Withanone and Withaferin-A are predicted to interact with transmembrane protease serine 2 (TMPRSS2) and block entry of SARS-CoV-2 into cells

Abstract

Coronavirus disease 2019 (COVID-19) initiated in December 2019 in Wuhan, China and became pandemic causing high fatality and disrupted normal life calling world almost to a halt. Causative agent is a novel coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/2019-nCoV). While new line of drug/vaccine development has been initiated world-wide, in the current scenario of high infected numbers, severity of the disease and high morbidity, repurposing of the existing drugs is heavily explored. Here, we used a homology-based structural model of transmembrane protease serine 2 (TMPRSS2), a cell surface receptor, required for entry of virus to the target host cell. Using the strengths of molecular docking and molecular dynamics simulations, we examined the binding potential of Withaferin-A (Wi-A), Withanone (Wi-N) and caffeic acid phenethyl ester to TPMRSS2 in comparison to its known inhibitor, Camostat mesylate. We found that both Wi-A and Wi-N could bind and stably interact at the catalytic site of TMPRSS2. Wi-N showed stronger interactions with TMPRSS2 catalytic residues than Wi-A and was also able to induce changes in its allosteric site. Furthermore, we investigated the effect of Wi-N on TMPRSS2 expression in MCF7 cells and found remarkable downregulation of TMPRSS2 mRNA in treated cells predicting dual action of Wi-N to block SARS-CoV-2 entry into the host cells. Since the natural compounds are easily available/affordable, they may even offer a timely therapeutic/preventive value for the management of SARS-CoV-2 pandemic. We also report that Wi-A/Wi-N content varies in different parts of Ashwagandha and warrants careful attention for their use.Communicated by Ramaswamy H. Sarma.

Keywords: Ashwagandha; COVID-19; Withaferin-A; Withanone; binding; caffeic acid phenethyl ester; honey bee; inhibition; molecular docking; propolis; transmembrane protease serine 2 (TMPRSS2).

Figure 1.

Molecular structures of (A) Camostat mesylate, (B) CAPE, (C) Withaferin-A and (D) Withanone.

Figure 2.

(A) RMSD of the protein backbone along the simulation trajectory for the protein alone and all the docked complexes. The overall structure of TMPRSS2 did not change much after the binding of Wi-N or Wi-A when compared to Camostat mesylate. (B) RMSF of the amino acids comprising the interacting domain of TMRSS2. No abrupt fluctuations were observed in any region of the protein with or without the three ligands. (C) Superimposition of the three docked complexes with Apo-TMPRSS2. All the three small molecules – Camostat mesylate, Wi-N and Wi-A were bound in the same site suggesting their similar mechanism of action. Conformational change from loop to helix was observed in region Arg316 to Tyr322 in case of Camostat mesylate and Wi-N. (D) Surface representation of TMPRSS2 showing all the ligands embedded in its catalytic pocket.

Figure 3.

Hydrogen bond occupancy of various important residues of TMPRSS2 during the simulation run in case of binding with Wi-N (A), Wi-A (B) and Camostat mesylate (C).

Figure 4.

Molecular interactions of Camostat mesylate (A), Wi-A (B) and Wi-N (C) within the catalytic site of TMPRSS2. Residues involved in hydrogen bond interactions are shown in sticks while the ones with non-polar interactions have been represented by lines. In all the three complexes, catalytic residues were participating in interaction with the ligands thereby blocking the site for priming of S protein, suggesting the therapeutic potential of Ashwagandha derived Wi-N and Wi-A.

Figure 5.

Dose dependent cytotoxicity of Wi-N to MCF7 cells. Cell morphology and viability, as determined by MTT and QCV assays, are shown (A). TMPRSS2 mRNA expression was determined by real time quantitative PCR (RT-PCR) in control and Wi-N (40 μM) treated cells. The data represents mean ± SD from three experiments. Statistical significance was calculated by Unpaired t test (GraphPad Prism, GraphPad Software, San Diego, CA. ** and ***represent p value < 0.05 and < 0.001 for significant and very significant changes, respectively).

Figure 6.

Content of Wi-A and Wi-N in root, stem and leaves of Ashwagandha as detected by high pressure liquid chromatography. Dry powders of each of the sample obtained from the same plant/harvest of Ashwagandha were extracted with β-CD water (Kaul et al., 2016). Content of Wi-A and Wi-N was determined by HPLC with respect to the standards shown on the top. X-axis and Y-axis show minutes and absorbance units, respectively.