Coronil, a Tri-Herbal Formulation, Attenuates Spike-Protein-Mediated SARS-CoV-2 Viral Entry into Human Alveolar Epithelial Cells and Pro-Inflammatory Cytokines Production by Inhibiting Spike Protein-ACE-2 Interaction

Abstract

Purpose: Coronil is a tri-herbal formulation containing extracts from Withania somnifera, Tinospora cordifolia, and Ocimum sanctum. Recently, it was shown that Coronil rescued humanized zebrafish from SARS-CoV-2 induced pathologies. Based on reported computational studies on the phytochemicals present in Coronil, it could be a potential inhibitor of SARS-CoV-2 entry into the host cell and associated cytokines' production.

Methods: Through an ELISA-based biochemical assay, effects of Coronil on interaction between ACE-2 and different mutants of viral spike (S) protein, crucial for viral invasion of host cell, were evaluated. Additionally, using recombinant pseudoviruses having SARS-CoV-2 spike (S) protein in their envelopes and firefly luciferase reporter in their genomes, effects of Coronil on virus entry into human alveolar epithelial cells were evaluated through luciferase assay. UHPLC profiled Coronil also modulated S-protein mediated production of pro-inflammatory cytokines in A549 cells, like interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), as evaluated through RT-qPCR and ELISA.

Results: Coronil effectively inhibited the interaction of ACE-2 not only with the wild-type S protein (S^WT) but also with its currently prevalent and more infectious variant (S^D614G) and another mutant (S^W436R) with significantly higher affinity toward ACE-2. Treatment with Coronil significantly reduced the increased levels of IL-6, IL-1β, and TNF-α in A549 cells incubated with different S-protein variants in a dose-dependent manner. Likewise, it also prevented the SARS-CoV-2 S-protein pseudotyped vesicular stomatitis virus (VSVppSARS-2S) mediated cytokine response in these cells by reducing entry of pseudoviruses into host cells.

Conclusion: Coronil prevented SARS-CoV-2 S-protein mediated viral entry into A549 cells by inhibiting spike protein-ACE-2 interactions. SARS-CoV-2 S protein induced inflammatory cytokine response in these cells was also moderated by Coronil.

Keywords: ACE-2; Coronil; SARS-CoV-2; pro-inflammatory cytokines; pseudovirus; spike protein.

Figure 1

Chemical composition of Coronil. Overlap HPLC chromatogram of standard mix (black line) and Coronil (blue line). Cordifolioside A, Magnoflorine, Withanoside IV, Withaferin A, Withanoside V and Withanone were quantified at 227 nm, Rosmarinic acid and Palmatine at 325 nm, and Betulinic and Ursolic acids at 210 nm wavelength.

Figure 2

Inhibitory effect of Coronil on interactions between human ACE-2 receptor and viral S proteins. (A–D) Through an ELISA based assay (A), the dose-dependent effect of Coronil on the interactions between human ACE-2 receptor and different types of SARS-CoV-2 S proteins, namely, S^WT (B), S^D614G (C) and S^W436R (D) were evaluated and represented as percent (%) inhibition relative to the extent of ACE-2-S protein interactions observed in the reaction mix without any inhibitor. Data are represented as mean ± SEM from three independent experiments. The statistical significance of the observed differences in the means compared to the no inhibitor group (first column in each case from (B–D)) was analyzed through one-way ANOVA followed by Dunnett’s multiple comparison test and represented as *, ** or *** depending on whether the calculated p value was <0.05, <0.01 or <0.001. IC₂₀ doses of Coronil against ACE-2 and each variant of S protein were determined.

Figure 3

Coronil inhibits viral S protein induced cytokine response. (A) Schematic representation of the experimental plan. (B–D) Activation of pro-inflammatory cytokines, IL-6 (B), TNF-α (C), and IL-1β (D) in alveolar epithelial A549 cells treated with different SARS-CoV-2 S proteins and dose-dependent effect of Coronil treatment thereof were measured through ELISA. Observations are depicted as levels of secreted cytokines. Data are represented as mean ± SEM from three independent experiments. The statistical significance of the differences observed between the means was analyzed through one-way ANOVA followed by Dunnett’s multiple comparison test and represented as ^### for p<0.001 when compared to normal cells without Coronil treatment and exposure to S protein and as *, **, and *** for p< 0.05, 0.01, and 0.001, respectively, when compared to cells exposed to S protein induction but not treated with Coronil.

Figure 4

Coronil moderates VSVppSARS-2S pseudovirus induced expressions of inflammatory cytokines. (A) Pictorial depiction of the experimental plan showing the steps involved in pseudotyping VSV with SARS-CoV-2 protein to obtain VSVpp2S viruses, subsequent treatments and end-point readouts. Representative bright field images of HEK293 cells before (panel a) and after (panel b) being induced for pseudotyping. Manifestation of successful pseudotyping was observed as cytopathic effects in the form of cytopathic islands (demarcated with brown dotted lines), plaques and syncytia (red arrow heads) in panel (b). (B) ACE-2 level was assessed through RT-qPCR in normal, infected and Coronil treated A549 cells and represented as mRNA expression relative to the housekeeping gene, peptidyl-prolyl cis-trans isomerase (PPIA). (C) Schematic representing the difference in the genomes of regular VSV and recombinant VSV [VSV*ΔG(Luc)] used in pseudotyping. (D) VSVppSARS-2S entry into the transduced A549 cells and the effect thereof due to treatment with Coronil and positive control Camostat mesylate, assessed through luciferase assay, were represented as fold change in luminescence units relative to the normal cells, taken as background. (E–H) Expression levels of pro-inflammatory cytokines, IL-6 (E), TNF-α (F), IL-1β (G) and IL-8 (H) represented as fold change with respect to the untreated, non-transduced normal control. Data represented as mean ± SEM from three independent experiments. The statistical significance of the observed differences between the means was analyzed through one-way ANOVA followed by Dunnett’s multiple comparison test and represented as ns, ^## and ^### for p which was non-significant, <0.01 and 0.001, respectively, when compared to non-transduced cells without any treatment and as ** and *** for p< 0.01 and 0.001, respectively, when compared to untreated virus transduced ones.

Figure 5

Coronil reduced pseudovirus elicited cytokine response in A549 cells. (A) The experimental plan involving the transduction of A549 cells with VSVpp2S viruses and subsequent measurement of levels of secreted pro-inflammatory cytokines in the medium through ELISA are shown in this schematic. (B–D) Measured levels of secreted IL-6 (B), TNF-α (C) and IL-1β (D) show the evoked cytokine responses and their reduction upon Coronil treatment. Data are represented as mean ± SEM from three independent experiments. The statistical significance of the differences observed between the means was analyzed through one-way ANOVA and represented as ### for p<0.001 when compared to non-transduced cells and as *** for p<0.001 when compared to untreated virus transduced ones.

Figure 6

Proposed mechanism for anti-viral activity of Coronil against SARS-CoV-2. The proposed model shows the pathogenesis of COVID-19 infection and the steps at which Coronil can inhibit COVID-19 pathogenesis.