Withanone from Withania somnifera Attenuates SARS-CoV-2 RBD and Host ACE2 Interactions to Rescue Spike Protein Induced Pathologies in Humanized Zebrafish Model
Authors: Monali Joshi
Hoshiyar Singh
Acharya Balkrishna
Vallabh Prakash Mulay
Swati Haldar
Anurag Varshney
Subarna Pokhrel
Journal: Drug Design Development and Therapy (2021)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: ACE2-RBD complex; ELISA; SARS-CoV-2 S-protein; Withania somnifera; docking and MD simulation
Affiliations: Drug Discovery and Development Division, Patanjali Research Institute, Haridwar, 249405, Uttarakhand, India., Department of Allied and Applied Sciences, University of Patanjali, Haridwar, 249405, Uttarakhand, India.

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 (MPro) 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. 

Retracted: No

PubPeer comments:
 Figs 2C, 4B.  Transwell assays were used to evaluate cell ... invasion using crystal violet staining . ![file] For comparison, Figs 2C and 5 from [Xiao, Liu Zhao (2015)] .  Cell ... invasion were then examined by conducting a ... Transwell assay in each group . ![file] *[upper]* Figs 4 C D and 5C from [Yang et al (2016)] .  Transwell assays were conducted to examine the cell invasion capacities ofA549 cells transfected with (C) miR-133b mimics and (D) FSCN1 plasmids. Non-transfected A549 cells were used as control ....  Transwell assay were conducted to examine the cell migration and invasion in each group . *[lower]* Fig 5B from [Wang et al (2016)] .  Transwell assay to determine the invasive capacity of U2OS cells transfected with miRu2011101 mimics or cu2011FOS siRNA, or cou2011transfected with miRu2011101 mimics and cu2011FOS plasmid. Magnification, x100. Control: untransfected U2OS cells . ![file] , 
 Retracted [13 September 2021] . ------------------------------------ Following the publication of this paper, it was drawn to the Editorsu2019 attention by a concerned reader that certain of the Transwell cell migration data shown in Figs. 2C and 4B were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to *Experimental and Therapeutic Medicine*, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive any reply. The Editor apologizes to the readership for any inconvenience caused. ,