# Structurally Modified Bioactive Peptide Inhibits SARS-CoV-2 Lentiviral Particles Expression
## 4 Discussion  
The global spread of SARS-CoV-2 and mounting cases of COVID-19 necessitates the study of potential therapeutic agents ([[https://www.fda.gov/drugs/coronavirus-covid-19](https://www.fda.gov/drugs/coronavirus-covid-19)]([https://www.fda.gov/drugs/coronavirus-covid-19](https://www.fda.gov/drugs/coronavirus-covid-19)) -drugs/coronavirus-treatment-acceleration-program-ctap, accessed on 10 August 2022). The genetic sequencing of SARS-CoV-2 and the study of the pathophysiology of COVID-19 has triggered intense global research efforts to develop pharmaceuticals to counter the disease. A striking feature of the drug development for COVID-19 is the range of research platforms being explored, including the development of repurposed drugs and bioactives against COVID-19 [51]. Rationale bioactive repurposing, owing to its low toxicity, can provide targeted drug candidates in a relatively short period. Given that the inhibitors of SARS-CoV-2 can aim at multiple targets such as RBD, ACE2 TMPRSS2, furin, and others, the bioactive repurposing effort has a consistent wider range and probability of success [15]. Our findings show that chemical modulation of IRW, an ACE2 activating tripeptide, can have a direct impact on the SARS-CoV-2 RBD. Structurally modified IRW, A9 (Acetylation-Ile-Arg-Trp-Amide) and A14 (Formylation-Ile-Arg-Trp-Amide) inhibited the interaction in situ and SARS-CoV-2 S Protein expression in HEK293T cells (Figures 2B and 6A). The lowering of SARS-CoV-2 S Protein expression is a vital pharmacological aspect of the modified peptides, A9 and A14. This feature of both peptides indicates their ability to possibly counter viral infection by inhibiting SARS-CoV-2's virion envelope involved in the receptor recognition [52]. These structural modification(s) led to the efficient free energy of binding leading to favorable interaction with the SARS-CoV-2 RBD. To obtain IRW analogs with high biostability, chemical modifications at its N- and C-terminus, including acetylation, formylation, fatty acid conjugation, glycosylation, and others, were carried out (Figure 1). Among these modifications, N-terminal acetylation, and formylation with the addition of the C-terminus amide group boosted the pharmacological impact. The acetylation of the bioactive compounds may favor the in vivo biological activity by increasing their hydrophobicity and cellular uptake [53]. It also makes the peptide closely mimic the charge state in the native protein [54]. A recent report has shown that N-terminal acetylation significantly enhanced the antimicrobial activity of peptide L163 against multiple pathogens [55]. Likewise, N-terminal acetylation of peptide cathelicidin LL-37 improved its antimicrobial activity as well [56]. N-formylated peptides play an important role in host defense against microbial agents via modulation of the immune system [57]. Therefore, the N-terminal formylation of IRW boosted its ability to interact with SARS-CoV-2 RBD. Another consideration is the addition of C-terminal amidation, a common feature of wild type membrane disrupting antimicrobial peptides [58]. Figure 8: The root mean square deviation curves of A9 and A14 complexes with the RBD. The initial system is displayed on the left showing (**A**) A9 and (**B**) A14 in the binding site of the RBD. YRB scheme was used to highlight the hydrophobic regions. The plot shows the RMSD of the Ca, side chains, and heavy atoms of the RBD. ‘Iig fit Prot’ displays the RMSD of a ligand after aligning the protein-ligand complex on the protein backbone of the initial configuration and measuring the RMSD of the ligand-heavy atoms. C-terminus amidation stabilizes \(\alpha\)-helical confirmation by additional hydrogen [59; 60]. This modification can yield a higher cationic charge leading to enhanced peptide binding with membranes of microbes [61]. Further, this modification makes the resulting peptide more stable towards enzymatic degradation resulting from exopeptiddases [62; 63].