Characterization of Bacterial Lectin-Carbohydrate Binding and Development of Anti-Adhesion Inhibitors

细菌凝集素-碳水化合物结合的表征和抗粘附抑制剂的开发

• 批准号: 10625679
• 负责人: Caitlin M McMahon
• 金额: $ 37.77万
• 依托单位: UNIVERSITY OF NORTH CAROLINA ASHEVILLE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625679
• 关键词: Adhesions; Affinity; Amides; Amino Acids; Antibiotic Resistance; Area; Bacterial Adhesins; Bacterial Adhesion; Bacterial Infections; Binding; Binding Sites; Biochemistry; Biological Assay; Biological Process; Carbohydrates; Cell Adhesion Molecules; Characteristics; Chemicals; Collection; Competitive Binding; Crystallography; Data; Development; Diagnostic; Diarrhea; Docking; Elements; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Future; Gastric ulcer; Goals; Helicobacter Infections; Helicobacter pylori; Hydrogen Bonding; Hydrophobic Surfaces; Hydrophobicity; Immobilization; Individual; Infection; Interruption; Knowledge; Lectin; Lectin Inhibitor; Libraries; Ligand Binding; Ligands; Mammals; Mediating; Microbial Biofilms; Modification; Molecular; Molecular Biology; Molecular Probes; Organic Chemistry; Outcome; Polysaccharides; Positioning Attribute; Proteins; Pseudomonas aeruginosa; Reporting; Research; Research Design; Research Personnel; Resistance; Resistance development; Roentgen Rays; Role; Sialic Acids; Site-Directed Mutagenesis; Specificity; Spectrum Analysis; Structure; Structure-Activity Relationship; Students; Surface Plasmon Resonance; Techniques; Testing; Time; Training; Uropathogenic E. coli; Virulence; Virulence Factors; Visualization; Work; X-Ray Crystallography; adhesion process; anomer; carbohydrate binding protein; carbohydrate receptor; career; design; effective therapy; enterotoxigenic Escherichia coli; experience; experimental study; inhibitor; insight; malignant stomach neoplasm; pathogen; pressure; rational design; sialic acid-binding lectin; sialyl Lewis x; skills; small molecule; tool; undergraduate student; virtual

PROJECT SUMMARY Lectins are carbohydrate-binding proteins that mediate a range of biological processes including bacterial adhesion and biofilm formation. Bacterial lectins involved in adhesion, termed adhesins, are one type of virulence factor pathogens use to infect and persist in a host. As rates of antibiotic resistance continue to rise, new strategies are needed to treat infection, including the use of anti-virulence therapies, which target bacterial virulence factors such as adhesins, to weaken resistance and facilitate treatment and clearance. This strategy is proving to be successful for a few lectins in uropathogenic Escherichia coli and Pseudomonas aeruginosa, but no effective inhibitors of adhesins in enterotoxigenic E. coli or Helicobacter pylori have been developed. This is in part due to a lack of characterization of the binding sites and structural basis for recognition in these lectins, including the F17G adhesin on enterotoxigenic E. coli which binds N-acetylglucosamine (GlcNAc) and the SabA adhesin on H. pylori which binds sialic acid and sialyl Lewis x. There is also incomplete structural data regarding the carbohydrate binding site of SabA - no crystal structure has been reported in the presence of a ligand. This project will engage undergraduate researchers in the characterization of the molecular basis for F17G and SabA binding and adhesion and the synthesis of high-affinity ligands. In Aim 1, F17G ligands will be rationally designed, harnessing structural information from molecular docking screens and protein X-ray crystal structure analysis. Small libraries of GlcNAc derivatives will be synthesized, making modifications at both the anomeric position and the amide position on the carbohydrate aimed at increasing hydrogen bonding, hydrophobic, and π-stacking interactions. Ligands will be tested in competitive binding assays using enzyme-linked immunosorbent assays (ELISA) and surface plasmon resonance spectroscopy (SPR). Effective compounds will lead to powerful probes and inhibitors of E. coli adhesion and infection. In Aim 2, the binding site of the H. pylori adhesin SabA will be interrogated using site-directed mutagenesis, X-ray crystallography, and binding assays. These experiments will lead to a clear understanding of the amino acid residues involved in binding, the first crystal structure with SabA bound to a ligand, and structural insight toward development of H. pylori adhesion inhibitors and probes. Together, the work proposed will provide structure-activity relationships and a detailed molecular basis for ligand binding of two bacterial lectins. This foundational data will lead to the development of potent lectin inhibitors which can be used to interrupt bacterial adhesion as anti-virulence treatments. The structural information generated from this work will also provide the basis for the manipulation of lectins for use as probes of lectin function and glycan structure.

项目概要 凝集素是碳水化合物结合蛋白，介导一系列生物过程，包括细菌 粘附和生物膜形成。参与粘附的细菌凝集素，称为粘附素，是一种毒力 病原体用于感染宿主并在宿主体内持续存在的因子。随着抗生素耐药率持续上升，新的 需要治疗感染的策略，包括使用针对细菌的抗毒力疗法 毒力因子，如粘附素，以削弱抵抗力并促进治疗和清除。这个策略 事实证明，对于泌尿道致病性大肠杆菌和铜绿假单胞菌中的一些凝集素来说是成功的，但是 目前尚未开发出产肠毒素大肠杆菌或幽门螺杆菌中粘附素的有效抑制剂。这是 部分原因是缺乏这些凝集素的结合位点特征和识别结构基础， 包括产肠毒素大肠杆菌上的 F17G 粘附素，可结合 N-乙酰氨基葡萄糖 (GlcNAc) 和 SabA 幽门螺杆菌上的粘附素，结合唾液酸和唾液酸路易斯 x。还存在不完整的结构数据 SabA 的碳水化合物结合位点 - 在配体存在下没有晶体结构的报道。这 该项目将让本科生研究人员参与 F17G 和 SabA 分子基础的表征 结合和粘附以及高亲和力配体的合成。在Aim 1中，F17G配体将被合理设计， 利用分子对接屏幕和蛋白质 X 射线晶体结构分析中的结构信息。 将合成 GlcNAc 衍生物的小文库，在异头位置和 碳水化合物上的酰胺位置旨在增加氢键、疏水性和 π 堆积 互动。将使用酶联免疫吸附测定在竞争性结合测定中测试配体 （ELISA）和表面等离子共振光谱（SPR）。有效的化合物将产生强大的探针 以及大肠杆菌粘附和感染的抑制剂。在目标 2 中，幽门螺杆菌粘附素 SabA 的结合位点是 使用定点诱变、X 射线晶体学和结合测定进行研究。这些实验将 有助于清楚地了解参与结合的氨基酸残基，这是与 SabA 的第一个晶体结构 与配体结合，以及对幽门螺杆菌粘附抑制剂和探针开发的结构洞察。 总之，所提出的工作将为配体提供结构-活性关系和详细的分子基础 两种细菌凝集素的结合。这些基础数据将促进有效凝集素抑制剂的开发 可用于阻断细菌粘附作为抗毒力治疗。结构信息 这项工作产生的结果也将为操作凝集素作为凝集素探针提供基础 功能和聚糖结构。