Genetic code expansion for the construction of beyond rule-of-5 compliant macrocyclic peptide libraries

修改后的项目摘要/摘要部分 许多细胞内的靶标涉及细胞内蛋白质-蛋白质的相互作用，这些相互作用是“不能下药的”，因为结合表面太大，没有特征，无法被标准的符合5规则的小分子所阻止。最近，有人试图对口服生物可用但超出5(BRo5)规则的分子进行分类，以访问这些靶点。大环肽可以存在于这个bRo5空间中，而将多肽用作bRo5分子的一个关键优势是，有许多成熟的技术可以从大量的文库中寻找肽结合蛋白。可以说，这些技术中最强大的是信使核糖核酸显示，它允许创建包含超过10万亿个变种的多肽库，比在珠子上制备的标准多肽库大6-7个数量级。这些文库的极端多样性使抑制剂的开发取得了许多成功。然而，这些成功与真正的药物发现是脱节的，因为发现的多肽太大了，不符合bRo5。短序列和符合bRo5的文库可以通过mRNA显示来创建，但这些文库缺乏发现有效抑制剂所需的多样性，因为标准的mRNA显示受遗传密码的限制，每个位置只能有20个变异体。在这项提案中，将采取加强这种职位多样性的战略。这将首先涉及通过分离完全修改的tRNA等位受体来打破标准遗传密码的简并。根据密码子阅读规则，预测这将允许在密码中添加10个非规范氨基酸(NCAA)。第二个目标是将这些新定义的密码子阅读规则和先前描述的tRNA与非自然碱基对相结合，以允许将遗传密码扩展到每个位置使用40个单体。这一目标还将侧重于开发和测试一组经过精选的氨基酰tRNA，用于未来创建符合bRo5标准的文库，该文库包含数十亿个用于药物发现的变体。

Modified Project Summary/Abstract Section Many intracellular targets involve intracellular 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) to access these targets. 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. In this proposal, strategies to enhance this positional diversity will be pursued. This will first involve breaking the degeneracy of the standard genetic code through isolation of fully modified tRNA isoacceptors. Based on codon reading rules it is predicted that this will allow the addition of 10 non-canonical amino acids (ncAAs) to the code. The second aim focuses on combining these newly defined codon reading rules and previously described tRNAs with unnatural base pairs to allow expansion of the genetic code to the use of 40 monomers at each position. This aim will also focus on developing and testing a curated group of aminoacyl-tRNAs for the future creation of bRo5 compliant libraries containing billions of variants for use in drug discovery.