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

冠状病毒被包裹在膜内，并具有突出的蛋白质刺突。刺突帮助病毒锚在细胞上。然后，病毒膜与细胞膜融合，感染开始。引入将结合蛋白质刺突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，称为肽，在抑制这些病毒的感染方面特别有效。如果我们了解这些肽是如何相互连接和连接到刺突蛋白上的，以及这些作用如何影响它们的有效性，我们将能够更好地设计出高效的抑制剂。这可能会对SARS-CoV-2病毒抑制剂的设计产生重大影响，SARS-CoV-2病毒会引起COVID-19疾病，并有助于感染患者的康复。在这个项目中，将研究亲和力对C肽与SARS-CoV-2刺突蛋白N端七肽重复（HRN）结构域结合的影响。该项目将测试多聚化可以降低解离速率从而增加C肽在刺突蛋白的HRN结构域上的停留时间的假设。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，评估多价类似物的结构（例如连接体的灵活性和长度）如何影响SARS-CoV-2 HRN的六螺旋束（6 HB）形成。这些参数将通过时间分辨的福斯特共振能量转移（TR-FRET）试验获得。后者将使用与C肽缀合的Lumi 4-Tb荧光团（Lumi 4-Tb）作为供体和与SARS-CoV-2 HRN结构域缀合的荧光素作为受体的已知组合。这种组合具有10- 15 nm的接近极限，足以检测6 HB。从本研究中获得的动力学速率常数和平衡结合常数将告知C肽-HRN系统响应C肽浓度变化的速度以及其在HRN上的停留时间。我们希望这个项目的结果提供一个机制的理解，如何亲合力可以用来调整与SARS-CoV-2刺突蛋白融合machinery.This奖项的C-肽的相互作用反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.