清除尿路6,9,11的细菌。然而， 设计到达尿路的抑制剂以降低复发感染的风险（例如，长时间 半衰期且不会破坏共生细菌）仍然具有挑战性。我们的目标是使用镜像噬菌体 显示以鉴定UPEC粘附的D肽抑制剂，以消除尿路中的细菌。我们 假设可以防止与宿主细胞附着附着的D肽抑制剂可以用作新颖 抗生素比例的UTI治疗。 D肽是此应用的理想选择，因为它们通过肾脏清除 过滤（积聚在尿液中），具有长的半衰期，可以作为长效注射配制。 为了筛选D肽抑制剂，我们将首先化学合成镜像D-target蛋白 （FIMH/FMLH）使用固相肽合成（SPP）与D-氨基酸和天然化学结扎（NCL）。 这些D蛋白将通过与重组L蛋白进行比较来验证二级结构和结合 活动。使用镜像噬菌体显示器，我们将筛选L肽粘合剂的不同噬菌体库 合成的D靶蛋白。下一代测序（NGS）将用于识别高亲和力的命中 然后将在d-中合成以抑制天然L靶向。 我们将通过直接结合和竞争研究来确定抑制剂的亲和力。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。体外测定将使用40个UPEC菌株面板进行验证D- 肽细胞附着，血凝和生物膜形成的肽抑制。我们将确定 小鼠中最有希望的D肽的药代动力学（PK）谱。最后，我们将评估 小鼠急性和慢性UTI模型的处理。该项目将贡献有关UPEC的宝贵信息 Pili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 牵头进行精确的UTI治疗。