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

用于构建超五规则合规大环肽库的遗传密码扩展

基本信息

批准号：
10810404
负责人：
Matthew C Hartman
金额：
$ 1.1万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2025-05-31
项目状态：
未结题

项目摘要

Project Summary Many intracellular cancer 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). 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.

项目摘要许多细胞内癌症靶点涉及细胞内蛋白质-蛋白质相互作用，因为结合表面太大且无特征而不能被符合标准的5规则阻挡小分子。最近，已经尝试对口服生物可利用但在体内不存在的分子进行分类。超越五规则（bRo5）。大环肽可以占据该bRo5空间，并且大环肽的一个关键优势是，使用肽作为bRo5分子的另一个优势是，有许多成熟的技术用于从bRo5分子中发现肽结合剂。巨大的图书馆可以说，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，比标准肽大6 - 7个数量级在珠上制备的文库。这些文库的极端多样性使抑制剂的许多成功成为可能。发展然而，这些成功与真实的药物发现是脱节的，因为肽未覆盖的太大而不符合bRo5。序列短且符合bRo5的文库可以通过mRNA展示创建，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展示受遗传密码的限制，在每个位置只有约20个变体。在这建议将采取两项战略来加强这种位置多样性。第一个涉及打破标准遗传密码的简并性，通过分离完全修饰的tRNA异源受体。基于根据密码子阅读规则，预测这将允许添加10个非规范氨基酸（ncAA），密码第二种方法是在代码中插入一个非自然碱基对（UBP）。添加单一 UBP进入遗传密码的单个密码子位置打开了32个新的空密码子，可以用于将新的ncAA引入代码。将制备读取这些密码子中的每一个的tRNA，并将这些密码子将验证阅读首选项。将这两项战略放在一起，遗传密码在每个位置使用40个单体。通过精心选择的构建模块，这将允许用于创建包含数十亿个用于药物发现的变体的bRo5兼容文库。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Expansion of the genetic code through reassignment of redundant sense codons using fully modified tRNA.

DOI：
10.1093/nar/gkac846
发表时间：
2022-10-28
期刊：
NUCLEIC ACIDS RESEARCH
影响因子：
14.9
作者：
McFeely, Clinton A. L.;Dods, Kara K.;Patel, Shivam S.;Hartman, Matthew C. T.
通讯作者：
Hartman, Matthew C. T.

Extensive breaking of genetic code degeneracy with non-canonical amino acids.

DOI：
10.1038/s41467-023-40529-x
发表时间：
2023-08-17
期刊：
NATURE COMMUNICATIONS
影响因子：
16.6
作者：
McFeely, Clinton A. L.;Shakya, Bipasana;Makovsky, Chelsea A.;Haney, Aidan K.;Cropp, T. Ashton;Hartman, Matthew C. T.
通讯作者：
Hartman, Matthew C. T.