Genetically encoded bicyclic peptide libraries for the discoveryof novel antiviral agents

项目摘要 双环肽是由两个大环组成的构象受限的肽。由于其 刚性增加，双环肽可以以比其线性肽更大的亲和力和选择性结合蛋白质靶标。 和单环对应物。因此，这些分子是用于开发 基于肽的疗法。噬菌体展示是一种实验室进化技术，可以发现高活性的 亲和肽配体从大的，组合肽库。虽然最初限于线性肽， 该技术已被用于双环肽配体的发现。大多数情况下，噬菌体展示双环 肽是通过使用半胱氨酸反应性小分子化学修饰线性肽产生的;如何- 然而，这种方法在技术上是有挑战性的。因此，这一领域的进展有限。最近有数 研究已经使用遗传密码扩展来协同安装半胱氨酸反应性非规范氨基， 酸（ncAA）转化为噬菌体展示肽以产生环肽文库。这一战略意义重大 这是相对于化学环化方法的优点，但目前仅限于单环肽。过度- 该提议的第二目的是开发能够使噬菌体展示双环PEP的技术， 利用遗传密码扩展的潮汐我们的中心假设是，双功能ncAA，即含有ncAA的 两个半胱氨酸反应性官能团，可用于产生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 目的1（K99阶段）我们将工程化氨酰-tRNA合成酶，其识别双功能ncAA，所述双功能ncAA含有- 两个半胱氨酸反应性部分。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/R 00阶段）中，我们将开发与共噬菌体展示系统相容的噬菌体展示系统。 翻译安装的双功能ncAA，我们将优化该系统的双环肽的形成。我们 然后将通过选择和表征模型靶点的配体来验证该系统。在目标3（R 00阶段）中，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， 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个简易的路线，产生双- 环肽文库，从而大大加速了治疗肽先导物的发现。候选人， 博士Jeffery Tharp的长期职业目标是建立一个独立的研究项目， 编码扩增和噬菌体展示来开发用于诊断、治疗和预防 传染病在此，我们提出了一个详细的五年职业发展计划，由导师监督 Drs. Dieter Söll和克雷格Wilen，以及一组主题专家。这个计划将加强以前的训练 并解决关键的培训差距，以准备萨普博士实现他的长期职业目标。

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.