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

项目摘要 许多细胞内靶点涉及细胞内蛋白质与蛋白质的相互作用，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)访问这些目标。大环肽可以存在于这个bRo5空间，这是一个关键的优势 使用多肽作为bRo5分子是因为有许多成熟的技术可以从 巨大的图书馆。可以说，这些技术中最强大的是mRNA显示，它允许创建 包含超过10万亿个变异体的多肽库，比标准多肽大6-7个数量级 用珠子准备的图书馆。这些文库的极端多样性使得在Inhibitor中取得了许多成功 发展。然而，这些成功与真正的药物发现脱节，因为这些多肽 未被覆盖的太大了，不符合bRo5。序列较短且符合bRo5标准的库 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 因为标准的mRNA显示受到遗传密码的限制，每个位置只能有20个变异体。在这 建议将采取两项战略，以加强这种职位多样性。第一个涉及打破 通过分离完全修饰的tRNA同种受体，标准遗传密码的简并性。基于 密码子阅读规则预测，这将允许将10个非规范氨基酸(NCAA)添加到 密码。第二个涉及在编码中插入非自然碱基对(UBP)。增加了一首单曲 在单个密码子位置进入遗传密码的UBP打开了32个新的空密码子，可用于 将新的NCAA引入到代码中。将准备阅读这些密码子中的每个密码子的tRNA和密码子 将验证阅读首选项。将这两种战略结合在一起，应该可以扩大 遗传密码在每个位置使用40个单体。通过精心选择的构建块，这将允许 用于创建包含用于药物发现的数十亿个变体的符合bRo5的文库。

Project Summary 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 two strategies to enhance this positional diversity will be pursued. The first involves 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 involves insertion of an unnatural base pair (UBP) to the code. The addition of a single UBP into the genetic code at a single codon position opens 32 new empty codons that can be exploited for the introduction of novel ncAAs to the code. tRNAs that read each of these codons will be prepared and the codon reading preferences will be validated. Putting the two strategies together should allow expansion of the genetic code to the use of 40 monomers at each position. With carefully chosen building blocks, this will allow for the creation of bRo5 compliant libraries containing billions of variants for use in drug discovery.