Ribosomal Synthesis of Peptides with Unnatural Building Blocks

描述(申请人提供)：我们将尝试使用我们的非天然氨基酸突变方法，对包含多种非天然氨基酸的环状和线状多肽进行遗传编码。这些多肽文库将在细菌细胞内和以噬菌体展示的形式产生，以分别允许体内选择和基于展示的亲和力平移。这一策略将实现对分子结构的直接遗传编码、随机或定向突变、选择和生物放大，同时允许化学多样性超出标准的20个氨基酸谱系所能提供的范围。将非天然氨基酸整合到基因编码的多肽库中，应该会促进进化实验和产生具有新生物活性的多肽，这些多肽最终可能会找到治疗用途。这将通过以下具体目标来实现：1.具有独特反应活性的新的非天然氨基酸的制备和与能够靶向疾病状态中涉及的特定蛋白质(例如丝氨酸蛋白酶、金属蛋白酶等)的官能团的结合2.使用基于内含子的细胞内环肽合成方法与非天然氨基酸技术相结合来产生新的环肽文库和选择特定的酶抑制剂3.使用噬菌体展示系统来生成和筛选含有非天然氨基酸的多肽库以用于特定蛋白质靶标的抑制剂。 与公共健康相关：在生物选择和多肽库筛选中使用非天然氨基酸的能力应该补充目前产生生物活性多肽的合成和生物学方法。重要的是，它将使我们能够将模板化生物合成、突变、扩增和选择的能力与化学家合成具有典型氨基酸中没有的特定空间和电子性质的氨基酸的能力结合起来。这些文库可以被有效地选择或筛选来识别特定的蛋白质功能抑制物，这一证明最终可能为肽疗法的发展提供一种新的途径。

DESCRIPTION (provided by applicant): We will attempt to use our unnatural amino acid mutagenesis methodology to genetically encode libraries of cyclic and linear peptides containing a diverse array of unnatural amino acids. These peptide libraries will be generated both intracellularly in bacteria and in a phage-displayed format to allow in vivo selections and display-based affinity panning, respectively. This strategy will enable direct genetic encoding of molecular structure, random or directed mutagenesis, selection, and biological amplification, while at the same time allowing chemical diversity beyond what the canonical 20 amino acid repertoire can offer. The integration of unnatural amino acids into genetically encoded peptide libraries should facilitate both evolutionary experiments and the generation of peptides with novel biological activities that may ultimately find therapeutic use. This will be achieved through the following specific aims: 1. The preparation and incorporation of novel unnatural amino acids with unique reactivities and with functional groups capable of targeting specific proteins involved in disease states (e.g., serine proteases, metalloproteases, etc.) 2. The use of intein-based intracellular cyclic peptide synthesis methodologies together with unnatural amino acid technology to generate novel cyclic peptide libraries and select specific enzyme inhibitors 3. The use of phage display systems to generate and screen libraries of peptides containing unnatural amino acids for inhibitors of specific protein targets. PUBLIC HEALTH RELEVANCE: The ability to employ unnatural amino acids in biological selections and screens of peptide libraries should complement current synthetic and biological methods for generating biologically active peptides. Importantly, it will allow us to combine the power of templated biological synthesis, mutation, amplification, and selection with the chemist's ability to synthesize amino acids with defined steric and electronic properties not found in the canonical amino acids. The demonstration that these libraries can be efficiently selected or screened to identify specific inhibitors of protein function may ultimately provide a new approach to the development of peptide therapeutics.