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.

项目总结 凝集素是一种碳水化合物结合蛋白，调节包括细菌在内的一系列生物过程。 黏附和生物膜的形成。参与黏附的细菌凝集素，称为黏附素，是一种毒力。 病原体用来感染并在宿主体内存活的因子。随着抗生素耐药率的持续上升，新的 需要采取策略来治疗感染，包括使用针对细菌的抗毒力疗法 毒力因子，如粘附素，以削弱耐药性，促进治疗和清除。这一战略 已被证明对几种凝集素在致尿路疾病的大肠杆菌和铜绿假单胞菌中成功，但 目前还没有针对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 部分由于缺乏对这些凝集素中的结合部位和识别的结构基础的表征， 包括与N-乙酰氨基葡萄糖(GlcNAc)和SABA结合的产肠毒素大肠杆菌上的F17G粘附素 幽门螺杆菌上的粘附素，它与唾液酸和唾液酸基Lewis x结合。 在配体存在的情况下，已报道了Saba-NO晶体结构中的碳水化合物结合部位。这 该项目将邀请本科生研究人员描述F17G和Saba的分子基础 结合和黏附以及高亲和力配体的合成。在目标1中，将合理设计F17G配体， 利用来自分子对接屏幕和蛋白质X射线晶体结构分析的结构信息。 将合成小的GlcNAc衍生物文库，在异构体位置和 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。配体将在竞争结合分析中使用酶联免疫吸附分析进行测试 (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 以及抑制大肠杆菌黏附和感染的药物。在目标2中，幽门螺杆菌粘附素Saba的结合部位为 使用定点突变、X射线结晶学和结合分析进行询问。这些实验将 导致清楚地了解氨基酸残基参与结合，第一晶体结构与撒巴 与配体结合，以及对幽门螺杆菌黏附抑制剂和探针开发的结构洞察力。 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。这一基础数据将导致有效的凝集素抑制剂的开发 它可以用来中断细菌黏附作为抗毒力治疗。结构信息 这项工作产生的凝集素也将为操作用作凝集素探针的凝集素提供基础 功能和糖链结构。