Cationic peptides inhibit SARS-CoV-2 cell entry

By Michael Greenwood, M.Sc.Jun 29 2021

Heparan sulfate (HS) is a cell surface glycosaminoglycan that has been identified as a key entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in association with the well-known angiotensin-converting enzyme 2 (ACE2) receptor. Some preliminary studies have suggested that competitive inhibition or removal of HS can reduce the ability of SARS-CoV-2 to enter cells and thus lessens infection.

In a paper recently published in Pathogens, in silico affinity studies by researchers at the University of Illinois, USA, reveal several potential peptide binding sites required for HS-spike protein and ACE2-spike protein bond formation, and synthesis of suitable peptide inhibitors reveals impaired SARS-CoV-2 cell entry.  

Study: Heparan Sulfate Binding Cationic Peptides Restrict SARS-CoV-2 Entry. Image Credit: Kateryna Kon / Shutterstock

In silico docking studies

HS contains a large number of negatively charged sulfate groups and therefore binds to positively charged peptide species with high affinity. Two peptides generated by the group are termed G1 and G2 (LRSRTKIIRIRH and MPRRRRIRRRQK, respectively), the first of which possesses alternating positively charged residues, while G2 bears strings of positive residues. The SARS-CoV-2 spike protein receptor binding domain (RBD), the ACE2 receptor, and the G1 and G2 peptides were modeled computationally, and the bonding interactions between the molecules were explored.

Both peptides were seen to bond with either the receptor binding domain or ACE2 in positions that would interfere with bonding between the proteins, particularly G1. Similar disruptive interactions were also observed between HS and the spike protein, which motivated the group to perform in vitro studies to test viral entry.

In vitro studies

A SARS-CoV-2 pseudovirus expressing the spike protein was generated and exposed to HEK (kidney) cells that were incubated with either peptide G1 or G2 one hour before infection. This prophylactic treatment significantly reduced pseudovirus entry at 48 hours post-infection at concentrations as low as 6.1 µg /mL, with similar results for Lund human mesencephalic (neuronal) cells. Untreated HEK cells were burdened with around 5 times as great of a viral load than those treated with 6.1 µg /mL of either peptide, and those treated with 50 µg /mL of peptide bore approximately 12.5 fold less.

LUHMES cells, in comparison, exhibited dozens fold less viral cell entry when incubated with either of the peptides, perhaps due to a differing rate of ACE2 or HS expression compared with HEK cells. The cytotoxicity of the peptides (IC50) towards HEK cells was ascertained to be 1.3 mg/mL and 1.09 mg/mL for G1 and G2, respectively, which suggests good specificity and is promising for a preclinical drug candidate.

To explore the influence of G1 and G2 on HS expression, the glycosaminoglycan was fluorescently tagged on the surface of HEK cells and subsequently exposed to the peptides. Small quantities of HS are naturally found in the nucleus, and G1-treated cells had only this source of HS remaining following treatment, having shed their external HS. Treatment with G2, however, caused internalization of HS rather than shedding. Further studies will be needed to investigate the complete mechanism behind this phenomenon.

Targeting glycoproteins such as HS is a broad-spectrum antiviral strategy, and G1 and G2 peptides have also shown efficacy in inhibiting cell entry in herpes simplex virus type 1. They bind with HS at the 3-O-sulfated heparan sulfate site. G1, in particular, demonstrates high affinity towards the TYR453 amino acid residue located on the SARS-CoV-2 receptor binding domain, which has high affinity towards the ACE2 receptor, and therefore G1 is likely to inhibit bonding. Additionally, G1 appears to induce the complete removal of HS from the cell surface, severely impairing SARS-CoV-2 cell entry.