Label-Free Analysis of Binding and Inhibition of SARS-Cov-19 Spike Proteins to ACE2 Receptor with ACE2-Derived Peptides by Surface Plasmon Resonance

Abstract

SARS-CoV-2 has been shown to enter and infect human cells via interactions between spike protein (S glycoprotein) and angiotensin-converting enzyme 2 (ACE2). As such, it may be possible to suppress the infection of the virus via the blocking of this binding interaction through the use of specific peptides that can mimic the human ACE 2 peptidase domain (PD) α 1-helix. Herein, we report the use of competitive assays along with surface plasmon resonance (SPR) to investigate the effect of peptide sequence and length on spike protein inhibition. The characterization of these binding interactions helps us understand the mechanisms behind peptide-based viral blockage and develop SPR methodologies to quickly screen disease inhibitors. This work not only helps further our understanding of the important biological interactions involved in viral inhibition but will also aid in future studies that focus on the development of therapeutics and drug options. Two peptides of different sequence lengths, [30-42] and [22-44], based on the α 1-helix of ACE2 PD were selected for this fundamental investigation. In addition to characterizing their inhibitory behavior, we also identified the critical amino acid residues of the RBD/ACE2-derived peptides by combining experimental results and molecular docking modeling. While both investigated peptides were found to effectively block the RBD residues known to bind to ACE2 PD, our investigation showed that the shorter peptide was able to reach a maximal inhibition at lower concentrations. These inhibition results matched with molecular docking models and indicated that peptide length and composition are key in the development of an effective peptide for inhibiting biophysical interactions. The work presented here emphasizes the importance of inhibition screening and modeling, as longer peptides are not always more effective.

Keywords: COVID-19; SARS-CoV-2; biosensing; molecular docking; peptide inhibitors; surface plasmon resonance.

Figure 1.

(A) Schematic of the competitive assay. (B) Relative location of [30–42] peptide (purple) and [22–44] peptide (tan) corresponding to PDB ID: 6m0j

Figure 2.

Evaluation of blocking efficiency of the ACE2-derived peptides to the S-protein using competitive SPR. A) Specific binding measured for the S-protein on the surface with ACE2. B) SPR sensorgrams with [22–44] peptide. C) The change of SPR binding signal as a function of peptide cocentration. D) SPR sensorgrams with [30–42] peptide.

Figure 3.

A) and B) Dose-dependent blocking of the S-protein with [22–44] and [30–42] peptides. C) structural alignment of both the docked result of the peptide/RBD complex and the α 1 helix/RBD complex, constructed from the ACE2/RBD complex (PDB ID 6m0j).

Figure 4.

An illustration of the interacting interface of the SARS-CoV-2 receptor-binding domain (RBD) (cyan) and hACE2 (green) from PDB-ID: 6M0J. The key interacting residues are shown in close-up as insets. The table shows the interacting residues within a 3A° region analyzed using the PyMOL tool.

Figure 5.

Interaction of the A) [22–44] peptide B) [30–42] peptide (pink) with the SARS-CoV-2 receptor-binding domain (RBD) (gray). Molecular docking complex obtained with PachDock. The key interacting residues are shown in close-up as insets. The table shows the interacting residues within a 3A° region analyzed using the PyMOL tool.