key: cord-0254347-t14oiolb
authors: Ghalib, Mohammed; Parekh, Yash; Banu, Sarena; Ram, Sushma; Nagaraj, Ramakrishnan; Kumar, Bokara Kiran; Idris, Mohammed M
title: Gramicidin S and Melittin - Potential anti-viral therapeutic peptides to treat SARS-CoV-2 infection
date: 2021-10-22
journal: bioRxiv
DOI: 10.1101/2021.10.21.465254
sha: 6176cf738e5cad58115cd3eee18a5a57d853fc66
doc_id: 254347
cord_uid: t14oiolb

The COVID19 pandemic has resulted in 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 gramicidin S and melittin peptides obtained from Bacillus brevis and bee venom respectively. Our in vitro antiviral assay results showed significant decrease in the viral load compared to the untreated group with no/very less cytotoxicity. The EC50 values for gramicidin S and melittin are calculated as 1.571μg and 0.656μg respectively. Both the peptides treated to the SARS-CoV-2 infected Vero cells showed viral clearance from 12 hours onwards with a maximal clearance after 24 hours post infection. Based on proteome analysis it was found that more than 250 proteins were found to be 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 hours 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.

The pandemic caused by SARS-CoV-2 has led to intense research not only on the biology of the virus but also therapeutic intervention with a multi-pronged approach [1] . Vaccines have been developed at "warp" speed and have played a major role in controlling the disease [2] .

However, vaccines are not available universally and there have been cases of infection with SARS-CoV-2 even in vaccinated individuals, though not severe [3] . There is clearly a need for development of therapeutic agents in addition to vaccines. In the area of anti-infective agents against SARS-CoV-2, efforts have been taken to generate therapeutic antibodies that would neutralize the virus and prevent its interaction with cellular receptors to gain entry into cells [4] . Considering the time scales in developing a drug de novo, there have been several attempts to re-purpose drugs to treat COVID19 [1, 5, 6] . However, repurposed drugs have had very limited success in treating SARS-CoV-2 infection including remdesivir [7] . There is no drug to-date that can be used specifically to treat COVID19 disease.

Infection in the case of SARS-Cov-2 is initiated by binding of the spike protein to ACE2 receptor followed by a series of steps leading to fusion and internalization of the virus and propagation [1, 8] . If the binding of the spike protein to ACE2 is prevented, then the virus will no longer be able to enter cells and propagate. SARS-CoV-2 is an enveloped virus where the RNA is encapsulated within a lipid vesicular structure with the spike protein decorating on the external side giving the "corona" appearance [1, 8] . Disruption of the lipid structure would lead to the disintegration of the virus. Naturally occurring membrane-active peptides have potent antimicrobial activity which stems from their ability to disrupt bacterial membranes [9, 10] . We have explored the antiviral activity of two extensively studied peptides, gramicidin S having the sequence: [cyclo-(Val-Orn-Leu-D-Phe-Pro)2] [11] and the against several viruses has been investigated extensively [16] . Nano-conjugates of melittin with sitagliptin have been investigated for anti-SARS-CoV-2 activity [17] . Other pharmacological activities of melittin have also been investigated [18, 19] . Gramicidin S has been used therapeutically to treat dental applications in humans [20] . We have investigated the antiviral activity of gramicidin S and melittin against SARS-CoV-2 in vitro in detail. We have observed that both the peptides have the ability to neutralize the virus in an in vitro assay using Vero cells. At the EC 50 value, there is no cytolytic activity. The viral load had decreased drastically in the treatment group as seen by confocal microscopic images.

Proteomic analysis indicates that there is also a metabolic effect and not merely viral lysis.

Both the peptides could be attractive candidates for development as therapeutic agents to treat SARS-CoV-2 infection. As the viral membrane would be a likely target, mutant strains may likely be susceptible to the peptides.

The SARS-CoV-2 viral particles enumerated by the RT-qPCR showed that treatment of melittin and gramicidin S effectively reduced viral load in vitro(Log EC 50 value of gramicidin S (0.1963) 

The effect of the peptides on the ability of virus to infect Vero cells was studied by proteomics. iTRAQ based quantitative proteomics analysis identified 7 SARS-CoV-2 proteins as up regulated in the control Vero Cells. Nsp9, ORF1ab, ORF10 and nucleocapsid phosphoprotein were found to be up-regulated in the Vero cells after 24 hours of infection, whereas the same proteins were found to be down regulated in the cells upon gramicidin S and melittin treatment (Table 1) . Similarly, at 48 hours post infection (hpi), ORF1ab, S protein, nucleocapsid phosphoprotein, helicase and RNA-dependent RNA polymerase were found to be up-regulated in the infected Vero cells which were found to be down regulated in the gramicidin S and melittin treated Vero cells (Table 1) .

Based on proteomic analysis it was found that a total of 254 proteins were found to be associated with differentially regulated in Vero cells (Table 2 and Figure 5a ). It was found that majority of down and up-regulated proteins were upturned with gramicidin S and melittin treatment at 24 and 48 hpi. RS28, K22E, K2C1, RL17 are few of the major down-regulated proteins which were found to be reversing their expression post peptide treatment.

NPM, ACLY, CALX and F184B were found to be up-regulated in Vero cells 24hpi, whereas peptide treatment showed reversal of the protein expression (Table 2) . Based on heat map and cluster analysis it is very interesting to see the tight association of peptide treated Vero cell protein expression against control infected Vero cell protein expression for both the time points post infection (Figure5a).

A total of 125 proteins were selected for the Network and pathway analysis involving STRING v11.5. Based on Gene ontological functional enrichment analysis it was found that cellular process, biological regulation and regulation of biological process are the most highly associated biological process; binding, proteins binding and heterocyclic compound binding are the most associated molecular functions and cellular anatomical entity, intracellular and organelle are the most occurred cellular components. Carbon metabolism (Blue color nodes), pentose phosphate pathway (Blue color nodes) and mRNA processing (Red color nodes) were most prominent local network cluster associated with STRING analysis (Figure 5b ).

Both gramicidin S and melittin are membrane-active peptides and exert their antimicrobial activity by interacting with membrane components (11, 12) . Both the peptides may exert their antiviral activity by targeting multiple regions of the virus. The ability of the peptides to bind to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein was examined by molecular docking using web version of the program ZDOCK (21) . The structures shown in Figure 6 show that both gramicidin S and melittin can bind to the RBD binding domain.

Panel A shows the crystal structure of RBD-ACE2 complex. The interface between RBD and ACE are shown in violet color and stick representation respectively. The models of gramicidin S and melittin are shown in panels B and C respectively. The LigPlot [22] of the interacting amino acids are shown in panels D for gramicidin S and E for melittin. The residues highlighted in yellow are involved in RBD binding to ACE2 [8] . The modeling study indicates that both the peptides can bind to RBD although their sequences are considerably different from the RBD binding region of ACE2.

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 [23, 24] . They belong to the group β -coronavirus and have the ability to cross animal-human barriers and cause serious illness in humans. The timely development of specific antivirals is of utmost importance.The development of vaccines at "warp speed" has led to decrease in mortality and serious illness caused by SARS-CoV-2 [2] . However, it is still not well established that whether vaccines are equally effective against the several variants that are emerging or the time frame when immunity will be present. [17] . Binding of sitagliptin and melittin nano conjugates to the active site of SARS-CoV-2 3CLpro (a protease) was also observed through molecular docking [17] .

Gramicidin S has potent antibacterial and fungicidal activity [11] . 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 [27].

SARS-Cov-2 is an enveloped virus, with the viral membrane essential for its integrity and function [1, 8] . We reasoned that membrane-active peptides would disrupt the viral membrane and render the virus ineffective. We have investigated the antiviral activity of two well studied membrane-active antibacterial peptides gramicidin S and melittin.

Our in vitro studies using gramicidin S and melittin showed EC 50 value of 1.571µg for gramicidin S and 0.656µg for melittin ( Figure 1 ). The results were comparable with remdesivir which we used as the assay controlin our study ( Non-oxidative pentose phosphate pathways (PPP) are also involved in viral replication;

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Research (CSIR MLP0056). RN is Indian national Academy (INSA) Senior Scientist.

The Anti-SARS CoV-2 study was approved from Institutional Bio-safety Committee of CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India.

The authors declare that they have no competing interests.