Anthranilate-Based Fungal Nonribosomal Peptide Synthetase Assembly Lines

描述(由申请人提供)：该申请的广泛目标是为非核糖体多肽合成酶(NRPS)生物合成药用相关真菌多环化合物提供新的分子见解。两大类氨基酸衍生的多环真菌代谢物是苯并二氮杂环酮类和喹唑烷酮类。核心结构是由β-氨基酸邻氨基苯甲酸酯(邻氨基苯甲酸酯)与蛋白质来源的氨基酸结合而成的，从而形成具有强大生物活性的支架，既作为治疗药物(如Asperlicin)，也作为毒素(如乙酰aszonalenin)。含邻氨基苯甲酸酯的真菌代谢物Asperlicin是一种纳摩尔效力的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；CCK受体拮抗剂为治疗胃肠道和神经系统疾病提供了一条途径。基于NRPS的线性多肽链的变形为具有不同取代基的结构受限的稠环系统，这是观察到这类化合物具有高亲和力和生物选择性的基础。以前的研究已经提供了许多这类化合物的结构和合成路线，但除了通过前体喂养研究构建块组成外，对它们的生物合成知之甚少。这项研究的目的是提供一个基本的理解：1)真菌NRPS对邻氨基苯甲酸酯的选择、激活和负载，以及2)生物合成酶介导的化学过程，以环化和定制线性多肽成为最终的生物活性产品。我们预测，多肽骨架中邻氨基苯甲酸酯构建块的数量和顺序是构建多环支架的关键决定因素。拟议研究的完成将提供可应用于将邻氨基苯甲酸酯定向并入多肽骨架以设计非天然环化模式和取代基的知识。通过基因组挖掘、生化实验和结构分析，我们期望表征NRPS选择和装载邻氨基苯甲酸酯的分子基础。通过生物合成机械活动的解构和重建，我们将探索一种基于NRPS的多肽骨架环化的预测新模式，用于构建多环支架。 公共卫生相关性：了解含有邻氨基苯甲酸酯的真菌天然产物的生物组装将为生产分子以筛选治疗药物提供基础，并将从毒素产生途径中识别生物靶点用于药物设计。

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.