4.  Discussion
The current outbreak of SARS-CoV-2, a life threatening zoonotic coronavirus has made us painfully realize that existing and available options for its treatment are limited. Although, several efforts were made to treat SARS in 2002 and MERS in 2013, none of the past or existing efforts to treat the ongoing pandemic of 2020 has been found to be fruitful till date. Existing therapeutic options include the use of repurposed/repositioned drugs for treatment of COVID-19 pandemic. Development of effective vaccine candidates is also underway but would take considerable time and effort to produce fruitful results as shown in Figure 13. Prospective therapeutic discovery against coronavirus can be subdivided into two groups depending on the target: one acting upon the virus and the other acting on the human innate immune system. The latter plays a significant role in controlling the viral replication and secretion of cytokines are expected to improve the immune system functioning.
Figure 13.  Stages in drug discovery and development. In our every tryst with nature, we always perceive and find more than what we seek. Ancient Vedic literature has described nature as the ‘mother of all healings’. Nature is known to have its own antidote against all natural and mankind activity induced maladies such as COVID-19. The benefits of ‘Ecotherapy’ and ‘healing power’ of nature have led to the discovery of several miraculous systems of healing amongst which Ayurveda holds a significant position.
Withanolides are a group of at least 300 naturally occurring steroids built on an ergostane skeleton that are produced as secondary metabolites in several plant species. They are composed specifically of triterpenoids bearing around 28-carbon backbone (Cai et al., 2015). Given the importance of WS in Ayurveda as well as ethnopharmacology, majority of the studies reported in literature are confined to its antitumor and rejuvenating properties. There is scanty information available regarding the antiviral activity of WS. In the present paper, an attempt has been made to explore the antiviral potential of WS against COVID-19 using molecular and chemoinformatic tools and in silico methods.
Generally, an orally active drug candidate cannot have more than one violation of Lipinski’s criteria otherwise it might compromise its bioavailability. Good drug candidates with MW < 500 can be administered easily and are readily diffusible and absorbed. For optimal biological activity, the number of rotatable bonds should be <10, indicating a higher amount of molecular stability. Similarly, total polar surface area (TPSA) should coincide with hydrogen bonding of a molecule and characterize the delivery properties of the drug which should be <160 Å2. For a high oral bioavailability, the absorption rate determined from TPSA should be >50% (Balakrishnan et al., 2014). Interestingly, none of the phytocomponents of WS exhibited Lipinski’s violation, however, the standard drugs cinacalcet and poziotinib displayed 1 violation of Lipinski’s rule of five (Table 2). In addition, the selected phytoconstituents exhibited no violations of Veber, Egan and Muegge filters thereby indicating their druglike character (Table 3).
Prior to selection of WS phytoconstituents, their ADMET properties were calculated using online database (admetSAR), which provides latest and most inclusive manually created data for various chemicals with known ADMET properties (Cheng et al., 2012). Interestingly, in the present study, most of the WS phytoconstituents exhibited positive results of ADME and none of the phytoconstituents were predicted to have any mutagenic effect. Further, in case of Caco-2 permeability, anaferine and withasomnine exhibited positive results indicating Caco-2 permeability. Positive results for Caco-2 indicate good permeability characteristics of compounds under evaluation since Caco-2 cells express a number of transporter and efflux proteins as well as Phase II conjugation enzymes for metabolic transformation of test substances (van Breemen & Li, 2005). Ames test is a short-term bacterial reverse mutation assay used for evaluating compounds for their ability to induce genetic damage and frame shift mutations (Ames et al., 1975; Mortelmans & Zeiger, 2000). Mutagenic effects bear a close connection to carcinogenesis (Xu et al., 2012). Interestingly, in the present study, none of the chosen WS phytoconstituents were predicted to have any mutagenic effect. The 3CL-pro of CoVs is necessary for the proteolytic maturation of the virus and a potential drug target to prevent infection from spreading by inhibiting the cleavage of viral proteins (Tian et al., 2015). Therefore, inhibition of such proteases which have a role in virus replication are often used as treatment strategies in antiviral drug therapeutics (Delaney, 2004). Human ACE2 expression in the airway epithelia appears to be critical as an entry receptor for SARS-CoV and SARS-CoV-2 (Morris et al., 1998). The transmembrane spike (S) glycoprotein present on the surface of coronaviruses facilitates their entry into the cell via ACE2 and is considered to be another prime target for antiviral agents against coronaviruses. These findings have implications for understanding disease pathogenesis and opportunity to identify potential drug candidates for treatment.
Nelfinavir and lopinavir are viral protease inhibitors used against HIV infection but are reported to possess high cytotoxic effects. Lopinavir and ritonavir are viral protease inhibitors recommended for the treatment of SARS and MERS having similar mechanisms of action as nelfinavir and lopinavir. (Miyamoto and Kollman, 1992). To elucidate the binding affinity, docking studies of various withanolides found in WS were carried out on human ACE2 receptor, SARS-CoV and SARS-CoV-2 specific proteins. Among various phytoconstituents, withanolide A displayed strong binding affinity to SARS-CoV spike glycoprotein (BE: −9.78 kcal/mol, Kd: 67.23 nM), SARS-CoV-2 spike glycoprotein (BE: −7.18 kcal/mol, Kd: 5.48 µM), SARS-CoV 3CL-pro main protease (BE:-8.93 kcal/mol, Kd: 285.01 nM) and SARS-CoV-2 Nsp10/Nsp-16 complex (BE: −10.38 kcal/mol, Kd: 24.67 nM). On the basis of binding energy, withanolides A and B and withanone were found to be the most effective phytocomponents in WS. The present study reports for the first time the antiviral efficacy of medicinal herbs like Withania somnifera that form the crux of Ayurveda, the Indian traditional system of medicine as a viable alternative to chemosynthetic drugs for preventing/blocking entry of SARS-CoV-2 into host cells and also inhibiting viral main protease.
In conclusion, the most effective withanolides viz. withanolide A, withanolide B and withanone can be exploited and studied in future both in vitro and in vivo as prospective first choice antiviral agents for curbing COVID-19 infection. This preliminary study provides validation for plausible inhibitory potential of major withanolides found in WS.