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.

项目摘要 凝集素是一种碳水化合物结合蛋白，介导一系列生物过程，包括细菌凝集素和植物凝集素。 粘附和生物膜形成。参与粘附的细菌凝集素，称为粘附素，是一种毒性类型 病原体用于感染并在宿主中持续存在的因素。随着抗生素耐药性的持续上升，新 治疗感染的策略是必要的，包括使用针对细菌的抗病毒疗法。 毒力因子，如粘附素，以削弱抵抗力，促进治疗和清除。这一战略 在致尿路感染的大肠杆菌和铜绿假单胞菌中， 在肠出血性大肠杆菌中没有有效的粘附素抑制剂。大肠杆菌或幽门螺杆菌。这是 部分由于缺乏对这些凝集素中识别的结合位点和结构基础的表征， 包括F17 G粘附素在肠出血性大肠杆菌上的表达。结合N-乙酰葡糖胺（GlcNAc）和SabA的大肠杆菌 粘附素在H.结合唾液酸和唾液酸刘易斯的幽门螺杆菌x.还有不完整的结构数据， 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 该项目将吸引本科研究人员参与F17 G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。目的1：合理设计F17 G配体， 利用分子对接屏幕和蛋白质X射线晶体结构分析的结构信息。 将合成GlcNAc衍生物的小文库，在异头位置和末端位置进行修饰。 碳水化合物上的酰胺位置旨在增加氢键、疏水性和π堆积 交互.将使用酶联免疫吸附试验在竞争性结合试验中检测配体 （ELISA）和表面等离子体共振光谱（SPR）。有效的化合物将导致强大的探针 和E.大肠杆菌粘附和感染。在Aim 2中，H. pylori adhesin SabA将 使用定点诱变、X射线晶体学和结合测定进行询问。这些实验将 导致清楚地了解参与结合的氨基酸残基，第一个晶体结构与SabA 与配体结合，以及对H发展的结构洞察。幽门粘附抑制剂和探针。 总之，所提出的工作将提供配体的结构-活性关系和详细的分子基础 两种细菌凝集素的结合。这一基础数据将导致开发有效的凝集素抑制剂 其可用于中断细菌粘附作为抗毒性治疗。结构信息 从这项工作中产生的也将提供基础的操作凝集素作为探针的凝集素 功能和聚糖结构。