Anthranilate-Based Fungal Nonribosomal Peptide Synthetase Assembly Lines

基于邻氨基苯甲酸的真菌非核糖体肽合成酶装配线

• 批准号: 8013850
• 负责人: Brian Douglas Ames
• 金额: $ 4.56万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2011-12-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8013850
• 关键词: Acyltransferase; Affinity; Alkaloids; Amino Acids; Anabolism; Aspergillus; Aspergillus fumigatus; Bacteria; Benzodiazepines; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Assay; Biological Factors; Biology; Brain; C-terminal; Characteristics; Chemicals; Chemistry; Cholecystokinin Receptor; Cloning; Coenzyme A; Collaborations; Crystallography; Cyclization; Data; Drug Design; Engineering; Environment; Enzymatic Biochemistry; Enzymes; Event; Fermentation; Foundations; Fungal Genome; Fungal Proteins; Gastrointestinal Diseases; Gastrointestinal tract structure; Gene Cluster; General Hospitals; Generations; Genes; Genome; Homology Modeling; In Vitro; Investigation; Knowledge; Literature; Mass Spectrum Analysis; Massachusetts; Mediating; Methods; Mining; Molecular; Molecular Cloning; NFIA gene; NMR Spectroscopy; Nature; Neuraxis; Oxygenases; Pathway interactions; Pattern; Peptides; Pharmacology; Physical condensation; Polycyclic Compounds; Process; Protein Isoforms; Publications; Publishing; Quinazolines; Reporting; Research; Research Personnel; Resources; Role; Route; Sequence Analysis; Solid; Structure; System; Targeted Toxins; Therapeutic; Therapeutic Agents; Time; Toxin; Universities; Validation; Vertebral column; anthranilate; asperlicin; base; chemical synthesis; enzyme activity; experience; feeding; fumiquinazoline A; fungus; insight; medical schools; nervous system disorder; peptide synthase; plant fungi; professor; protein expression; protein purification; protein structure; public health relevance; reconstitution; research study; scaffold; small molecule; tool

DESCRIPTION (provided by applicant): The application's broad objective is to provide new molecular insights into the biosynthetic assembly of medicinally relevant fungal polycyclic compounds by nonribosomal peptide synthetases (NRPSs). Two major classes of amino acid-derived multicyclic fungal metabolites are the benzodiazepinones and quinazolinones. Core structures are derived from combination of the beta amino acid anthranilate (ortho-aminobenzoate) with proteinogenic amino acids, resulting in the formation of scaffolds with potent bioactivities, both as therapeutics (such as the asperlicins) and toxins (such as acetylaszonalenin). The anthranilate-containing fungal metabolite asperlicin is a cholecystokinin (CCK) receptor antagonist of nanomolar potency. CCK receptor isoforms are found in the brain, central nervous system and alimentary canal; CCK receptor antagonists provide a route to treat both gastrointestinal and neurological disorders. The NRPS-based morphing of linear peptide chains into architecturally constrained fused-ring systems with diverse substituents underlies the high affinities and biological selectivities observed for these classes of compounds. Previous studies have provided structures and synthetic routes for many compounds of these classes, but little is known regarding their biosynthesis beyond building block composition through precursor feeding studies. The proposed research aims to provide a fundamental understanding of: 1) the selection, activation, and loading of anthranilate by fungal NRPSs, and 2) the chemical processes mediated by the biosynthetic enzymes to cyclize and tailor the linear peptide into the final bioactive product. We predict that the number and order or anthranilate building blocks incorporated into the peptide backbone are key determinants to multicylic scaffold construction. Completion of the proposed research will provide knowledge that may be applied to the directed incorporation of anthranilate into peptide backbones to engineer non-natural cyclization patterns and substituents. Through genome mining, biochemical experimentation, and structural analysis we expect to characterize the molecular basis of anthranilate selection and loading by NRPSs. Through deconstruction and reconstitution of the biosynthetic machinery activity, we will explore a predicted new mode of NRPS-based cyclization of the peptide backbone for multicylic scaffold construction. PUBLIC HEALTH RELEVANCE: Understanding the biological assembly of anthranilate-containing fungal natural products will provide a foundation for producing molecules to screen as therapeutic agents, and will identify biological targets from toxin producing pathways for drug design.

描述（由申请人提供）：该申请的广泛目标是通过非核糖体肽合成酶（NRPS）为医学相关真菌多环化合物的生物合成组装提供新的分子见解。氨基酸衍生的多环真菌代谢物的两个主要类别是苯并二氮杂卓酮类和喹唑啉酮类。核心结构衍生自β氨基酸邻氨基苯甲酸酯（邻氨基苯甲酸酯）与蛋白质氨基酸的组合，导致形成具有有效生物活性的支架，既作为治疗剂（如曲霉素）又作为毒素（如乙酰aszonalenin）。含邻氨基苯甲酸盐的真菌代谢物曲霉素是一种纳摩尔效力的胆囊收缩素（CCK）受体拮抗剂。CCK受体亚型存在于大脑、中枢神经系统和消化道中; CCK受体拮抗剂提供了治疗胃肠道和神经系统疾病的途径。基于NRPS的线性肽链变形为具有不同取代基的结构上受约束的稠环系统是对这些类别的化合物观察到的高亲和力和生物选择性的基础。以前的研究已经提供了这些类别的许多化合物的结构和合成路线，但很少有人知道他们的生物合成以外的积木组合物通过前体喂养的研究。拟议的研究旨在提供对以下内容的基本理解：1）真菌NRPS对邻氨基苯甲酸酯的选择，活化和装载，以及2）由生物合成酶介导的化学过程，以将线性肽环化并定制为最终的生物活性产物。我们预测，纳入肽骨架的邻氨基苯甲酸酯构建块的数量和顺序是多环支架构建的关键决定因素。完成拟议的研究将提供的知识，可应用于邻氨基苯甲酸酯的肽骨架的工程师非天然环化模式和取代基的定向掺入。通过基因组挖掘，生物化学实验和结构分析，我们希望表征邻氨基苯甲酸酯选择和加载NRPS的分子基础。通过对生物合成机制活性的解构和重构，我们将探索一种预测的基于NRPS的肽骨架环化的新模式，用于多环支架的构建。 公共卫生相关性：了解含邻氨基苯甲酸的真菌天然产物的生物组装将为产生分子以筛选作为治疗剂提供基础，并将从毒素产生途径中识别用于药物设计的生物靶标。