Genetic code expansion to enable the development of short, diverse peptide libraries

遗传密码扩展以实现短的、多样化的肽库的开发

• 批准号: 10353426
• 负责人: Matthew C Hartman
• 金额: $ 7.49万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-15 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10353426
• 关键词: Address; Amino Acids; Amino Acyl Transfer RNA; Amino Acyl-tRNA Synthetases; Anticodon; Area; Base Pairing; Binding; Catalogs; Charge; Code; Codon Nucleotides; Collection; DNA; Development; Diversity Library; Fluorescence; Genetic Code; Genetic Transcription; Goals; Hydrogen Bonding; In Vitro; Lead; Libraries; Ligands; Malignant Neoplasms; Messenger RNA; Oral; Peptide Library; Peptides; Pharmaceutical Preparations; Phase; Positioning Attribute; Proteins; Research; Solid; Surface; System; Techniques; Terminator Codon; Testing; Transfer RNA; Transfer RNA Aminoacylation; Translations; Variant; Work; base; c-myc Genes; drug discovery; inhibitor; mutant; new technology; novel; peptidomimetics; preference; prevent; protein protein interaction; ras Proteins; small molecule; success; transcription factor; translation assay; trend

Project Summary Many intracellular cancer targets involve protein-protein interactions that are “undruggable” because the binding surfaces are too large and featureless to be blocked by a standard rule-of-5 compliant small molecule. Recently, there have been attempts to catalog molecules that are orally bioavailable but lie beyond the rule of five (bRo5). Macrocyclic peptides can inhabit this bRo5 space, and a key advantage to using peptides as bRo5 molecules is that there are many mature techniques for finding peptide binders from vast libraries. Arguably, the most powerful of these techniques is mRNA display, which allows creation of peptide libraries containing over 10 trillion variants, 6-7 orders of magnitude larger than a standard peptide library prepared on beads. The extreme diversity of these libraries has enabled many successes in inhibitor development. Yet these successes are disconnected from real drug discovery, because the peptides uncovered are much too large to be bRo5 compliant. Libraries that are short in sequence and bRo5 compliant can be created by mRNA display, but these libraries lack the diversity needed to uncover potent inhibitors because standard mRNA display is limited by the genetic code to ~20 variants at each position. Addition of unnatural base pairs (UBP)s offers great potential to address this problem. In fact, the addition of a single UBP into the genetic code at a single codon position opens 32 new empty codons. In principle, these codons can be exploited to encode novel non-canonical amino acids which in turn will dramatically enhance the potential diversity of short macrocyclic peptide libraries. Still, genetic code expansion on this scale has not before been attempted, and, therefore, the key goals of this proposal are to prepare, validate, and optimize the mRNAs, tRNAs, and non- canonical amino acids required to build this system. The validated system will be able to create macrocyclic peptide libraries that are short, yet contain billions of variants for the discovery of bRo5 compliant inhibitors to undruggable cancer targets.

项目概要 许多细胞内癌症靶标涉及“不可成药”的蛋白质-蛋白质相互作用，因为 结合表面太大且无特征，无法被符合标准 5 规则的小分子封闭。 最近，有人尝试对口服生物可利用但超出规则的分子进行分类。 五个 (bRo5) 大环肽可以占据这个 bRo5 空间，这是使用肽作为肽的一个关键优势。 bRo5分子的优点是有许多成熟的技术可以从庞大的文库中寻找肽结合物。 可以说，这些技术中最强大的是 mRNA 展示，它允许创建肽库 包含超过 10 万亿个变体，比标准肽库大 6-7 个数量级 这些库的极端多样性使得抑制剂的开发取得了许多成功。 这些成功与真正的药物发现脱节，因为发现的肽太多了 序列短且符合 bRo5 的文库可以通过 mRNA 创建。 显示，但这些文库缺乏发现有效抑制剂所需的多样性，因为标准 mRNA 显示受到遗传密码的限制，每个位置最多有 20 个变体添加非自然碱基对 (UBP)。 事实上，将单个 UBP 添加到遗传密码中具有解决这一问题的巨大潜力。 单个密码子位置打开 32 个新的空密码子 原则上，这些密码子可用于编码。 新型非规范氨基酸，这反过来将极大地增强短链氨基酸的潜在多样性 尽管如此，之前还没有尝试过这种规模的遗传密码扩展，并且， 因此，该提案的关键目标是准备、验证和优化 mRNA、tRNA 和非 构建该系统所需的典型氨基酸经过验证的系统将能够创建大环。 肽库虽短，但包含数十亿个变体，可用于发现 bRo5 兼容抑制剂 不可药物治疗的癌症目标。