Genetically encoded bicyclic peptide libraries for the discoveryof novel antiviral agents

项目总结 双环肽是由两个大环环组成的构象受限多肽。由于他们的 刚性增加，双环肽与蛋白质靶标结合的亲和力和选择性比线性多肽更强 和单环对应的化合物。因此，这些分子是非常理想的支架，用于开发 基于多肽的疗法。噬菌体展示是一种实验室进化技术，它能够发现高密度的 来自大型组合多肽文库的亲和多肽配体。虽然最初仅限于线型多肽，但这 双环肽配体的发现采用了新的技术。最常见的是噬菌体展示的双环 多肽是通过使用半胱氨酸反应性小分子对线性多肽进行化学修饰而产生的；如何- 无论如何，这种方法在技术上是具有挑战性的。因此，这一领域的进展有限。最近，有几个 研究已经使用遗传密码扩展来共翻译安装半胱氨酸反应性非正则氨基 将氨基酸(NCAA)转化为噬菌体展示的多肽，以产生环肽文库。这一战略具有重要的意义 比化学环化方法更具优势，但目前仅限于单环肽。已经过去了- 这项提案的主要目标是开发能够在双环植物中展示噬菌体的技术。 潮汐利用遗传密码扩展。我们的中心假设是双功能NCAA，即含有 两个半胱氨酸反应性官能团可用于合成核糖体合成的双环肽 与半胱氨酸残基的分子内反应。为了实现我们的目标，我们将追求三个具体目标。在……里面 目标1(K99阶段)我们将设计一种氨基酰基-tRNA合成酶，它能识别双功能NCAA含有- 两个半胱氨酸反应部分。这将使用传统的和最先进的方法来完成 定向进化。在目标2(K99/R00阶段)，我们将开发一个与Co. 双功能NCAA的翻译安装，我们将优化该系统以用于双环肽的形成。我们 然后将通过选择和表征模型靶标的配体来验证该系统。在目标3(R00阶段)，我们 将使用噬菌体展示来鉴定与人类冠状病毒刺突蛋白结合的双环肽和 抑制病毒-宿主膜融合。通过针对各种冠状病毒的蛋白质，我们将努力识别 具有广谱抗病毒活性的抑制剂。拟议的工作将提供一条简单的路线来产生双 从而极大地加快了发现治疗性多肽先导化合物的速度。候选人， 杰弗里·萨普博士的长期职业目标是建立一个独立的研究项目，利用基因 编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多肽 传染病。在此，我们提出了一个由导师指导的详细的五年职业发展计划 迪特尔·S博士和克雷格·威伦博士，以及一个主题专家团队。这项计划将加强先前的培训 并解决关键的培训差距，为萨普博士实现长期职业目标做好准备。

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.