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 许多细胞内靶标涉及细胞内蛋白质-蛋白质相互作用，这种相互作用是“不可成药的”，因为结合表面太大且无特征，无法被标准的 5 规则兼容小分子阻断。最近，有人尝试对口服生物可利用但超出五法则（bRo5）的分子进行分类，以访问这些靶标。 大环肽可以占据这个 bRo5 空间，使用肽作为 bRo5 分子的一个关键优势是，有许多成熟的技术可以从庞大的库中寻找肽结合物。 可以说，这些技术中最强大的是 mRNA 展示，它允许创建包含超过 10 万亿个变体的肽库，比在珠子上制备的标准肽库大 6-7 个数量级。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，这些成功与真正的药物发现脱节，因为发现的肽太大，不符合 bRo5 标准。 序列短且符合 bRo5 的文库可以通过 mRNA 展示来创建，但这些文库缺乏发现有效抑制剂所需的多样性，因为标准 mRNA 展示受到遗传密码的限制，每个位置只能有约 20 个变体。 在本提案中，将寻求加强这种职位多样性的战略。 这首先涉及通过分离完全修饰的 tRNA 同受体来打破标准遗传密码的简并性。 根据密码子阅读规则，预计这将允许在代码中添加 10 个非规范氨基酸 (ncAA)。第二个目标侧重于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。 该目标还将重点开发和测试一组精选的氨酰基-tRNA，以便将来创建包含数十亿个变体的 bRo5 兼容库，用于药物发现。

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.