Fig 1.
SARS-CoV-2 receptor binding motif–ACE2 protein-protein interaction binding interface.
Amino acids in red highlight SARS-CoV-2 receptor binding motif (RBM) contact residues with ACE2.–adapted from (Lan, et al. 2020) [7]. ACE2 receptor–Grey. SARS-CoV-2 RBD–Orange. Green structures represent glycoproteins. Structure generated using PyMol software.
Table 1.
Primers.
Fig 2.
Selecting a potential RBD–ACE2 PPI inhibitor peptide derived from SARS-CoV-2 RBM S438 –K462 peptide.
(A) N-terminal and (B) C-terminal stepwise truncation peptide array of RBM S438 –K462, overlaid with ACE2-Fc human purified protein. Underlined (blue) sequence represents shorter sequence peptide selected for assessment in vitro. (C) Structure of S443 –K458 SARS-CoV-2 RBM and the respective binding interface of ACE2 receptor; RBM interacting residues highlighted in red–adapted [7].
Fig 3.
Selecting a potential RBD–ACE2 PPI inhibitor peptide derived from SARS-CoV-2 RBM E484 –Y508.
(A) N-terminal and (B) C-terminal stepwise truncation peptide array of RBM E484 –Y508, overlaid with ACE2-Fc human purified protein. Underlined (blue) sequence represents shorter sequence peptide selected for assessment in vitro. (C) ACE2 binding to short sequence RBM 20mer; F486 –Y505. (D) Structure of F486 –Y505 SARS-CoV-2 RBM and the respective binding interface of ACE2 receptor; RBM interacting residues highlighted in red–adapted [7].
Fig 4.
Direct binding assay for ACE2 and RBM-derived peptides (FP; fluorescent polarisation).
Increasing concentrations of ACE2-Fc or GST purified recombinant protein (0.012 nM– 6 μM) were incubated with 500 nM of RBM peptide (FITC labelled) at room temperature, FP measured at 2 hours and binding affinities calculated via non-linear regression analysis (A). Binding saturation curves of (B) RBM1, (C) RBM2A and RBM2A-Sc, (D) RBM2B binding. GST represents negative protein control. Binding affinity (Kd, red vertical line) measurements represented as MEAN ± SEM, N = 3 independent experiments.
Table 2.
ACE2 interacting SARS-CoV-2 RBM peptides.
Peptides synthesised with and without a C-terminal FITC tag.
Fig 5.
SRBD internalisation in human ACE2 overexpressing human cell lines.
(A) western immunoblot detection of internalised SRBD protein in ACE2 overexpressing (stable) A549 cells following 30 minutes pre-treatment with (i) vehicle only (lane 4), (ii) RBM2A-Sc (lane 5), (iii) RBM2A (lane 6), (iv) RBM2B (lane 7), (v) RBM1 (lane 8), RBM1 + RBM2B (lane 9). (B) Changes to SRBD mediated internalisation in transiently overexpressing ACE2 (luciferase) HEK293T cells were measured following co-treatment with SRBD (100 nM) and (i) RBM1, (ii) RBM2A, (iii) RBM2B peptides (0.5, 5 and 50 μM). Levels of internalised SRBD measured as % difference of vehicle only control. All data represented as MEAN ± SEM, N = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. not significant.
Fig 6.
Spike protein (full-length trimer, Wuhan-Hu-1 strain) pseudovirus neutralisation assay in human ACE2 overexpressing HEK293T cells.
RBM peptides were pre-treated (1 hour) at 300 μM, 100 μM, 30 μM, 10 μM, 3 μM and 1 μM, followed by 48-hour co-incubation with spike protein PsV (luciferase labelled) and subsequent luminescence detection. sACE2 (human ACE2 protein) represents positive PsV neutralisation control. Data represented as MEAN % difference of DMSO only negative control (100%) ± SEM, N = 3 independent experiments. PsV, Pseudovirus.
Fig 7.
Effect of RBM peptides on recombinant S1 protein-induced inflammation in ACE2 endogenously expressing human coronary microvascular endothelial cells.
Total RNA extracted from CMEC and gene expression of (A) IL-1β, (B) IL-6, (C) VCAM-1, (D) MCP-1 pro-inflammatory markers determined via RT-PCR (normalised to GAPDH). RBM peptides treated alone (10 μM) or pre-treated (10 μM, 1 hour) following S1 protein 5-hour incubation (9 nM). Data represented as MEAN ± SEM, N ≥ 3 independent experiments. * P < 0.05, *** P < 0.001, **** P < 0.0001, n.s. not significant. C, Control; S1, spike protein S1 subunit; V, vehicle only control; 1, RBM1 peptide; 2B, RBM2B peptide; Sc, RBM2A-Scrambled peptide.