RAPID: Effect of Avidity on Association of Fusion Inhibitory Peptides with the HRN Domain of SARS-CoV-2 Spike Protein

RAPID：亲合力对融合抑制肽与 SARS-CoV-2 刺突蛋白 HRN 结构域关联的影响

• 批准号: 2031167
• 负责人: Christopher Alabi
• 金额: $ 17.69万
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2031167&HistoricalAwards=false
• 关键词: RAPID; Effect; Avidity; Association; Fusion

Coronaviruses are enclosed within a membrane and have protruding protein spikes. The spikes help the virus anchor to the cell. Then, the viral membrane fuses with the cell membrane and the infection begins. Introducing a molecule that will bind to the protein spikes can reduce or prevent attachment of the virus particle to the cell. Small protein chains, called peptides, are particularly effect at inhibiting infection for these viruses. If we understand how these peptides attach to each other and to the spike proteins, and how those actions influence their effectiveness, we will be in a better position to design highly effective inhibitors. This could have a significant impact on the design of inhibitors of the SARS-CoV-2 virus, which gives rise to COVID-19 disease, and aid in the recovery of infected patients.In this project, the effect of avidity on the association of C-peptides with the N-terminal heptad repeat (HRN) domain of the SARS-CoV-2 spike protein will be investigated. This project will test the hypothesis that multimerization can decrease the off-rate and thus increase the residence time of the C-peptide on the HRN domain of the spike protein. Furthermore, this project will evaluate how the structure (e.g. linker flexibility and length) of the multivalent analogs affect six-helix bundle (6HB) formation with SARS-CoV-2 HRN by measuring the kinetic and thermodynamic parameters associated with binding. These parameters will be obtained via a time-resolved Forster resonance energy transfer (TR-FRET) assay. The latter will use a known combination of Lumi4-Terbium fluorophore (Lumi4-Tb) conjugated to the C-peptides as the donor and fluorescein conjugated to the SARS-CoV-2 HRN domain as the acceptor. This combination has a proximity limit of 10-15nm that is more than sufficient for detection of the 6HB. The kinetic rate constants and equilibrium binding constants obtained from this study will inform how fast the C-peptide-HRN system responds to changes in concentration of the C-peptide as well as its residence time on HRN. We expect the results of this project to provide a mechanistic understanding of how avidity can be used to tune the interaction of C-peptides with the SARS-CoV-2 spike protein fusion machinery.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

冠状病毒被包裹在膜内，有突出的蛋白质尖峰。这些尖峰帮助病毒锚定在细胞上。然后，病毒膜与细胞膜融合，感染开始。引入一种与蛋白质尖峰结合的分子可以减少或防止病毒颗粒附着到细胞上。小的蛋白质链，称为多肽，在抑制这些病毒的感染方面特别有效。如果我们了解了这些多肽是如何相互附着以及如何附着到尖峰蛋白上的，以及这些作用是如何影响它们的有效性的，我们将更好地设计高效的抑制剂。这可能会对导致新冠肺炎病的SARS-CoV-2病毒抑制剂的设计产生重大影响，并有助于感染患者的康复。在本项目中，将研究亲和力对C-肽与SARS-CoV-2刺突蛋白N-末端七肽重复结构域关联的影响。这个项目将检验这样的假设，即多聚体可以降低失败率，从而增加C肽在Spike蛋白HRN结构域上的停留时间。此外，该项目将通过测量与结合相关的动力学和热力学参数来评估多价类似物的结构(例如接头灵活性和长度)如何影响与SARS-CoV-2 HRN形成的六螺旋束(6HB)。这些参数将通过时间分辨Forster共振能量转移(TR-FRET)分析获得。后者将使用已知的与C-肽结合的Lumi4-Tb荧光团(Lumi4-Tb)作为供体，与SARS-CoV-2 HRN结构域结合的荧光素作为受体。这种组合具有10-15 nm的接近极限，对于6HB的检测绰绰有余。由本研究得到的动力学速率常数和平衡结合常数将反映C-肽-HRN系统对C-肽浓度变化的响应速度以及C-肽在HRN上的停留时间。我们期望该项目的结果能够提供一个机械上的理解，即如何利用亲和力来调整C肽与SARS-CoV-2刺突蛋白融合机器的相互作用。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。