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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要尿路致病性大肠杆菌 (UPEC) 菌毛覆盖表面上的粘附蛋白是附着所必需的宿主细胞以促进尿路感染（UTI）的定植和疾病进展。初级与 UPEC 毒力相关的粘附蛋白是 FimH，它与尿上皮细胞上的 D-甘露糖结合，并且 FmlH，与肾脏和发炎的膀胱细胞上的 N-乙酰半乳糖胺结合。该提案旨在发展阻止 UPEC 在尿路上皮细胞中附着和随后内化的 UTI 治疗，消除尿道中的细菌。阻断高度保守的结合口袋的治疗 FimH/FmlH 阻止 UPEC 附着于宿主细胞并清除尿道中的细菌6,9,11。然而，设计到达尿路的抑制剂，以降低复发感染的风险（例如，通过长时间的感染）半衰期并且不破坏共生细菌）仍然具有挑战性。我们的目标是使用镜像噬菌体显示识别 UPEC 粘附的 D 肽抑制剂，以消除尿道中的细菌。我们假设阻止与宿主细胞附着的 D 肽抑制剂可以作为新型药物少用抗生素的尿路感染治疗。 D 肽非常适合此应用，因为它们通过肾脏清除过滤（在尿液中积累），半衰期长，可配制成长效注射剂。为了筛选D-肽抑制剂，我们首先化学合成镜像D-靶蛋白 (FimH/FmlH) 使用 D-氨基酸固相肽合成 (SPPS) 和天然化学连接 (NCL)。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。使用镜像噬菌体展示，我们将筛选不同的噬菌体文库，以寻找 L 肽结合物合成D-靶蛋白。下一代测序（NGS）将用于识别高亲和力命中然后将在 D- 中合成以抑制天然的 L-靶标。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。将使用一组 40 种 UPEC 菌株进行体外测定，以验证 D- 肽抑制宿主细胞附着、血凝和生物膜形成。我们将确定最有前途的 D 肽在小鼠体内的药代动力学 (PK) 概况。最后，我们将评估其功效小鼠急性和慢性尿路感染模型的治疗。该项目将提供有关 UPEC 的宝贵信息菌毛结合相互作用，推进我们的化学蛋白质合成知识，并生成 D 肽抑制剂精准治疗尿路感染的线索。