Identification of Leading Compounds from Euphorbia neriifolia (Dudsor) Extracts as a Potential Inhibitor of SARS-CoV-2 ACE2-RBDS1 Receptor Complex: An Insight from Molecular Docking ADMET Profiling and MD-simulation Studies

INTRODUCTION

Early in December 2019, Wuhan, China, reported that the respiratory disease known as “coronavirus disease-19 (COVID-19)” was caused by the Severe acute respiratory syndrome–related coronavirus 2 (SARS-CoV-2) that has since spread around the globe.¹ This dreadful virus has evolved as one of the most fatal human illnesses on earth since its first outbreak in China.² The 30 kb single-stranded positive-sense coronaviruses have a diameter of 65–125 nm.³ The β-coronaviruses of the Coronaviridae family are enveloped RNA viruses that fall into five classes: α, β, γ, δ, and ο coronavirus.^4,5 Because of the disease’s widespread distribution and severeness around the world, the World Health Organization (WHO) has designated it as a public health issue of worldwide concern (Fig. 1).^6,7 There is a correlation between age, smoking status, ethnicity, and male sex with SARS-CoV-2 infection.^8,9 It’s interesting to note that patient age and underlying health issues affect COVID-19 persistence and prognosis.¹⁰ The etiology of this sickness is SARS-CoV-2 infection, which induces hyperinflammation that increases alveolar permeability. This hyperinflammation leads to a quickly fatal hypercytokinemia with multiorgan failure, and angiotensin converting enzyme 2 (ACE2) enzyme activity is suppressed.^11,12

Little is known about the human immune response to SARS-CoV-2 infection, despite mounting fears about the potentially catastrophic worldwide spread of COVID-19.^13,14 Severe acute respiratory syndrome–related coronavirus infections may spread to other people via the nasal epithelium. Recently, the use of Euphorbia neriifolia leaf juice in human trials has shown a significant improvement in COVID-19 illness recovery in Bangladesh.¹⁵ The rate of viral replication and severity of the sickness are significantly impacted when the protein of SARS-S CoV-2 attaches to ACE2 as a receptor for cellular entrance).^14,16,17 A number of methods have been proposed to interfere with the SARS-CoV-2 protein S-ACE2 binding. The N501Y mutation shown in the RBD-S1 complex enhances the interaction in all strains of variants that have been found, including B117, P1351, and P1 in the UK, South Africa, and Brazil, respectively. In this instance, addressing the ACE2 receptor could be an alternative to focusing on RBD. The SARS-CoV-2 spike (S) protein plays a crucial role in the fusion of the host cell membrane with the viral membrane during infection.^18,19 When the receptor-binding domain (RBD) of ACE2 is linked to the peptidase domain (PD), host proteases that split the S1 and S2 subunits at their N and C terminals activate the S protein.^20,21 Scientists have previously raised concerns about the use of drugs that target the renin-angiotensin signaling (RAS) system.^22–24 Nonetheless, Jia et al. detailed the present efforts to target the ACE2 receptor for SARS-CoV-2 therapy (Fig. 1).²⁵

The persisting SARS-CoV2 pandemic, as well as a lack of thorough understanding of the course of COVID-19, has limited our capacity to create effective therapies for afflicted individuals. However, blocking the SARS-CoV-2 protein S-ACE2 connection by targeting the ACE2 receptor might be a potential technique. In a recent study, the COVID-19 ο variation has prompted worldwide worry about altering vaccination programs and also slow vaccine rollout.⁵ Furthermore, this critical situation makes patients psychologically discomfort; the resulting public health continues to be threatened by COVID-19.^26,27 Coronavirus disease-19 patients are solely eligible for curative and supportive care; also as infections continue to increase on a regular schedule, there is an immediate want for effective treatment medications. For this pure, several scientific organizations globally are now focused to identify new effective antiviral drugs utilizing bioinformatics approaches of different organic plants like tea, Vernonia amygdalina, neem (Azadirachta indica) and some phytochemicals including, kolaviron, apigenin, and fisetin.^28–31 During the 2003 SARS outbreak, the efficacy of a therapy based on traditional medicinal herbs was documented.³²

Several potential phytochemicals found in Euphorbia neriifolia were tested for their ability to inhibit COVID-19 ACE2 protein complex (6VW1), investigated in our study. Native to India, Euphorbia neriifolia was scientifically proven to treat bronchitis, tumors, inflammation, and other diseases as well as to be hazardous to human beings.³³ It has been studied for several decades from a phytochemical perspective. Euphorbias have traditionally been used as a folk medicine to treat cough and asthma due to its anti-inflammatory, antibacterial, and antiparasitic characteristics.^34,35 Scientists think that natural chemicals originating from plants can protect against viral infection by functioning as therapeutic secondary metabolites in medicinal plants and as novel scaffolds for the creation of new drugs. Several types of secondary metabolites including triterpenes, anthocyanins, and diterpenes were isolated from Euphorbia neriifolia plants by different methods. Principal components have been identified including abietane, isopimarane, kaurane, and lathyrane as diterpenoids showed anti-HIV activity.^36–41 Currently, several phytochemicals from the terpenoids, alkaloids, and flavonoids families are being used to treat medical disorders such as medications, cycloartenol, euphol, neriifoliol, euphorbol, delphin, tulipanin, antiquorin and ingenol triacetate were isolated from latex, bark, root, whole plant and leaf.^36,42–46 All of these compounds are reclaimed for the aim of reducing SARS-CoV-2 pathogenesis, therefore reducing the alarming death rate.

In light of this, the current study will combine molecular docking, ADMET profiling, and molecular dynamics simulation (MDS) studies to assess the effectiveness of 17 bioactive compounds from Euphorbia neriifolia against the SARS-CoV-2 protein target ACE2 (PDB ID: 6VW1). This study aims to serve as a starting point for future research into specific COVID-19 treatment regimens.

RESULTS

DISCUSSION

Euphorbia neriifolia is a medicinal plant from the Euphorbiaceae family that has been used for millennia to control and cure a variety of infections. It is bitter, pungent, laxative, carminative, enhances appetite, beneficial in gastrointestinal problems, and causes loss of consciousness, and scientifically proven to the treatment of enlargement of the spleen, delirium, leucoderma, piles, tumors, bronchitis, anemia, ulcers, fever, cough, and asthma for being anti-inflammatory, antimicrobial and anti-parasitic properties.^{33–35,48,49} The current work investigates the potential inhibitory action of specific phytochemical ingredients (β-amyrin, taraxterol, delphin, tulipanin, antiquorin, euphorbol, cycloartenol, euphol, and nerifoliol) from the Euphorbia neriifolia leaf extract, against the SARS-COV2 receptor protein (6VW1).

Delphin had the highest affinity for 6VW1 as revealed in the results. Our result suggests that delphin might be a potential SARS-CoV-2 inhibitor. Delphin and tulipanin are anthocyanin phytochemicals, in particular, anthocyanins, a group of photosynthetic pigments that are hydrophilic in nature. In several investigations, such as anti-Influenza virus activity, anthocyanins have been demonstrated to have antiviral properties, virus adsorption and release from infected cells were both suppressed, through generating IL-2, reducing cytokine production in the lungs and boosting T cell function.^50–52 Delphin inhibits the attachment stage, virus particles are directly affected by the entry phase.^53,54 Delphinidin-3-rutinoside, a chemical produced from the Solanum monga plant, inhibits HSV-1 multiplication, viral proteins, and NOX4 transcription.⁵⁵ Tulipanin, derived from the rind of Punica granatum Var. nana can be used as an antidiabetic agent.⁵⁶ β-amyrin, on the other hand, is a phytochemical triterpene that has been proven in vitro and in vivo to have anti-inflammatory, anti-microbial, anti-fungal, and antiviral activities.⁵⁷ According to Vitor and colleagues, suppression of NF-kB and cAMP response element binding protein (CREB) activity is most likely the principal mechanism through which triterpenes exercise their anti-inflammatory effects.⁵⁸ Holanda Pinto et al. found that β-amyrin from Protium heptaphyllum reduces neutrophil infiltration, oxidative stress, and TNF-α expression in rats in another investigation.⁵⁹ According to Jabeen et al. research, beta-amyrin has the most potent antifungal action and antimicrobial activity against the growth of some oral microbial pathogens identified by Zheng et al. studies.^60,61 The IC (50) values for beta-amyrin in the A549 and HL-60 cancer cell lines were found to be 46.2 and 38.6 M, respectively.⁶² Ching et al. discovered that β-amyrin from leaf extract inhibits collagen-induced platelet aggregation more effectively than aspirin.⁶³ However, there exists a shortage of computational as well as clinical study information based on such medications for the treatment of SARS-CoV-2.

The ADMET profiles were evaluated as part of a systematic virtual drug and prospective drug screening can be an alternative to in vivo examination. The Lipinski rule is a critical criterion for identifying a molecule’s pharmacological and biological activity in humans (log P).⁶⁴ Only delphin and tulipanin showed 3 violations of the Lipinski rule. The Rule of 5 states that when there are more than 5 H-bond donors, 10 H-bond acceptors, and a molecular weight of more than 500, poor absorption or penetration is more likely to occur. In general, high MW is associated with low solubility, high lipophilicity, and high metabolism rate of drug absorption that slightly affect drug-likeness properties.^11,65 Only Tulipanin and delphin were able to maintain good lipophilicity properties at appropriate concentrations in our study, which was confirmed by Hughes et al., who demonstrated that the phytochemicals have comparatively good lipophilicity as the logP values were less than five.⁶⁶ A compound is regarded to be poorly absorbed if its absorbance is lower than 30%. Only delphin showed an intestinal absorption value of zero which indicates a lower absorption rate. This is not a good sign for selecting lead compounds only in silico analysis.⁶⁷ The final bioavailability of drug candidates is calculated by digestibility and Caco-2 penetrability. For predicting the absorption of drugs taken orally, Caco-2 monolayer cells are frequently utilized as an in vitro model of the human intestinal mucosa. If a substance has a Papp >8 × 10^–6, it is thought to have a high Caco-2 permeability. The tested phytochemicals (Tulipanin and Delphin) have a low Caco-2 permeability potential (<8 × 10^–6 cm/s) and could be assimilated through the human intestine. Except for tulipanin and delphin, all phytochemicals examined were anticipated to be P-glycoprotein inhibitors, which demonstrate that they do not disrupt the typical physiological actions of P-glycoprotein (an ATP-binding cassette transporter and efflux membrane transporter found primarily in epithelial cells) which reducing active uptake and drug distribution.⁶⁸ Cytochrome P450 is a group of enzymes that play an important role in drug activation as well as drug toxicity.^68,69 Only Tulipanin and Delphin are not CYP3A4 and CYP2D6 substrates, whereas all other phytochemicals tested were either CYP2D6 or CYP3A4 substrates. The drug’s lipophilicity appears to be adversely connected with metabolism-related toxicity. In addition, neither phytochemical tested was a substrate of the renal organic cation transporter, indicating they are eliminated by sweat, bile, or other mechanisms. The toxicological evaluation of the phytochemicals found that none of them are skin-toxic or hepatotoxic. Consequently, no one utilizes the AMES toxicity test to determine bacterial mutagenic potential medications. When compared to tulipanin and delphin, all compounds demonstrated a high level of toxicity in Tetrahymena pyriformis toxicity tests. However, hERG I am not inhibited by any of the phytochemicals, while all substances may inhibit hERG II. Several studies demonstrated that suppression of the hERG potassium channel might cause a delay in ventricular repolarization, disrupting the normal cardiac rhythm and causing hepatic dysfunction.^70,71

Molecular dynamics simulations can be used to demonstrate the consistency of a protein in a compound containing ligands.^72,73 It is also possible to measure the stiffness of protein molecules in a specific circumstance, like the human body.⁷² However, the RSMF values of the protein-ligand complex are used to define the complex’s mean fluctuations rather than the maximal stability of individual molecules.⁷⁴ The system RMSD was determined utilizing activities of the protein-ligand complex, which confirmed the minimum protein fluctuation. The decrease in RMSF fluctuation indicates that the molecule was stable with the target molecule. Through our research project, the compounds β-amyrin, delphin, and tulipanin had a high RMSD and RMSF value when combined with SARS-CoV-2 ACE2 protein. Baildya et al. were able to determine the molecule’s rotational inertia from its C and N termini while also investigating its overall structural stability using the Rg platform.⁷⁵ Specifically, Elebeedy et al. reported that a lower Rg value implies a more compact structure, whereas a larger Rg value indicates a more dissociative structure.⁷⁶ Higher SASA values indicate that water droplets and amino acid substances have less stable structures, whereas lower SASA values indicate that water particles and amino acid residues are more compacted.^77,78 In the study, the Rg, SASA, MolSA, and PSA values of the three ligand compounds were identified to have them at their optimal levels. Furthermore, MM/GBSA values of the studied compounds will be capable of maintaining a long-term interaction with the targeted ACE2 receptor of the SARS-CoV-2 protein.

β-amyrin, delphin, and tulipanin are three ligand molecules that were chosen for the present in silico investigation, which has shown high durability against the SARS-CoV-2 ACE2 protein complex in molecular docking, ADMET profiling, and molecular dynamic simulation studies at 100 numbers time intervals. These three phytochemicals had less variation with the protein complex when RMSD and RMSF were calculated. Furthermore, these three compounds demonstrated potential outcomes in MM/GBSA, SASA, Rg, MolSA, and PSA validation. However, there is completely inadequate drug trial evidence based on these compounds for the treatment of SARS-CoV-2. Following in vitro and in vivo confirmation of anti-SARS-CoV-2 interaction, these putative pharmacologically active polyphenols could be a potential treatment strategic approach for SARS-CoV-2 therapy.

MATERIALS AND METHODS

CONCLUSIONS

Natural products include a variety of bioactive compounds, and making them better candidates for multiple drug targets. The current work used a virtual screening strategy to identify natural phytochemicals as possible inhibitors of the SARS-CoV-2 ACE2 receptor, which is involved in viral attachment. Molecular docking to evaluate binding affinities and 100 numbers molecular dynamics analysis have been used to examine the ability of lead phytochemicals to form energetically and structurally stable complexes with the SARS-CoV-2 ACE2 protein. Because of its ability to effectively inhibit ACE2 protein, delphin (estimated binding affinity –10.4 kcal/mol) was recognized as a lead compound among the selected compounds and control drug (binding affinity –6.2 kcal/mol). Moreover, β-amyrin and tulipanin showed the ability to inhibit the ACE2 receptor with a binding affinity (–10.0 and –9.6 kcal/mol, respectively), and acceptable pharmacokinetics and physicochemical properties. Delphin is an attractive pharmaceutical option to treat COVID-19 because of its safety and minimal toxicity to normal cells, as well as its affinity and stability when forming complexes with the SARS-CoV-2 receptor protein. In-vitro investigations with delphin, β-amyrin, and tulipanin can be encouraged to identify a noble remedy for COVID-19 and to suggest a specific mechanism.

DATA AVAILABILITY

The data reported in this study can be found in the article. Moreover, the corresponding author may provide the reader with the data that were utilized to support the study’s findings upon request.

AUTHORS’ CONTRIBUTIONS

Conceptualization, MN Islam, MH Rahman, MS Hossen, MEA Pramanik, MMZ Miah, MJ Haque, AZM Hossain, Manzurul Islam, Mamun Mahtab, Sheikh Akbar and Ai Liang; Data curation, MEA Pramanik, MA Hossain, MA Islam, MH Rahman, MS Hossen, MJ Haque, RA Jahan, MS Hasan, Motiur Rahman, MM Kabir, PM Basak, MR Rahman, AKMA Prodhan, Ashik Mosaddik, Humayra Haque, Fahmida Fahmin, MS Islam, HS Das, Sheikh Akbar and Ai Liang; Formal analysis, MN Islam, MEA Pramanik, MA Hossain, MA Islam, MH Rahman, MS Hossen and RA Jahan; Funding acquisition, MEA Pramanik, MMZ Miah, Istiak Ahmed, MJ Haque, AKMM Islam, MN Ali, RA Jahan, ME Haque, Munzur Rahman, MS Hasan, Motiur Rahman, MM Kabir, PM Basak, MA Sarkar, AZM Hossain, AKMA Prodhan, Ashik Mosaddik, Fahmida Fahmin, Manzurul Islam, Mamun Mahtab, Sheikh Akbar and Ai Liang; Investigation, MN Islam, MEA Pramanik, AKMM Islam, MN Ali, Munzur Rahman, MA Sarkar, AZM Hossain, MR Rahman, AKMA Prodhan, Ashik Mosaddik, MS Islam, HS Das, Humayra Haque, Manzurul Islam, Mamun Mahtab, Sheikh Akbar and Ai Liang; Methodology, MN Islam, MEA Pramanik, MA Hossain, MA Islam, MH Rahman, MS Hossen, MMZ Miah, Istiak Ahmed, MN Ali, ME Haque, MS Hasan, Motiur Rahman, MM Kabir, PM Basak, AZM Hossain, Ashik Mosaddik, Manzurul Islam, Mamun Mahtab, Sheikh Akbar and Ai Liang; Project administration, MN Islam, MEA Pramanik, Istiak Ahmed, AKMM Islam, MN Ali, RA Jahan, Chandrima Emtia, MR Gofur, AKMA Prodhan, Ashik Mosaddik, Humayra Haque, Fahmida Fahmin, Manzurul Islam, Mamun Mahtab, Sheikh Akbar and Ai Liang; Resources, MN Islam, MEA Pramanik, AKMM Islam, AZM Hossain, MR Rahman, Ashik Mosaddik, Manzurul Islam, Mamun Mahtab, Sheikh Akbar and Ai Liang; Software, MN Islam, MEA Pramanik, MH Rahman and MA Sarkar; Supervision, MEA Pramanik, Ashik Mosaddik, Manzurul Islam, Mamun Mahtab, MR Gofur, Sheikh Akbar and Ai Liang; Validation, MEA Pramanik, Ashik Mosaddik, Manzurul Islam, Sheikh Akbar and Ai Liang; Visualization, MEA Pramanik, ME Haque, Chandrima Emtia, Sheikh Akbar and Ai Liang; Writing – original draft, MN Islam, MEA Pramanik, MA Hossain, MA Islam, Motiur Rahman, MR Gofur and PM Basak; Writing – review and editing, MN Islam, MEA Pramanik, MH Rahman, MS Hossen, MMZ Miah, Istiak Ahmed, MJ Haque, AKMM Islam, MN Ali, RA Jahan, ME Haque, Munzur Rahman, MS Hasan, MM Kabir, MA Sarkar, AZM Hossain, MR Rahman, AKMA Prodhan, Ashik Mosaddik, Humayra Haque, Fahmida Fahmin, Manzurul Islam, Mamun Mahtab, Sheikh Akbar and Ai Liang. The published version of the manuscript has been read and approved by all authors.

ACKNOWLEDGMENTS

This research is supported by ABEx Bio-Research Center, East Azampur, Dhaka 1230, Bangladesh.

ORCID

Md Enayet Ali Pramanik https://orcid.org/0000-0002-7276-331X

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