Chimeric ACE2 peptide ligand for diagnostic assays of SARS-CoV-2

用于 SARS-CoV-2 诊断分析的嵌合 ACE2 肽配体

• 批准号: 10262576
• 负责人: alexander wlodawer
• 金额: $ 11.2万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262576
• 关键词: 2019-nCoV; 3-Dimensional; Affinity; Antigens; Binding; Biodistribution; Biological Assay; Cell Surface Receptors; Cells; Chimera organism; Code; Complex; Coronavirus; Detection; Development; Diagnostic; Epidemiology; Epitopes; Image; Immobilization; Immune response; In Vitro; Knowledge; Ligands; Methods; Mucous Membrane; Pathogenicity; Patients; Peptides; Peptidyl-Dipeptidase A; Physiological; Protein Fragment; Proteins; Recombinants; Saliva; Sampling; Stains; Structure; Surface; Techniques; Testing; Toxic effect; Viremia; Virus; Work; base; bioimaging; design; diagnostic assay; improved; in vivo; novel; novel strategies; receptor; receptor binding; tool

Recently, angiotensin-converting enzyme 2 (ACE2) was shown to be a functional receptor for SARS-CoV-2, enabling the virus to enter host target cells. Using structure-based rational design, a chimeric ACE2 peptide was designed by us for use as a ligand for analytical quantitation of SARS-CoV-2. We aim to improve the initial design and examine the potential uses of a chimeric peptide displaying a 3-D discontinuous epitope representative of the binding surface of ACE2 recognized by the RBD of the corona virus spike protein, S1. This chimera was initially designed based on multiple structures of the S1 RBD and ACE2 complexes (PDB codes 6LZG, 6VSB, 6VXX, 6VYB, and 6LXT).The chimera was meant to be a physiologically relevant, high affinity probe specific for the epitope that is critically important for the virus binding to the target cell surface receptor. Functionality of the chimera in that respect can be easily proven by in vitro competition assays using recombinant S1 protein fragments (S1 ectodomain and S1 RBD) and recombinant ectodomain of the ACE2 protein. We will use tagged proteins that can be immobilized to use BLI for characterization of binding. The aim of this work is to adapt the chimeric ACE2 peptide for two diagnostic applications: 1) to develop and characterize an imuno-qPCR assay for the detection of SARS-CoV-2 in saliva and mucosal samples 2) to perform in vivo bioimaging, biodistribution, clearance and toxicity studies. The novel applications represent promising tools to advance coronavirus epidemiology, pathogenicity, and receptors related knowledge.

最近，血管紧张素转换酶2 （ACE2）被证明是SARS-CoV-2的功能性受体，使病毒能够进入宿主靶细胞。采用基于结构的合理设计，我们设计了一种嵌合ACE2肽作为配体用于分析定量SARS-CoV-2。我们的目标是改进初始设计，并研究一种嵌合肽的潜在用途，该嵌合肽显示一个3-D不连续表位，代表被冠状病毒刺突蛋白S1的RBD识别的ACE2结合表面。该嵌合体最初是基于S1 RBD和ACE2复合物（PDB代码6LZG、6VSB、6VXX、6VYB和6LXT）的多种结构设计的。嵌合体是一种生理相关的、高亲和力的探针，特异于对病毒与靶细胞表面受体结合至关重要的表位。嵌合体在这方面的功能可以很容易地通过使用重组S1蛋白片段（S1外结构域和S1 RBD）和重组ACE2蛋白外结构域的体外竞争分析来证明。我们将使用可固定化的标记蛋白来使用BLI来表征结合。这项工作的目的是将嵌合ACE2肽用于两种诊断应用：1)开发和表征用于检测唾液和粘膜样品中SARS-CoV-2的免疫qpcr方法；2)进行体内生物成像、生物分布、清除和毒性研究。这些新应用代表了推进冠状病毒流行病学、致病性和受体相关知识的有希望的工具。