Gramicidin S and melittin: potential anti-viral therapeutic peptides to treat SARS-CoV-2 infection

During the past two decades, the world had witnessed infection by three highly pathogenic human corona viruses namely, SARS-Co-V, MERS, SARS-CoV-2. They belong to the group of β-coronavirus and have the ability to cross animal-human barriers and cause serious illness in humans. The COVID19 pandemic has led to multipronged approaches for treatment of the disease. Since de novo discovery of drugs is time consuming, repurposing of molecules is now considered as one of the alternative strategies to treat COVID19. Antibacterial peptides are being recognized as attractive candidates for repurposing to treat viral infections. In this study, we describe the anti-SARS-CoV-2 activity of the well-studied antibacterial peptides gramicidin S and melittin obtained from Bacillus brevis and bee venom respectively. The EC50 values for gramicidin S and melittin were 1.571 µg and 0.656 µg respectively based on in vitro antiviral assay. Significant decrease in the viral load as compared to the untreated group with no/very less cytotoxicity was observed. Both the peptides treated to the SARS-CoV-2 infected Vero cells showed viral clearance from 12 h onwards with a maximal viral clearance after 24 h post infection. Proteomics analysis indicated that more than 250 proteins were differentially regulated in the gramicidin S and melittin treated SARS-CoV-2 infected Vero cells against control SARS-CoV-2 infected Vero cells after 24 and 48 h post infection. The identified proteins were found to be associated in the metabolic and mRNA processing of the Vero cells post-treatment and infection. Both these peptides could be attractive candidates for repurposing to treat SARS-CoV-2 infection.

Gramicidin S having the sequence: [cyclo-(Val-Orn-Leu-D-Phe-Pro)2]13 and the bee venom peptide, melittin having the sequence: GIGAVLKVLTTGLPALISWIKRKRQQ-amide. Several studies showed melittin is effective against diverse array of viruses such as coxsackievirus, enterovirus, influenza A viruses, human immunodeficiency virus (HIV), herpes simplex virus (HSV), Junín virus (JV), respiratory syncytial virus (RSV), vesicular stomatitis virus (VSV), and tobacco mosaic virus (TMV). The antiviral activity of gramicidin S (3.0 µg) and melittin (1.5 µg) at 12 and 24 h was examined along with remdesivir (1 µM) as assay control. The data shown in Fig. 3 indicates that the peptides show antiviral activity at 12 h and is more pronounced at 24 h. The gramicidin S and melittin showed 99% and 95% viral reduction respectively at 12 h compared with remdesivir (20%). At 24 h remdesivir showed 90% viral reduction whereas both gramicidin S and melittin showed 99% viral reduction. The SARS-CoV-2 antiviral activity of gramicidin S and melittin was compared with remdesivir by confocal microscopy.

Melittin is the main component of bee venom, and it is active against both enveloped and non-enveloped viruses by activating the Toll-like receptors (TLRs) pathway, which reduces inflammatory cytokines like nuclear factor-kappa B (NF-kB), extracellular signal-regulated kinases (ERK1/2), and protein kinase Akt. Gramicidin S has potent antibacterial and fungicidal activity13. Molecular docking revealed that gramicidin S has a binding affinity of 11.4 kcal/mol to the SARS-CoV-2 spike glycoprotein and SARS-CoV-2 papain like protease, implying that gramicidin S could be an effective drug against the SARS-CoV-2 virus.

Molecular docking
The receptor binding domain (RBD) of the SARS-CoV-2 spike protein was obtained by editing the crystal structure of the C-terminal domain of the SARS-CoV-2 spike protein in complex with human ACE2 (PDB id: 6zlg). The ID of the structure used for gramicidin S monomer is CCDC 626343. Monomeric melittin structure was obtained by editing the crystal structure of tetrameric melittin (PDB id: 2mlt). The structures were generated using Discovery Studio v19.1.0.18287 (2019). Interactions between amino acids were visualized using LigPlot. (Scientific Reports volume 12, Article number: 3446 (2022))