Expanding the Landscape of Amino Acid-Rich Antimicrobial Peptides: Definition, Deployment in Nature, Implications for Peptide Design and Therapeutic Potential

Unlike the α-helical and β-sheet antimicrobial peptides (AMPs), our knowledge on amino acid-rich AMPs is limited. This article conducts a systematic study of rich AMPs (>25%) from different life kingdoms based on the Antimicrobial Peptide Database (APD) using the program R. Of 3425 peptides, 724 rich AMPs were identified. Rich AMPs are more common in animals and bacteria than in plants. In different animal classes, a unique set of rich AMPs is deployed. While histidine, proline, and arginine-rich AMPs are abundant in mammals, alanine, glycine, and leucine-rich AMPs are common in amphibians. Ten amino acids (Ala, Cys, Gly, His, Ile, Lys, Leu, Pro, Arg, and Val) are frequently observed in rich AMPs, seven (Asp, Glu, Phe, Ser, Thr, Trp, and Tyr) are occasionally observed, and three (Met, Asn, and Gln) were not yet found. Leucine is much more frequent in forming rich AMPs than either valine or isoleucine. To date, no natural AMPs are simultaneously rich in leucine and lysine, while proline, tryptophan, and cysteine-rich peptides can simultaneously be rich in arginine. These findings can be utilized to guide peptide design. Since multiple candidates are potent against antibiotic-resistant bacteria, rich AMPs stand out as promising future antibiotics. (Int J Mol Sci. 2022 Nov; 23(21): 12874.)

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))

Hydrazine hydrate: A new reagent for Fmoc group removal in solid phase peptide synthesis

In solid-phase peptide synthesis using the Fmoc/tBu strategy (SPPS-Fmoc/tBu), an orthogonal protection scheme of amino acids is used; specifically, the alpha-amine group is protected by the 9-fluorenylmethyloxycarbonyl (Fmoc) group, which is removed by weak bases, while side chains are protected by groups that are acid labile. We demonstrated that hydrazine hydrate is an efficient reagent for eliminating the Fmoc group in SPPS-Fmoc/tBu. First, experimental conditions were established for Fmoc group removal from Fmoc-Val-OH in solution. It was determined that the Fmoc group was completely removed with 16% hydrazine hydrate in DMF after 60 min at rt. Second, SPPS-Fmoc/tBu using hydrazine hydrate for Fmoc group removal was standardized. The Fmoc group removal was completed using 16% hydrazine hydrate in DMF for 10 min at rt (twice). When the reaction of Fmoc group removal was microwave-assisted, the reaction only required 30 s to efficiently remove the Fmoc group in SPPS-Fmoc/tBu. The method reported here can be routinely used, and it is equivalent to conventional SPPS-Fmoc/tBu methodologies where 4-methylpiperidine or piperidine is used. (Tetrahedron Letters.,Volume 60, Issue 1, 3 January 2019, Pages 48-51.)