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-蛋白质相互作用，这些相互作用是“不可药物治疗的”，因为蛋白质-蛋白质相互作用是“不可药物治疗的”。 结合表面太大且无特征，以至于不能被标准的5规则顺应性小分子阻断。 最近，已经尝试对口服生物可利用的但超出口服生物可利用性规则的分子进行分类。 5（bRo5）。大环肽可以占据该bRo 5空间，并且使用肽作为 bRo 5分子的一个优势是，有许多成熟的技术可以从庞大的文库中找到肽结合剂。 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，比在上制备的标准肽文库大6-7个数量级。 珠这些文库的极端多样性使得抑制剂开发取得了许多成功。然而 这些成功与真实的药物发现是脱节的，因为发现的肽太多了， 大到符合bRo 5。序列短且符合bRo 5的文库可以通过mRNA 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 显示被遗传密码限制为在每个位置处约20个变体。添加非天然碱基对（UBP） 为解决这一问题提供了巨大的潜力。事实上，在遗传密码中添加一个单一的UBP， 单个密码子位置打开32个新的空密码子。原则上，这些密码子可以用来编码 新的非典型氨基酸，这反过来又将大大提高短的潜在多样性， 大环肽文库。尽管如此，这种规模的遗传密码扩展以前从未尝试过， 因此，本提案的关键目标是制备、验证和优化mRNA、tRNA和非RNA。 构建这个系统所需的典型氨基酸。经验证的系统将能够创建大环 肽库很短，但包含数十亿个变体，用于发现符合bRo 5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.