Structure and Ligand-based In Silico Studies towards the Natural Inhibitors against Receptor Recognition Spike Protein of SARS-CoV-2

1. INTRODUCTION

The global pandemic problem with SARS-CoV-2 and its variants is still a major health issue, causing huge mortality [1]. The spread of the virus has led to many questions about how to treat affected patients and keep them healthy. It all started in Wuhan, China, in December 2019, and since then, this dangerous virus, called SARS-CoV-2, has quickly spread around the globe, infecting people worldwide [2-6]. Taxonomically, the SARS-CoV-2 belongs to the Coronaviridae family and Nidovirales order and is a kind of virus with an Enveloped (E) protein and a single-stranded RNA (ssRNA). It is a tiny virus, about 40-60 nm in diameter, and it looks like it has a crown on its surface, hence the name coronavirus [7, 8]. The coronavirus family has four main groups: alpha (α), beta (β), gamma (γ), and delta (Δ). The alpha (α) and beta (β) types are the ones that usually infect healthy persons, but we are not sure how the gamma (γ) and delta (Δ) modes of action yet. Interestingly, the newly emerged coronavirus shares 80% of its genetic material with the well-known SARS-CoV-2 virus, which first appeared in China in 2002–2003 [9, 10]. Recently, there have been different types of coronaviruses, like 229E (α-CoV), HKU1 (β-CoV), OC43 (β-CoV), and NL63 (α-CoV), causing mild infections in individual’s upper respiratory tracts [11, 12]. However, there are two types of the virus, SARS-CoV and MERS-CoV, that have raised serious health concerns, and the latest one is SARS-CoV-2 (Beta-coronavirus) [13]. SARS-CoV-2 is highly contagious and can be harmful to the lungs and other important organs like the heart, liver, kidneys, gastrointestinal tract, and central nervous system. It often leads to failure of multiple organs [14, 15]. Additionally, SARS-CoV-2 has four main protein parts: spike (S) protein, envelope (E), membrane (M), and nucleocapsid (N) [16, 17]. Various types of polyproteins, both structural and non-structural, are required for SARS-CoV-2 to multiply into more copies of itself (Fig. 1). Interestingly, the spike (S) protein attaches to a receptor protein called angiotensin-converting enzyme (ACE-2) on the cells of the host who got infected. The spike (S) protein cleaved into smaller parts, namely PPLA and IAB through viral proteases during pathogenicity established respectively. Additionally, the spike (S) protein is more important due to its mode of action, it acts as a virulence factor by interacting and adhering to ACE-2 proteins of host cells. Therefore, the spike protein of SARS-CoV-2 like a significant target could be hit by natural bioactive compounds to interfere with the pathogenesis mechanism of SARS-CoV-2 [18-20]. Currently, there are no FDA-approved drugs identified specifically for treating SARS-CoV-2 (COVID-19). In cases of SARS-CoV-2 infection, whether it is mild or severe, doctors usually focus on managing preliminary symptoms. They were commonly prescribed FDA-approved drugs like Remdesivir, EIDD-2801, Hydroxychloroquine, Dexamethasone, Rito navir, Favipiravir, and Chloroquine. However, these drugs have not shown effective results, and they might have side effects for patients [21]. Using an overdose of these aforementioned drugs in affected persons with corona virus has been associated with lower levels of anti-inflammatory substances, causing a problem known as cytokine storm syndrome (CSS) and acute respiratory distress syndrome (ARDS)., Dexamethasone, a type of corticosteroid, is one drug that has been used to reduce mortality in the early stages of treatment.

While the adverse effects of dexamethasone are most frequently associated with anxiety, weight gain, fluid retention, hormonal imbalance, and insomnia [22]. The commonly used drugs such as hydroxychloroquine and chloroquine prevent virus and host cell interaction process and lead to control the activation of T-cells [23-25]. Drugs like Remdesivir, Favipiravir, and EIDD-2801 interfere with the activity of the RNA-dependent RNA polymerase of SARS-CoV-2, while the drug Ritonavir inhibits the activity of viral proteases, specifically cysteine protease PLpro and Mpro. Interestingly, natural herbs and their derivatives, known for minimal side effects and biocompatible, have also shown promising potential bioagents against viral infections [26-28]. Certain plants, like Ashwagandha (Withania somnifera), Neem (Azadirachta indica), and Giloy (Tinospora cordifolia), are reported in the literature and known to contain bioactive compounds with a wide range of therapeutic benefits like reducing inflammation, lowering fever, antioxidative properties, pain relief, immune system stimulation, and potentially antiviral effects [29-33]. These natural bioactive compounds might help lower mortality rates and block the chain of coronavirus infection. The aim of this study is to deliver a quick overview of the SARS-CoV-2 virus's therapeutic target sites. In addition, the research is aimed at exploring the potential efficacy of these natural chemicals as an alternative therapeutic agent through employing advanced computer-based techniques such as molecular docking and in silico high-throughput screening.

2. MATERIALS AND METHODS

3. RESULTS

3.2. Analysis of Physicochemical and Toxicity Properties/parameters of Screened Bioactive Compounds

In silico method to analyze the physicochemical and toxicity properties of the top five bioactive compounds: Withanolide B (CID: 14236711), Withanolide A (CID: 11294368), Withaferin A (CID: 16760705), Gedunin (CID: 12004512), and Berberine (CID: 2353). Subsequently, employing Discovery Studio 4.0, the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of these compounds were estimated (see Table 3). The ADME and toxicity assessments of selected bioactive compounds showed drug-like characteristics that make them suitable for human use. Mutagenicity, CYP2 inhibition activity, blood-brain barrier penetration, and intestinal absorption were included among the variables considered into consideration throughout the evaluation. Notably, Withanolide B (CID: 14236711), Withanolide A (CID: 11294368), Gedunin (CID: 12004512), and Withaferin A (CID: 16760705) exhibited high blood-brain penetration and were non-mutagenic. In contrast, the compound berberine (CID: 2353) exhibited minimum blood-brain penetration, while the selected bioactive compounds demonstrated neither mutagenic nor inhibitory effects on CYP450, with the exception of Berberine. These bioactive compounds were also tested for absorption and mutagenicity, and the results showed that they were highly permeable across cell membranes and exhibited nearly no mutagenicity. These compounds are considered non-carcinogenic and highly suitable for drug development, aligning with the Lipinski rule of five (RO5). The compound Withanolide B (CID: 1423611) possessed an evaluation score of -6.301 for ADMET and solubility potential, while Withanolide A achieved a value of -5.2±8. Importantly, none of the compounds that were evaluated had been found to be considered a risk to human health (Fig. 4A, B).

Table 3.

Sr. No.	Compound Name and Pubmed ID	ADMET Solubility	Absorption Level	ADMET BBB	BBB Level	CYP2 Inhibitor	PPB	PPB	Alog	PSA2D	Mutagencity
1	Withanolide B CID_1423611	-6.301	0	-0.05	1	False	4.62429	True	4.411	73.277	Non-mutagen
2	Withnolide A CID_11294368	-5.242	0	-0.593	3	False	2.83101	True	3.397	94.092	Non-mutagen
3	Withaferin A CID_16760705	-5.124	0	-0.5.96	3	False	2.68768	True	3.388	94.092	Non-mutagen
4	Gedunin CID_12004512	-5.994	0	-0.563	3	False	-1.29319	True	3.349	91.247	Non-mutagen
5	Berberine CID_2353	-5.541	0	-0.261	1	True	6.79215	True	3.446	41.068	Mutagen

4. DISCUSSION

The binding affinities and interaction patterns between the ligands and the receptor protein have been revealed by the molecular docking interactions [47]. The in-silico analysis offers valuable insights into the binding interactions between the spike (S) protein and screened ligands, providing potential therapeutic options for combating SARS-CoV-2. The ADMET analysis revealed favourable properties for the selected bioactive compounds, including Withanolide B, Withanolide A, Withaferin A, Gedunin, and Berberine, in terms of absorption, distribution, metabolism, and excretion. These compounds exhibited high blood-brain penetration, suggesting their potential to reach the central nervous system and exert therapeutic effects. Additionally, the non-mutagenicity and inhibitory activity against CYP450 enzymes in most of the selected bioactive compounds indicate their safety and minimum risk of adverse effects. However, it is important to note that Berberine showed lower blood-brain penetration, suggesting a potential limitation in its ability to reach the central nervous system. The assessment of the compounds' absorption and mutagenicity, along with their adherence to the Lipinski rule of five, further reinforces their potential as drug candidates. These compounds displayed high permeability through cell membranes and exhibited low mutagenicity, making them suitable for consumption and indicating their safety for use as novel drugs. In summary, the in-silico analysis of the physicochemical and toxicity properties of the selected bioactive compounds highlights their promising characteristics as potential therapeutic agents.These findings serve as a foundation for further experimental studies to validate their efficacy and safety profiles. While, the MD simulation analysis provided insights into the stability and flexibility of the docked complexes of Withanolide B and Withanolide A with the spike (S) protein of SARS-CoV-2. The calculated free binding energies demonstrated a strong affinity between the bioactive compounds and the spike protein, indicating their potential as effective binders to the target protein. The docked complexes of Spike+Withanolide B and Spike+Withanolide A preserved their structural integrity and stability for a considerable portion of the simulation time frame in accordance with the stability evaluations conducted via RMSD values. This consistency correlates with findings from other research [48]. The research reports persistent complex forms between the spike protein and bioactive compounds. On the contrary, the unbound spike protein of SARS-CoV-2 displayed greater variability in RMSD values, indicating its inherent flexibility and conformational changes. This finding is consistent with previous studies that have highlighted the dynamic nature of the spike protein in the absence of ligand binding. Moreover, the analysis of fluctuation and compactness in Withanolide B revealed significant changes at specific time points during the simulation. This dynamic behaviour suggests potential conformational adjustments of the compound, which could have implications for its binding interactions and pharmacological effects. Their results, which showed that the bound complexes had lower RMSD values than the unbound protein, were in line with our findings and suggested that the ligand had increased stability. The resemblance points to a widespread pattern of spike protein complexes became more stable. Additionally, studies conducted [49, 50] explored the RMSF patterns of different proteins in order to comprehend their flexibility. They determined that proteins with stable secondary structures had lower RMSF values, which is consistent with our findings. In our study, the bound complexes of Withanolide B and Withanolide A displayed significantly lower RMSF values than the unbound spike protein, indicating a more rigid and stable secondary structure. Regarding hydrogen bonding, our analysis revealed that the bound complex of Withanolide B formed four hydrogen bonds, while the bound complex of Withanolide A formed six hydrogen bonds. This insight correlates with the findings of other studies investigating interactions between proteins and ligands as a case study [51-53], which emphasized the importance of hydrogen bonding in stabilizing protein-ligand complexes. As compared to Withanolide A, Withanolide B's bound complex exhibits fewer hydrogen bonds, which might suggest that the first compound is more stable. Subsequently, to assess protein compactness, comparing the Rg values indicated no significant changes in Rg throughout the simulation, similar findings have been reported in other studies investigating protein dynamics, as seen in a study [54]. The analysis of SASA in this study revealed similar values for the unbound spike protein and the bound complexes of Withanolide B and Withanolide A. This suggests that the binding of these compounds did not significantly impact the folding and unfolding profiles of the protein surface. Therefore, further investigations are necessary to provide a more comprehensive understanding of the impact of ligand binding on protein surface accessibility and thoroughly explore the therapeutic potential of Withanolide B and Withanolide A against the virus.

CONCLUSION

This in silico study highlights the anti-viral potential of Withanolide B and Withanolide A and suggests their role in developing therapeutic agents against SARS-CoV-2 and other viral infections. These bioactive compounds demonstrate significant potential in inhibiting the activity of the spike (S) protein and disrupting the molecular interaction between the viral receptor and host recognition. Their scaffolds hold promise for future therapeutic agents against SARS-CoV-2 and other viral infections, emphasizing the importance of exploring alternative treatments, especially natural bioactive compounds, in the context of the COVID-19 pandemic. Additionally, experimental studies are necessary to establish their efficacy and safety compared to commonly used drugs during the COVID-19 pandemic. These bioactive compounds present a promising avenue for developing novel antiviral treatments, holding significant potential in addressing the ongoing challenges posed by SARS-CoV-2 and other viral pathogens.

REFERENCES