Targeting the interaction between the SARS-CoV-2 spike protein and human ACE2, its primary cell membrane receptor, is a promising therapeutic strategy to prevent viral entry. Recent in vitro studies revealed that the receptor binding domain (RBD) of the spike protein plays a prominent role in ACE2 binding, yet a simple and quantitative assay for monitoring this interaction in a cellular environment is lacking. Here, we developed an RBD-ACE2 binding assay that is based on time-resolved FRET, which reliably monitors the interaction in a physiologically relevant and cellular context. Because it is modular, the assay can monitor the impact of different cellular components, such as heparan sulfate, lipids, and membrane proteins on the RBD-ACE2 interaction and it can be extended to the full-length spike protein. The assay is HTS compatible and can detect small-molecule competitive and allosteric modulators of the RBD-ACE2 interaction with high relevance for SARS-CoV-2 therapeutics.

针对 SARS-CoV-2 刺突蛋白和人类 ACE2（其主要细胞膜受体）之间的相互作用，是一种有前途的预防病毒进入的治疗策略。最近的体外研究表明，刺突蛋白的受体结合结构域 (RBD) 在 ACE2 结合中发挥着重要作用，但缺乏用于监测细胞环境中这种相互作用的简单定量分析。在这里，我们开发了一种基于时间分辨 FRET 的 RBD-ACE2 结合测定，它可靠地监测生理相关和细胞环境中的相互作用。由于它是模块化的，因此该测定可以监测不同细胞成分（例如硫酸乙酰肝素、脂质和膜蛋白）对 RBD-ACE2 相互作用的影响，并且可以扩展到全长刺突蛋白。该测定与 HTS 兼容，可以检测与 SARS-CoV-2 治疗高度相关的 RBD-ACE2 相互作用的小分子竞争性和变构调节剂。