Peptides derived from the SARS-CoV-2 receptor binding motif bind to ACE2 but do not block ACE2-mediated host cell entry or pro-inflammatory cytokine induction.

SARS-CoV-2 viral attachment and entry into host cells is mediated by a direct interaction between viral spike glycoproteins and membrane bound angiotensin-converting enzyme 2 (ACE2). The receptor binding motif (RBM), located within the S1 subunit of the spike protein, incorporates the majority of known ACE2 contact residues responsible for high affinity binding and associated virulence. Observation of existing crystal structures of the SARS-CoV-2 receptor binding domain (SRBD)-ACE2 interface, combined with peptide array screening, allowed us to define a series of linear native RBM-derived peptides that were selected as potential antiviral decoy sequences with the aim of directly binding ACE2 and attenuating viral cell entry. RBM1 (16mer): S443KVGGNYNYLYRLFRK458, RBM2A (25mer): E484GFNCYFPLQSYGFQPTNGVGYQPY508, RBM2B (20mer): F456NCYFPLQSYGFQPTNGVGY505 and RBM2A-Sc (25mer): NYGLQGSPFGYQETPYPFCNFVQYG. Data from fluorescence polarisation experiments suggested direct binding between RBM peptides and ACE2, with binding affinities ranging from the high nM to low µM range (Kd = 0.207-1.206 µM). However, the RBM peptides demonstrated only modest effects in preventing SRBD internalisation and showed no antiviral activity in a spike protein trimer neutralisation assay. The RBM peptides also failed to suppress S1-protein mediated inflammation in an endogenously expressing ACE2 human cell line. We conclude that linear native RBM-derived peptides are unable to outcompete viral spike protein for binding to ACE2 and therefore represent a suboptimal approach to inhibiting SARS-CoV-2 viral cell entry. These findings reinforce the notion that larger biologics (such as soluble ACE2, 'miniproteins', nanobodies and antibodies) are likely better suited as SARS-CoV-2 cell-entry inhibitors than short-sequence linear peptides.

ACE2 and Proinflammatory Signaling by S1 Protein of SARS-Cov-2 in Human Endothelial Cells

Introduction COVID-19 is primarily a respiratory disease associated with cardiovascular risk. SARS-CoV-2, the virus causing COVID-19, uses ACE2, an important enzyme in the cardiovascular system that regulates the conversion of Ang II (deleterious/pro-hypertensive) to Ang 1-7 (protective/anti-hypertensive), as a receptor for host cell entry and infection. Considering the relationship between the viral S1-protein and the host's ACE2, it is unclear whether this interaction is merely a mechanism of infection or whether it also contributes to cardiovascular damage associated with COVID-19. We hypothesisedthat SARS-Cov-2-ACE2 interaction induces activation of vascular cell inflammatory responses that are influenced by ACE2 dependent and/or independent enzymatic Ang-(1-7) production. Methods Human microvascular endothelial cells (MEC) were used and stimulated with SARS-CoV-2 recombinant S1 protein (rS1p) (0.66 ?g/mL) at 10/30 min (acute) and 5/24h (chronic). Activation of pro-inflammatory signaling pathways (immunoblotting, real-time PCR), microparticle (MP) generation (NanoSight), and cytokine production (ELISA) were assessed. In some experiments, cells were pre-incubated with an ACE2 activator (DIZE ? 190 nM) and inhibitor (MLN-4760 ? 440 pM). Results rS1P increased NF?B activation (Control ?=0.99±0.06 vs. 1.38±0.19 AU;p<0.05) and MP formation (C=1.01±0.17 vs. 2.06±0.21, x109/mL;p<0.05), a marker of endothelial cell damage. mRA expression of IL-1? (C=1.07±0.13 vs. 50.04±4.63 2