Withanone from Withania somnifera Attenuates SARS-CoV-2 RBD and Host ACE2 Interactions to Rescue Spike Protein Induced Pathologies in Humanized Zebrafish Model

Abstract

Purpose: SARS-CoV-2 engages human ACE2 through its spike (S) protein receptor binding domain (RBD) to enter the host cell. Recent computational studies have reported that withanone and withaferin A, phytochemicals found in Withania somnifera, target viral main protease (M^Pro) and host transmembrane TMPRSS2, and glucose related protein 78 (GRP78), respectively, implicating their potential as viral entry inhibitors. Absence of specific treatment against SARS-CoV-2 infection has encouraged exploration of phytochemicals as potential antivirals.

Aim: This study aimed at in silico exploration, along with in vitro and in vivo validation of antiviral efficacy of the phytochemical withanone.

Methods: Through molecular docking, molecular dynamic (MD) simulation and electrostatic energy calculation the plausible biochemical interactions between withanone and the ACE2-RBD complex were investigated. These in silico observations were biochemically validated by ELISA-based assays. Withanone-enriched extract from W. somnifera was tested for its ability to ameliorate clinically relevant pathological features, modelled in humanized zebrafish through SARS-CoV-2 recombinant spike (S) protein induction.

Results: Withanone bound efficiently at the interacting interface of the ACE2-RBD complex and destabilized it energetically. The electrostatic component of binding free energies of the complex was significantly decreased. The two intrachain salt bridge interactions (K31-E35) and the interchain long-range ion-pair (K31-E484), at the ACE2-RBD interface were completely abolished by withanone, in the 50 ns simulation. In vitro binding assay experimentally validated that withanone efficiently inhibited (IC₅₀=0.33 ng/mL) the interaction between ACE2 and RBD, in a dose-dependent manner. A withanone-enriched extract, without any co-extracted withaferin A, was prepared from W. somnifera leaves. This enriched extract was found to be efficient in ameliorating human-like pathological responses induced in humanized zebrafish by SARS-CoV-2 recombinant spike (S) protein.

Conclusion: In conclusion, this study provided experimental validation for computational insight into the potential of withanone as a potent inhibitor of SARS-CoV-2 coronavirus entry into the host cells.

Keywords: ACE2-RBD complex; ELISA; SARS-CoV-2 S-protein; Withania somnifera; docking and MD simulation; humanized zebrafish model; withanone.

Figure 1

Proposed mechanisms to block the entry of SARS-CoV-2 into host cells. Notes: The mechanism behind SARS-CoV-2 entry into the host cell is schematically represented. Three proposed models are depicted where COVID-19 infection can be abrogated by blocking the interaction of RBD of spike (S) protein and ACE2. In one of the approaches, ACE2-RBD interaction can be destabilized by small molecules. In the second approach, ACE2 can be blocked with RBD mimetics or single-chain antibody fragment (scFv) against ACE2. In the third approach, RBD of SARS-CoV-2 S-protein can be blocked using the ACE-2 extracellular domain. An Fc domain fused to ACE-2 would facilitate prolonged circulation of the biologic (ACE2-Fc). The observations made in this study support the strategy to block or weaken the interaction between RBD and ACE2 by using phytocompounds of natural origin.

Figure 2

Withanone docks at the interface of the ACE2-RBD complex and shifts slightly towards the center of the interface, modulating several molecular interactions in the process. Notes: (A) Initial and final position of withanone in the ACE2-RBD complex (PDB ID: 6M17) and is predicted to move slightly toward the ACE2 side of the complex, as revealed by 50 ns molecular dynamic (MD) simulation. (B) The initial position of withanone (shown in golden yellow at 0 ns) and its final positioning as predicted from MD simulation after 50 ns (shown in green) is depicted as a magnified view. (C and D) Withanone at 0 ns (C), binds in the pocket forming three H-bonds, D30, N33, and Q96 of ACE2, in addition to alkyl and van der Waals interactions (D). (E and F) Withanone at 50 ns, (after MD simulation) with final trajectory zoomed in (E) and interactions of withanone within the ACE2-RBD complex as seen in the final trajectory (F). All atoms RMSD of withanone between initial and final positions are 2.166 Å. (G and H) Salt bridge interaction at the binding interface of ACE2-RBD in the final trajectory without withanone (G) and with withanone (H). (I) Percent occupancy of the salt bridge and long-range ion pair modulated by withanone incorporation as seen by analysis of the simulation trajectories.

Figure 3

Flexibility analysis of the ACE2-RBD complex docked with withanone. Notes: RMSD changes of backbone atoms (C, CA, N) of (A) the complex, (B and C) per residue RMSF (Cα atom only) of ACE2 (C) and RBD (D) during 50 ns simulation time, as observed in the presence and absence of withanone. (D) Comparison of electrostatic component of binding free energies in the ACE2-RBD complexes with and without withanone. Statistical significance was analyzed through Welch’s t-test and represented as * p<0.05.

Figure 4

Experimental validation of the computational insights. Notes: (A) Schematic representation of the experimental procedure employed in evaluating the inhibitory effect of withanone on the biochemical interaction between host ACE2 and viral RBD. Biotinylated ACE2 bound to RBD, immobilized to the substratum, is detectable through HRP-conjugated streptavidin due to oxidation of 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB). (B) Dose-response curve exhibiting the inhibitory effect of withanone on interaction between human ACE2 and RBD of SARS-CoV-2 S-protein. IC₅₀ is found to be 0.33 ng/mL.

Figure 5

Compositional analysis of extract prepared from leaves of W. somnifera. Notes: (A and B) HPTLC spectra of withanone and W. somnifera hydro-methanolic leaf extract both showing peaks at 230 nm. (C) Overlap chromatograms of standard mix (in black) and hydro-methanolic extract of W. somnifera leaves (in blue) showing the peak for withanone eluted at 227 nm. Insets show the individual peaks of standard withanone and that present in W. somnifera leaf extracts.

Figure 6

Experimental plan of the in vivo study conducted in xenotransplanted humanized zebrafish model. Notes: Overall timeline of in vivo experiment has been schematically shown along with the major study sttif with corresponding time points. The study included two screenings (at days 4 and 7) and one survival assay (until day 10) conducted in parallel. The establishment and subsequent, cytological confirmation of the xenotransplanted humanized zebrafish model (HZF) took 7 days from the day of transplantation. Human alveolar epithelial (A549) cells were intramuscularly injected into the posterior lobe of the swim bladder of zebrafish, incubated for 7 days, and adherence of the injected human cells to the fish swim bladder epithelium was confirmed through gross cytology of the swim bladder. A group of HZF subjects were maintained throughout the experiment as xenotransplant model control (XMC). Likewise, a group of zebrafish without any xenotransplantation was taken as normal control (NC). Successive to the model confirmation, recombinant SARS-CoV-2 protein was injected at the site of xenotransplantation to establish the disease model. A group of HZF subjects injected with viral S-protein without any treatment was included as disease control (DC). Three experimental groups of DC subjects, namely, 0.2X-HED, 1X-HED and 5X-HED, respectively, received 6, 28 and 142 µg/kg of withanone enriched W. somnifera extract (WiNeWsE) for 3 (for 1^st endpoint study), 6 (for 2^nd endpoint study) and 10 (for 3^rd endpoint study) days. The group of DC subjects (1X-Dexa-HED) receiving 0.08 µg/kg of dexamethasone for same period as experimental groups was included as a positive control. Doses used in all treatment are shown in Table 3. The screening endpoint studies included monitoring of behavioral fever, skin hemorrhage, and swim bladder and kidney necropsy studies. Survival assay endpoint study included Kaplan–Meier analysis, represented as survival curve.

Figure 7

WiNeWsE rescues zebrafish from SARS-CoV-2 S-protein induced behavioral fever. Notes: (A) Pictorial depiction of the chambered water bath maintained at different temperatures with free access between these chambers for zebrafish subjects to migrate to conducive temperature zones. The anterior-most and posterior-most chambers are connected to cooling and heating facilities respectively to maintain the temperature gradient along the experimental chambers. In addition, these flanking chambers are designed to prevent access to fish from experimental chambers. As predictive outcome of the treatments, the picture depicts three scenarios. The first scenario pertains to normal condition where the subjects, could be humanized or not, but have not been induced with viral S-protein and therefore, populate the chamber at 29°C, matching the body temperature of the zebrafish. In the second scenario, where the HZF subjects, injected with recombinant viral S-protein, experience a rise in their body temperature and thus, migrate to the chamber at 37°C. This phenomenon in which the zebrafish with higher body temperature than their physiological one migrate to a warmer surrounding matching their body temperature is called behavioral fever. In the third scenario, the fevered subjects are treated either with dexamethasone or WiNeWsE, and therefore, are seen migrating back to the chamber at 29°C. The time spent by each treated or untreated subject in a specified chamber is recorded and represented as a color-coded matrix, called a heat map. Increase in time spent is depicted as corresponding darkening of the chosen green color palate. (B) Heat map representing the effect of treatments on behavioral fever.

Figure 8

WiNeWsE ameliorates SARS-CoV-2 S-protein induced skin hemorrhages in zebrafish. Notes: (A and C) Pictorial flowchart depicting the establishment of HZF model (XMC) in normal zebrafish population (NC) and subsequent induction of skin hemorrhage (encircled with open red circles) with intramuscular injections of recombinant viral S-protein to develop the disease control (DC), and monitoring the effects of various treatments, namely, 0.08 µg/kg bodyweight of dexamethasone (1X-Dexa-HED) or different concentrations of WiNeWsE (6 µg/kg for 0.2X-HED, 28 µg/kg for 1X-HED and 142 µg/kg for 5X-HED) after 3 (A) and 6 (B) days. (B and D) Number of zebrafish subjects developing skin hemorrhages were counted and plotted separately for 3 (C) and 6-day (D) cohorts, for a quantitative understanding of the effect of treatments on this disease outcome.

Figure 9

WiNeWsE attenuates viral S-protein induced inflammation in zebrafish swim bladder. Notes: (A and C) Effects of xenotransplantation, subsequent induction of HZF subjects with recombinant viral S-protein and successive different ameliorative therapeutic ministrations on the morphology of swim bladders and infiltration of inflammatory cells therein, as monitored through histopathology, are depicted through pictorial flowcharts for 3- (A) and 6-day (C) treatments. (B and D) Infiltration of different inflammatory cells [macrophages (mud brown arrowheads), granulocytes (green arrowheads) and lymphocytes (blue arrowheads)] in response to xenotransplantation, disease model development and subsequent, treatments were quantified and graphically, represented separately for 3- (B) and 6-day (D) treatments. Data plotted are mean ±SEM of counts obtained from 24 individual subjects in each group. Statistical significance of the means of different groups was analyzed through one-way ANOVA followed by Dunnett’s post hoc test and marked as *p<0.05, **p<0.01 and ***p-<0.001, when compared to disease control (DC). Abbreviations: Sm, smooth muscle nuclei; E, epithelial nuclei.

Figure 10

WiNeWsE assuages detrimental effects of SARS-CoV-2 S-protein on zebrafish kidney.