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

项目摘要 许多细胞内癌症靶点涉及蛋白质-蛋白质之间的相互作用，这是“无法下药”的，因为 结合表面太大，没有特征，不能被标准的符合5规则的小分子所阻挡。 最近，有人试图对口服生物可用但不符合规则的分子进行分类。 五(BRo5)。大环肽可以存在于这个bRo5空间中，并且将其用作 BRo5分子的一个特点是，有许多成熟的技术可以从大量的文库中寻找多肽结合体。 可以说，这些技术中最强大的是信使核糖核酸显示，它允许创建多肽文库 包含超过10万亿个变异体，比标准多肽库大6-7个数量级 珠子。这些文库的极端多样性使抑制剂的开发取得了许多成功。还没有 这些成功与真正的药物发现脱节，因为发现的多肽太多了 大到符合bRo5标准。短序列和符合bRo5标准的文库可以通过mRNA来创建 显示，但这些文库缺乏发现有效抑制物所需的多样性，因为标准的 受遗传密码的限制，每个位置只能有20个变种。非天然碱基对的添加(UBP)S 为解决这一问题提供了巨大的潜力。事实上，将单个UBP添加到遗传密码中 单一密码子位置打开32个新的空密码子。原则上，这些密码子可以被利用来编码 新的非正则氨基酸反过来将极大地增强Short的潜在多样性 大环肽文库。尽管如此，这种规模的遗传密码扩展之前从未尝试过，而且， 因此，该提案的主要目标是准备、验证和优化mRNAs、tRNAs和Non 建立这个系统所需的标准氨基酸。经过验证的系统将能够创建大循环 短小的多肽文库，但包含数十亿个变体，用于发现符合bRo5标准的抑制剂 无法治愈的癌症靶点。

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.