# Structurally Modified Bioactive Peptide Inhibits SARS-CoV-2 Lentiviral Particles Expression
## 4 Discussion
Further, it is critical to mention that A1 and A6 peptides without C-terminal amide moiety failed to elicit any bioactivity even in presence of N-terminus N-terminal acetylation, and formylation (Figure 1). Hence, the collective alteration of terminal groups of IRW leads to potent changes in its pharmacological activity. Previous studies have also shown that antimicrobial activity of peptides (e.g., dermataseptin s3) is enhanced by multiple folds with the addition of an amide group instead of a free carboxylic acid at the C-terminus [64]. Our findings are in line with many recent studies to combat SARS-CoV-2 infection. Kobophenol A, a tetramer of resveratrol, blocked the interaction between the ACE2 receptor and S1-RBD in vitro [65]. Quinolines and terpen based inhibitors of Spike-RBD-ACE2 were identified using methods similar to ours [66; 67]. In another report, 3800 FDA approved drugs were screened for their ability to inhibit the RBD-ACE2 interface as a target [68]. Rationally designed, small peptide inhibitors with the ability to block the interaction of SARS-CoV-2 spike protein with ACE2 have been developed as well [69]. A study has shown that daily intranasal administration lipoprotein fusion inhibitors completely stopped the SARS-CoV-2 direct-contact transmission in animals [70]. EK1C4, a lipopeptide derived from EK1, a pan-coronavirus fusion inhibitor, has displayed exceptional ability to inhibit RBD-ACE2 interaction in vivo [71]. Apart from this first pan-CoV fusion inhibitor (EK1C4), 136 new peptides have been developed from ACE2, of which the best peptide interacts with Arg 403, Tyr453, Gln 493, and Tyr505 in RBD, a striking similarity to A9 and A14 IRW analogs [28]. This serves as a lead drug design template for upcoming molecules as well. Likewise, a 23-aa peptide SBP1 (IEEQAKTFLDKFNHEAEDLFYQS) from ACE2 successfully stably binds to SARS-CoV-2 RBD in a manner similar to A9 and A14 [72]. Other peptides targeting this Spike RBD are being developed as well [73]. It is vital to note that the RBD region is also a critical target for neutralizing antibodies, another therapeutic used in COVID-19 treatment [74]. To further validate these findings, we conducted the pseudotyped lentiviral particles with the SARS-CoV-2 Spike assay as described in an earlier report (Figure 7A) [47]. These results confirmed the potential ability of A9 and A14 to lower SARS-CoV-2 viral load, particularly owing to the marked reduction in lentivirusrus particles. Our findings corroborate the other published data on bioactive compounds and SARS-CoV-2 lentivirus studies. For example, brazilin, TF-3, and curcumin can reduce the fusion of spike-expressing cells to the hACE2 in cells [75]. Other bioactive molecules with the ability against pseudo-virus particles include dimethoxycurcumin [76], peptides AYp28 [KKKKKKVVEGFNCYFPLQS] and AYn1 [KKKKKKKDKFNHEAEDLFY] [77], and tannic acid [78], to name a few. Similar to the studies on natural bioactives, we are unsure if A9 and A14 either destroy viral particles, or whether they via affecting membrane fluidity. One idea is that these peptides being lipophilic molecules, can provoke morphological changes in the cellular membrane, which may modify the lentivirusrus production in cells. Additionally, neither of the two analogs had any impact on TMPRSS2, a serine protease located on the host cell membrane, which promotes virus entry into the cell by activating the S protein [22]. This possibly indicates the fusion inhibitory activity of the analogs rather than any impact on spike activation. As discussed previously, SARS-CoV-2 S hides a furin cleavage site (682-685 residues) at the S1/S2 boundary, thus, providing another drug target [15]. Among the two analogs, analog A14 exhibited a strong ability to inhibit furin which can help in lowering the efficacy of SARS-CoV-2 transmission [79]. As furin is in the trans-Golgi network and activated by acidic pH, it is possible that A14 changes the pH of the cytoplasm or directly inhibits the enzyme [80].