The COVID-19 pandemic, caused by SARS-CoV-2, necessitates a thorough understanding of viral entry into host cells. SARS-CoV-2, similar to SARS-CoV, utilizes its S-glycoprotein to bind to the host cell receptor ACE2. The S-glycoprotein comprises two subunits: S1 (containing the RBD responsible for receptor binding) and S2 (mediating membrane fusion). Previous studies identified ACE2 as the cellular receptor for SARS-CoV-2, with high-resolution cryo-electron microscopy structures revealing simultaneous binding of two S-glycoproteins to an ACE2 dimer. CoV entry is a complex process involving receptor binding and proteolytic activation of the S-glycoprotein, ultimately leading to virus-cell membrane fusion. However, direct evidence on the dynamics of S1-ACE2 binding at the single-molecule level was lacking. This study aimed to analyze the biophysical properties of SARS-CoV-2 S-glycoprotein binding to ACE2 using force-distance (FD) curve-based AFM, extracting kinetic and thermodynamic properties of the interaction and testing the efficacy of ACE2-derived peptides as binding inhibitors.

The literature review section covers existing knowledge about SARS-CoV-2 entry mechanism, focusing on the role of the S-glycoprotein and ACE2 receptor. It cites previous studies that identified ACE2 as a receptor for both SARS-CoV and SARS-CoV-2 and highlights the structural details of the S-glycoprotein and its interaction with ACE2. The review emphasizes the complexity of viral entry, involving receptor binding and proteolytic activation, and points out the gap in knowledge regarding single-molecule level dynamics of the S1-ACE2 interaction, which the current study addresses. The importance of understanding this interaction for developing antiviral strategies is also highlighted.

The study employed atomic force microscopy (AFM) to investigate the interaction between SARS-CoV-2 S-glycoprotein and the ACE2 receptor. Two approaches were used: (1) experiments on model surfaces, where ACE2 receptors were immobilized on a surface and the S1 subunit or RBD were attached to the AFM tip, and (2) experiments on living A549 cells (either unmodified or transfected to overexpress ACE2). Force-distance (FD) curves were recorded at various retraction rates and contact times to analyze the kinetics of the interactions. The Bell-Evans model was applied to analyze the binding strength and extract kinetic parameters (koff, kon, Kd). The binding probability was also measured at various contact times. Additionally, optical bioluminescence interference (BLI) was used to confirm the kinetic parameters obtained from AFM. To investigate the potential of ACE2-derived peptides as inhibitors, binding assays were performed on both model surfaces and living cells, measuring the binding probability before and after adding different concentrations of the peptides. The peptides' efficacy in inhibiting the interaction was assessed. Finally, molecular dynamics (MD) simulations were used to investigate the interactions between the ACE2-derived peptides and the S1 glycoprotein.

The key findings of the study include: (1) The RBD within the S1 subunit of SARS-CoV-2 S-glycoprotein is the primary binding interface with ACE2. Both S1 and RBD exhibited similar binding kinetics and thermodynamics on model surfaces, with a dissociation constant (Kd) around 120 nM, indicating high affinity. (2) Single-molecule force spectroscopy using AFM demonstrated that the interaction between the RBD and ACE2 involves single-bond ruptures. (3) The binding probability increased exponentially with contact time for both S1 and RBD on ACE2 model surfaces. (4) These interactions were also confirmed on living cells, with higher binding probabilities observed on cells overexpressing ACE2. However, some binding was observed even on cells without ACE2 overexpression, suggesting involvement of additional receptors. (5) ACE2-derived peptides, specifically those mimicking the N-terminal helix ([22–44] and [22–57]), showed significant anti-binding activity both on model surfaces and living cells, reducing the binding probability by >70%. These peptides have IC50 values in the µM range. (6) BLI assays confirmed the kinetic parameters obtained from AFM.

The study provides strong evidence that the RBD of the SARS-CoV-2 S-glycoprotein mediates high-affinity binding to the ACE2 receptor. The detailed kinetic and thermodynamic characterization of this interaction, obtained using AFM on both model surfaces and living cells, offers valuable insights into the viral entry mechanism. The finding that ACE2-derived peptides, particularly those mimicking the N-terminal helix, effectively inhibit this interaction highlights the potential for developing peptide-based therapeutics targeting the virus's initial attachment to the host cells. The observation of some S1 binding on cells without ACE2 overexpression warrants further investigation into the potential involvement of other receptors in the early stages of viral entry. The high affinity and long lifetime of the RBD-ACE2 interaction make it a challenging target for inhibitors; however, this study successfully identifies promising lead compounds for future development.

This study provides a detailed biophysical characterization of the SARS-CoV-2 S-glycoprotein-ACE2 interaction, confirming the critical role of the RBD and revealing the high affinity of this interaction. The identification of effective ACE2-derived peptides as potent inhibitors opens avenues for developing novel antiviral therapies. Future research should focus on optimizing these peptides for improved efficacy and exploring the potential roles of other receptors in viral entry.

The study primarily focuses on the interaction between the S-glycoprotein and ACE2. While it notes the potential involvement of other receptors, it doesn't fully characterize these alternative binding interactions. The use of undifferentiated A549 cells, which may not perfectly mimic the natural state of ACE2 expression in the lungs, could also be considered a limitation. Finally, the in vivo efficacy of the identified peptides remains to be demonstrated.