Genetically encoded bicyclic peptide libraries for the discoveryof novel antiviral agents

用于发现新型抗病毒药物的基因编码双环肽库

基本信息

批准号：
10189880
负责人：
Jeffery Micheal Tharp
金额：
$ 9.52万
依托单位：
YALE 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：
2021-09-02 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10189880
关键词：
2019-nCoV Address Adoption Affinity Amines Amino Acids Amino Acyl-tRNA Synthetases Antibodies Antiviral Agents Bacteriophage M13 Bacteriophages Binding Cells Chemicals Communicable Diseases Coronavirus Coronavirus Infections Coupled Cyclic Peptides Cyclization Cysteine Development Development Plans Diagnosis Diagnostic Directed Molecular Evolution Disease Drug Targeting Engineering Evolution Future Genetic Code Goals Human In Vitro Laboratories Libraries Ligands Lung diseases Mediating Membrane Fusion Mentors Methods Middle East Respiratory Syndrome Coronavirus Modeling Molecular Conformation Peptide Conformation Peptide Library Peptides Phage Display Phase Prevention Property Proteins Reaction Research Ribosomes Route SARS coronavirus Side Supervision Surface System Techniques Technology Therapeutic Training Transfer RNA Translations Tyrosine-tRNA Ligase Virus Work base career career development combinatorial functional group human coronavirus in vivo inhibitor/antagonist novel novel coronavirus novel therapeutics pandemic disease peptide drug prevent programs protein protein interaction scaffold screening skills small molecule technology development thioether tool

项目摘要

PROJECT SUMMARY Bicyclic peptides are conformationally constrained peptides comprised of two macrocyclic rings. Owing to their increased rigidity, bicyclic peptides can bind to protein targets with greater affinity and selectivity than their linear and monocyclic counterparts. As a result, these molecules are highly desirable scaffolds for the development of peptide-based therapeutics. Phage display is a laboratory evolution technique that enables the discovery of high- affinity peptide ligands from large, combinatorial peptide libraries. Although initially limited to linear peptides, this technique has been adapted for the discovery of bicyclic peptide ligands. Most often, phage-displayed bicyclic peptides are generated by chemically modifying linear peptides using cysteine-reactive small molecules; how- ever, this method is technically challenging. As a result, progress in this field has been limited. Recently, several studies have used genetic code expansion to co-translationally install cysteine-reactive noncanonical amino acids (ncAAs) into phage-displayed peptides to produce libraries of cyclic peptides. This strategy has significant advantages over the chemical cyclization approach, but is currently limited to monocyclic peptides. The over- arching objective of this proposal is to develop technology that enables phage display of bicyclic pep- tides using genetic code expansion. Our central hypothesis is that bifunctional ncAAs, i.e. ncAAs containing two cysteine-reactive functional groups, can be used to generate ribosomally synthesized bicyclic peptides by intramolecular reaction with cysteine residues. To realize our objective, we will pursue three Specific Aims. In Aim 1 (K99 Phase) we will engineer an aminoacyl-tRNA synthetase that recognizes bifunctional ncAAs contain- ing two cysteine-reactive moieties. This will be accomplished using traditional and state-of-the-art methods of directed evolution. In Aim 2 (K99/R00 Phase) we will develop a phage display system that is compatible with co- translational installation of bifunctional ncAAs and we will optimize this system for bicyclic peptide formation. We will then validate this system by selecting and characterizing ligands for model targets. In Aim 3 (R00 Phase) we will use phage display to identify bicyclic peptides that bind to the spike protein of human coronaviruses and inhibit virus-host membrane fusion. By targeting proteins from various coronaviruses, we will strive to identify inhibitors with broad-spectrum antiviral activity. The proposed work will provide a facile route for generating bi- cyclic peptide libraries thereby greatly accelerating the discovery of therapeutic peptide leads. The Candidate, Dr. Jeffery Tharp’s long-term career goal is to establish an independent research program that uses genetic code expansion and phage display to develop antiviral peptides for the diagnosis, treatment, and prevention of infectious diseases. Herein we propose a detailed five-year Career Development Plan supervised by mentors Drs. Dieter Söll and Craig Wilen, and a team of subject-matter experts. This plan will augment previous training and address key training gaps to prepare Dr. Tharp for accomplishing his long-term career goal.

项目概要双环肽是由两个大环组成的构象受限肽。由于刚性增加，双环肽可以比线性肽以更高的亲和力和选择性与蛋白质靶标结合因此，这些分子是开发非常理想的支架。基于肽的疗法。噬菌体展示是一种实验室进化技术，能够发现高水平的药物。来自大型组合肽库的亲和肽配体虽然最初仅限于线性肽，但这该技术已被用于发现双环肽配体，最常见的是噬菌体展示的双环肽配体。肽是通过使用半胱氨酸反应性小分子对线性肽进行化学修饰而产生的；然而，这种方法在技术上具有挑战性，因此最近该领域的进展受到限制。研究已使用遗传密码扩展来共翻译安装半胱氨酸反应性非规范氨基酸这种策略具有重要意义。与化学环化方法相比具有优势，但目前仅限于单环肽。该提案的首要目标是开发能够实现双环 pep-噬菌体展示的技术我们的中心假设是双功能 ncAA，即包含的 ncAA。两个半胱氨酸反应性官能团，可用于生成核糖体合成的双环肽为了实现我们的目标，我们将追求三个具体目标。目标 1（K99 阶段）我们将设计一种氨酰基-tRNA 合成酶，该酶可识别双功能 ncAA，其中包含- 这将使用传统和最先进的方法来完成。在Aim 2（K99/R00阶段）中，我们将开发一种与co-兼容的噬菌体展示系统。双功能 ncAA 的翻译安装，我们将优化该系统以形成双环肽。然后，我们将通过选择和表征模型靶点的配体来验证该系统。将使用噬菌体展示来识别与人类冠状病毒的刺突蛋白结合的双环肽，通过针对各种冠状病毒的蛋白质，我们将努力识别抑制病毒-宿主膜融合。具有广谱抗病毒活性的抑制剂。拟议的工作将为产生双-提供一种简便的途径。环肽文库从而大大加速了候选肽的发现。 Jeffery Tharp 博士的长期职业目标是建立一个利用基因技术的独立研究项目代码扩展和噬菌体展示，以开发用于诊断、治疗和预防的抗病毒肽在此，我们提出了由导师监督的详细五年职业发展计划。 Dieter Söll 博士和 Craig Wilen 博士以及主题专家团队将加强之前的培训。并解决关键的培训差距，为撒普博士实现其长期职业目标做好准备。