Inhibitory Effect of Phytochemicals from Azadirachta indica A Juss. and Tinospora cordifolia (Thunb.) Miers against SARS-CoV-2 Mpro and Spike Protease- An In Silico Analysis


 COVID 19 caused by SARS-CoV-2 is spreading worldwide and affected 10 million people with a mortality rate between 0.5 % to 5%. Medicinal plants from China, Morocco, Algeria, Africa and India were tested for antiviral efficacy in SARS-CoV-2. Ayurveda Medicine described many medicinal plants. The Nimba (Azadirachta indica A. Juss) is used in fever, bacterial and viral infections, and Amrita (Tinospora cordifolia (Thunb.) Miers) is used as antiviral, antipyretic, and anti-inflammatory purposes. The combination of both these plants is called Nimbamritam, and it is widely used in pyrexia, dermatitis, viral infections, etc. Spike protease (PDB ID 6VXX) and Mpro (PDB ID 6LU) were retrieved from RCSB and 16 ligands from A. indica and 6 ligands from T. cordifolia were obtained from IMPPAT and PubChem. AutoDock Vina embedded PyRx was used for docking. Remdesivir was taken as a reference drug. In silico study of Cordifolide A of T cordifolia showed the highest scores with -8.2 Kcal/mol and -10.3Kcal/mol with Mpro protease and Spike protease respectively. Cordifolide A had 4 H bonds and Kaempferol had 7 non-conventional bonds, including van der Waal with Mpro (6LU7) protease. The interactions with 6VXX had 5 H bonds in each ligand Cordifolide A and Azadirachtin B. The prevention of virus entry by targeting spike protease host receptor ACE2 and restricting replication of the viral genome by targeting Mpro residues were identified in our study. A. indica and T. cordifolia are promising therapeutic agents in COVID 19.


Introduction
Ayurveda, a traditional medical system of India practiced worldwide is based on Tri-Humoral theory known as Tridosha which consists of Vata, Pitha, and Kapha. They have pathophysiological functions in the human body and diseases are caused due to the derangement of them. Drugs, herbs, or minerals make vitiated doshas into normalization to bring Health. The traditional Chinese medical system also has a similar strategy in health care. Medicinal plants are being used for health problems and many of these plant-derived drugs have been tested in vitro, in vivo, and clinical studies to prove their e cacy.
A virus, initially named as novel coronavirus 2019 (nCov209), now known as Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) was identi ed at Wuhan, China. The disease caused by this virus is named as COVID 19, and the WHO has announced it as a pandemic [1][2][3][4][5][6]. Till the end of June 2020 nearly 10 million people were affected by COVID 19 disease with a mortality rate between 0.5 % and 5% in different countries. Despite in vitro, in vivo and clinical studies are being carried out internationally to combat the COVID-19 best cure is yet to be identi ed. Antiviral drugs Remdesivir, favipiravir, ribavirin; antimalarial chloroquine and hydroxychloroquine showed anti-SARS-CoV-1 and SARS-CoV-2. Vaccines and Plasma therapyantibodies from COVID 19 recovered subjects are being tried in COVID 19 but awaited for satisfactory results [7][8][9].
The SARS-CoV-2 is a β coronavirus enveloped with RNA and consists of four structural proteins viz. Spike protein(S), Envelop protein(E), Membrane protein(M). and Nucleocapsid protein(N) [10]. Spike glycoprotein acts as a viral antigen and binds to host cell receptors Angiotensin-Converting Enzyme 2 (ACE2) which is an initial entry point [11]. The 3-Chymotrypsin like protease (3CLpro) also called as Main protease (Mpro) and Papain Like protease (PLpro) are important proteases to transcribe and replicate the viral genome encodes. Since 3CLpro has a key role in replication, it is considered for studying as a drug target [12].
Mpro and PLpro are cysteine proteases responsible for the segregation of viral polypeptides into functional proteins for replication and aggregation in host cells [7]. Since the surface Spike glycoprotein fusions with the cellular membrane through ACE2, drug candidates should prevent the protein-cell binding. Therefore, targeting ACE2 through drug candidates shall block SARS-CoV-2 from entering into the host cells and prevent COVID 19 infection. Considering the activities of these protease drugs, drugs having e cacy to prevent the binding and to inhibit virus replication are to be explored. Worldwide investigations on plant extracts are being undertaken through in vitro, in vivo, in silico or clinical trials.
The Anti-in ammatory action of berberine, and the antiviral activity of tinosporin, jatrorrhizine cordifolioside A, magno orine and tinocordiside, and cordifolide were identi ed. Stems of T. cordifolia contain an appreciable quantity of zinc [32].
A diterpenoid, tinosporin showed activity against HIV, HTLV and other viral diseases for its immunomodulatory and selective inhibition of the virus to target T helper cells [40]. T. cordifolia has been used as an excellent immune-stimulant and serves as a remedy against various microbial infections. With a polyclonal B cell mitogen, G1-4A on binding to macrophages have been reported to enhance the immune response in mice by inducing secretion of IL-1 together with activation of macrophages [41].
Its extract has shown to result in the up-regulation of IL-6 cytokine, resulting in acute reactions to injury, in ammation, activation of cytotoxic T cells, and B cell differentiation [42]. Stem and leaves extracts have shown a hepatoprotective effect in Swiss albino male mice against lead nitrate induced toxicity [43]. The anti-bacterial activity was assayed against Escherichia coli, Staphylococcus aureus etc [44]. This plant decreased the recurrent resistance of HIV to antiretroviral therapy (ART) and improve the outcome of the therapy [45].
Molecules-Curcumin, nimbin, withaferin A, piperine, mangiferin, thebaine, berberine, and andrographolide showed signi cant binding a nity towards spike glycoprotein of SARS-CoV-2 and ACE2 receptor. Ligands berberine and nimbin from T. cordifolia and A indica respectively were docked with Mpro (PDB 6LU7) and exhibited good a nities [46]. IL-6, TNF-a, and IFNs were found to be elevated in patients with SARS-CoV-2 and drugs which are regulators of interleukins, chemokines, and cytokines are helpful in as adjuvant in COVID 19 [42].

Proteins:
Spike Protease PDB ID 6VXX and Mpro PDB ID 6LU7 were retrieved from the RCSB protein data bank [47,48].

Docking:
AutoDock Vina embedded software PyRx was used for docking purposes [52]. Two chains A and C were found in the PDB 6LU7 monomer retrieved from RCSB. The C chain N- [53].The C chain N3 inhibitor in Mpro protein PDB 6LU7 was deleted through AutoDock 4.2 and saved as.pdb le which used for docking purposes [54]. The pdb of Spike protein was directly docked with ligands in AutoDock vina in PyRx.
The protein in.pdb format was loaded in the PyRx and converted into Macromolecule and saved in.pdbqt format. Ligands in 2D SDF format were imported and modi ed with minimization and then converted into AutoDock ligand.pdqbt format through Open Babel embedded in PyRx. Further, in the AutoDock Vina wizard docking was done with blind docking in the grid box. The molecular docking calculations have been performed as blind, i.e., covered the entire protein surface, not any speci c region of the protein as the binding pocket to avoid sampling bias [52].
Results of docking a nities were saved in.csv format and bonding with residues were saved in.dsv format for further analysis. Binding energies with the highest negative scores were considered for good docking between protein and ligand. Each proteinligand docking emerged with 9 poses and the highest score from these poses was noted for its e cacy.

Analysis:
Bonding analysis of ligand-residue conformations was done with Discovery Studio Visualizer 2020 of BIOVIA software and their target residues were recorded. The receptor-ligand interactions were documented along with 2D diagrams. The conventional Hydrogen bonds, non -conventional van der Waal, Pi Alkyl, etc., were recorded [55].

Drug-likeliness:
Lipinski's Rule of Five is adopted for all ligands for their drug-likeness. More than two violations among ve rules disqualify for drug utility. Lipinski's rule of Five includes Molecular mass less than 500 Dalton, high lipophilicity (expressed as LogP less than 5), less than 5 hydrogen bond donors, less than 10 hydrogen bond acceptors, Molar refractivity should be between 40-130 [56,57].
Best a nities with root mean square deviation (RMSD) obtained in the PyRx AutoDock Vina were recorded. Among the 9 poses from each ligand best a nity of docking with RMSD was taken for analysis. All ligands were docked successfully with Mpro (6LU7) protease and a nities were found between -4 4 Kcal/mol and -8.2 Kcal/mol. Phytochemicals from A. indica, Kaempferol and Azadirachtin B showed high a nities with -7.8Kcal/mol, and -7.7Kcal/mol respectively.   Conventional Hydrogen bonds, van der Waals, carbon bonds, Pi Alkyl, etc. with amino acids in 2D are depicted in Fig.2 Table 3, Table 4 The pharmacophore of a molecule includes the Hydrogen bond acceptor, H bond donor, negative and positive functional features with hydrophobic, aromatic groups. The present molecules were studied with a reference ligand-Remdesivir and showed similar essential features of the reference drug. The lesser energy showed the greater possibility of the drug candidate for prevention as well as to cure. In the present study, with computational docking tools AutoDock Vina, it is established that all tested ligands successfully docked against the inhibitory region of the main protease of the SARS-CoV-2 virus with docking scores between -4 Kcal/mol and -10 Kcal/mol. Further ligands were also had the best a nities with Spike protease which are responsible for viral entry at host ACE2. In a multidrug therapy, molecules contemporize and produce synchronized synergetic action. It is evidenced from our in silico study that ligands had common H bond residue interactions. A combination of molecules from A. indica and T. cordifolia may synchronize and prognosticate to establish an effective therapy in COVID 19.

MET165
These active molecules might inhibit the viral pathogenesis with a more e cient inhibitory effect against viral replication. IL-6, TNF-a, and IFNs were found to be elevated in patients with SARS-CoV-2 and drugs which are regulators of interleukins, chemokines, and cytokines are helpful in as adjuvant in COVID 19 [42]. T cordifolia immune-regulatory effect may intervene with the IL 6. TNF-a etc and regulate cytokines to produce a therapeutic effect in COVID 19.  [18]. Nimbin, berberine, mangiferin of A indica showed signi cant binding a nity towards spike protein of SARS-CoV-2 and ACE2 receptor [46]. Molecules from A. indica and T. cordifolia may be useful as a prophylactic as well as therapeutic agents due to restricting viral attachment to the host cells and prevent replication of viral RNA.
The tinosporin of T. cordifolia showed activity against HIV, HTLV and other viral diseases for its immunomodulatory and selective inhibition of the virus to target T helper cells [40]. Dry cough, Fever, Dyspnoea and Myalgia are the main symptoms, and in severely affected COVID 19 subjects, Acute Respiratory Distress is observed. Apart from antiviral e cacy, A. indica and T. cordifolia have anti-in ammatory, analgesic and antipyretic actions. Therefore, a combination of these plants may improve clinical conditions such as fever, myalgia and cough in COVID 19 subjects.
The role of the immune system was explained in SARS-CoV-2 infection [58]. In the immune depleted older subjects COVID 19 produces severe symptoms, and comorbidities may increase the mortality rate in senior citizens. The immune-enhance effect of the T cordifolia may help in reducing the severity of symptoms.

Conclusion
Neem A. indica and Amrita T. cordifolia have antiviral, antipyretic and anti-in ammatory actions, and a combination of these two plants is promising drug therapy for prevention and intervention in SARS-CoV-2 infection.
Our in silico study revealed that 22 molecules of these plants had good binding a nities with Spike protease and Mpro protease, and substantiate the claim for anti-SARS-CoV-2 by preventing the spike protease-ACE2 target. Further docking with Mpro protease by the ligands producing a nities reiterate that replication of the viral genome will be prevented. Protein-ligand docking of these phytochemicals, on comparison with reference synthetic antiviral Remdesivir, showed equivalent to Remdesivir. In addition to the antiviral effect, this combination has a role in symptomatic relief from fever, cough, myalgia.
Therefore, with the multidrug therapy containing A. indica and T. cordifolia promises an effective alternate solution in COVID 19. However, since the present study conducted in silico we need to establish antiviral activity in vitro, in vivo, and clinical studies in COVID 19.

Declarations
Con ict of Interest: There is no con ict of interest with this work.