Discovery and characterization of biocatalysts for ribosomally encoded alpha-N-methylated peptide natural products

项目摘要/摘要 自从20世纪20年代青霉素被发现以来，生物活性多肽天然产物一直被用作 抗生素、抗病毒、免疫抑制和抗癌药物。其中许多生物活性多肽都含有 主干α-N-甲基化和/或大环化，因为这些剪裁显著改善了多肽 药物动力学。正如在轰动一时的免疫抑制剂环孢素A中所看到的那样，α-N甲基化的多肽 增加了结构刚性、抗蛋白水解性和膜通透性。此外，环状α-N- 甲基化的多肽能够以高亲和力结合大而平的表面，使它们成为具有吸引力的靶标 破坏蛋白质之间的相互作用。尽管有这些优点，但低效的合成和体外过程 阻碍α-N-甲基化多肽的生产、筛选和优化。此外，自然来源的 酰胺骨架甲基化的多肽被认为完全限于刚性的非核糖体多肽。 生物合成途径。这项工作的目标是梳理出α-N的机械和结构约束。 我们新发现的核糖体编码多肽天然产物家族中的甲基化生物催化剂 那就是冰片。我们的第一个目标是确定一种在担子菌中编码的有效生物活性模型系统。 梳理出α-N-甲基化、N-到-C大环化和生物活性的规律和局限性。我们的 第二个目标集中在结构上不同的硼素家族成员，在那里我们将执行详细的动力学 梳理出化学挑战α-N-甲基化的机制的措施。我们还概述了我们的 开发研究担子菌真菌中的多肽天然产物的方法以及 我们努力梳理出硼素代谢物的潜在生物学作用。这项研究将创造一个多样化的 灵活高效的催化剂工具箱，用于生物生产、筛选和优化基因 模板化生物活性α-N甲基化多肽。

PROJECT SUMMARY / ABSTRACT Since the discovery of penicillin in the 1920s, bioactive peptide natural products have been used as antibiotic, antiviral, immunosuppressive, and anti-cancer agents. Many of these bioactive peptides harbor backbone α-N-methylations and/or macrocyclizations, since these tailorings significantly improve peptide pharmacokinetics. As seen in the blockbuster immunosuppressant cyclosporin A, α-N-methylated peptides have increased structural rigidity, proteolytic resistance, and membrane permeability. Additionally, cyclic α-N- methylated peptides are able to bind large, flat surfaces with high affinity, making them attractive targets for disrupting protein-protein interactions. Despite these advantages, inefficient synthetic and in vitro processes hinder the production, screening, and optimization of α-N-methylated peptides. In addition, natural sources of amide backbone-methylated peptides were thought to be completely limited to inflexible nonribosomal peptide biosynthetic pathways. The goal of this work is to tease out the mechanistic and structural constraints of α-N- methylating biocatalysts within our newly discovered ribosomally encoded peptide natural product family called the borosins. Our first objective identifies a potent bioactive model system encoded in a basidiomycete fungus to tease out the rules and limitations of borosin α-N-methylation, N-to-C macrocyclization, and bioactivity. Our second objective focuses on structurally distinct borosin family members, where we will perform detailed kinetic measurements to tease out the mechanism for chemically challenging α-N-methylation. We also outline our longer-term visions to develop methods for studying peptide natural products in basidiomycete fungi as well as our efforts to tease out the potential biological roles of borosin metabolites. This research will create a diverse toolbox of flexible and efficient catalysts for the biological production, screening, and optimization of genetically templated bioactive α-N-methylated peptides.