Fig 2: Impact of peptides A9 and A14 on SARS-CoV-2 Spike-pseudotyped Lentiviral Particles. Lentiviral pseudotyping schematic showing the use of (A) 293T cells as packaging cells for producing lentivirus particles using Viral Entry Protein (S D614G Glycoprotein), Lentiviral Backbone (Luc2; ZsGreen), and helper plasmids expressing the other HIV proteins needed for virion formation (Tat, Gag-Pol, and Rev). The transfected 293T cells produce lentiviral particles with surface Spike and can infect (B) 293T-ACE2.TMPRSS2 (mCherry) cells (C) Bright-field and fluorescence microscopy images; and (D) flowcytometry analysis showing changes in ZsGreen expression in 293T-ACE2.TMPRSS2 cells at 24 h after incubation with Spike-pseudotyped lentiviral particles with the ZsGreen backbone in presence of A9 and A14 at 50 µM and (E) the spike lentivirus count in the 293T-ACE2.TMPRSS2 cells transfected with the Spike lentiviral particles with the ZsGreen backbone in presence of A9 and A14 at 50 µM. Flow cytometry was used to measure Spike by staining with Recombinant Anti-SARS-CoV-2 Spike Glycoprotein S1 antibody followed by staining (1:500 dilution) with an APC AffiniPure Goat Anti-Human IgG, Fcγ fragment specific antibody (Jackson Labs, 109-135-098) (1:100 dilution).

Fig 3: Impact of IRW and its analogs on SARS-CoV-2 S1 RBD-ACE2 interaction. (A) An in vitro assay was conducted to assess the possible (B) inhibitory impact of IRW and its analog peptides (50 µM) on SARS-CoV-2 S1 RBD-ACE2 interaction and (C) toxicity analysis of the selected peptides. The results are expressed as percentage inhibition with respect vehicle control (nuclease free water). Data expressed as mean ± SEM of n = 3–6. Experiments were reproduced three times with independent tips and samples. p values were determined by Analysis by one-way ANOVA followed by Bonferroni’s post hoc test for the vehicle. * p < 0.05, ** p < 0.01, **** p < 0.001 versus vehicle and ns: nonsignificant.

Fig 4: Effect of ACE2-Fc on SARS-CoV-2-GFP infection and inhibition of SARS-CoV-2 primary isolates. a ACE2-IgG4-Fc reduces SARS-CoV-2-GFP replication. Representative fluorescent images of Vero E6 cells infected with SARS-CoV-2-GFP (multiplicity of infection (MOI) = 0.6 IU/cell) pre-incubated with ACE2-IgG4-Fc fusion construct 1 (632 nM). b ACE2-Fc fusion proteins potently neutralize coronaviruses. Serial dilutions of ACE2-Fc fusion proteins were pre-incubated with different coronaviruses and tested for their ability to neutralize the virus before infection of Vero E6 cells. Neutralization of SARS-CoV (top), SARS-CoV-2-Jan (middle) and SARS-CoV-2-April (bottom) by ACE2-IgG4-Fc constructs (left) and ACE2-IgG1-Fc constructs (right) is shown. Data given are means ± SEM of three independent experiments each. 50% inhibitory concentrations (IC50) determined as well as the 95% confidence interval (CI 95%) are given for each construct. The dashed lines indicate the IC50 values on the corresponding curves.

Fig 5: Impact of peptides A9 and A14 on ACE2 levels in cells. (A,B) Immunoblots showing the changes in ACE2 levels of HEK293T cells after treatment with A9 and A14 at the different tested concentrations and (C,D) impact of A9 and A14 on cellular levels of ACE2 using an activity assay Kit (ab273297, Abcam, Toronto, ON). HEK293T cells were grown in DMEM complete media containing 10% FBS with antibiotics. The cells were treated with vehicle (nuclease free water) or A9 or A14 at different concentrations (25, 50, and 100 µM) for 24 h. Thereafter, protein was extracted using RIPA buffer and was stored at −20 °C till further analysis. For immunoblotting, the results are expressed as change in ACE2 fold change with respect vehicle control (nuclease free water). For ACE2 activity assay, the results were expressed as nmol of ACE2 per µL of cell extract. Data expressed as mean ± SEM of n = 4–6. p values were determined by Analysis by one-way ANOVA followed by Bonferroni’s post hoc test for vehicle. * p < 0.05 and *** p < 0.001 indicate versus vehicle. The error bar indicates S.D. of the mean value. µM: micromolar, ns: nonsignificant.