Stapled ACE2 peptidomimetics designed to target the SARS-CoV-2 spike protein do not prevent virus internalization

Abstract

COVID-19 is caused by a novel coronavirus called severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Virus cell entry is mediated through a protein-protein interaction (PPI) between the SARS-CoV-2 spike protein and angiotensin-converting enzyme 2 (ACE2). A series of stapled peptide ACE2 peptidomimetics based on the ACE2 interaction motif were designed to bind the coronavirus S-protein RBD and inhibit binding to the human ACE2 receptor. The peptidomimetics were assessed for antiviral activity in an array of assays including a neutralization pseudovirus assay, immunofluorescence (IF) assay and in-vitro fluorescence polarization (FP) assay. However, none of the peptidomimetics showed activity in these assays, suggesting that an enhanced binding interface is required to outcompete ACE2 for S-protein RBD binding and prevent virus internalization.

Keywords: SARS‐CoV‐2; peptidomimetic; protein‐protein interaction; stapled peptides; virus.

FIGURE 1

The structure of the SARS‐CoV‐2‐S receptor binding domain (RBD) (orange) and a segment of the ACE2 receptor (pink) (PDB: 6M07). A, Front view. B, Side view. α1 (cyan), appears to interact with the RBD of SARS‐CoV‐2 (orange). C, A section of the α1 peptide sequence, showing the interacting residues (green)

FIGURE 2

A helix wheel showing the positioning of the amino acids in the sequence with respect to the helix structure. The right side shows the amino acids on the back, nonbonding face of the helix, where we elected to position our staples (colored bridges)

FIGURE 3

Circular dichroism spectra of the native ACE2 peptide and the stapled peptidomimetics. A, CD data of the native peptides compared to the stapled analogues. B, CD data of the truncated analogues and their stapled variants. Conditions: peptides 100 μM in PBS, pH 7.4. Spectra recorded between 190 and 260 nm

FIGURE 4

Neutralization data for the positive control—soluble ACE2, and data for peptide 1, 8 and the negative controls

FIGURE 5

Immunofluorescence data shows that cells treated with ACE2 can block the entry of His‐tagged SARS‐CoV RBD (green). The cells treated with peptides 1, 8 show entry of the His‐tagged SARS‐CoV RBD into cells. Stapled peptides (10 μM concentration) or soluble hACE2 (~100 nM) were incubated with 50 nM S_RBD for 30 min at 37 °C and later added to A549 cells for 3 hours. Scale bar, 20 μm

FIGURE 6

Direct binding assay (fluorescent polarization (FP) data for peptide 1, 8 and the negative controls—Fluorescent polarization measurements taken at 30 minutes post‐incubation at room temperature

FIGURE 7

The ACE2 PD helix α1 (cyan) plus the discontinuous fragment (green). A, Front view. B, Side view. In previous work by Han et al. on SARS‐CoV, the peptide fragments were joined by a glycine (dashed red line). C, The longer G‐link peptides were conformationally constrained using an i, i + 7 alkene staple. (PDB: 6M07)

FIGURE 8

A, Neutralization data for the second generation peptides. B, Immunofluorescence data shows that cells treated with ACE2 can block the entry of His‐tagged SARS‐CoV RBD (green). The cells that were treated with the second gen. Peptides showed entry of the His‐tagged SARS‐CoV RBD into cells. Stapled peptides (10 μM concentration) or soluble hACE2 (100 nM) were incubated with 100 nM S‐RBD for 30 minutes at RT and later added to A549 cells overexpressing ACE2 for 3 hours. S‐RBD induces ACE2 internalization but recognition by His antibody is very weak. Scale bar, 20 μm. C, The Western Blot indicates that the peptides show no ability to block entry of the SARS‐CoV‐2 RBD

FIGURE 9

Direct binding assay (fluorescent polarization (FP) data for the second generation peptides—Fluorescent polarization measurements taken at 30 minutes post‐incubation at room temperature