key: cord-0970285-1xh04st3
authors: Kumar, Suresh; Kashyap, Priya; Chowdhury, Suman; Kumar, Shivani; Panwar, Anil; Kumar, Ashok
title: Identification of phytochemicals as potential therapeutic agents that binds to Nsp15 protein target of coronavirus (SARS-CoV-2) that are capable of inhibiting virus replication
date: 2020-09-03
journal: Phytomedicine
DOI: 10.1016/j.phymed.2020.153317
sha: 0ae65a58a2f89861530ea3dffdad6995df523dc1
doc_id: 970285
cord_uid: 1xh04st3

BACKGROUND: : Coronavirus disease 2019 (COVID-19) playing havoc across the globe caused 585,727 deaths and 13,616,593 confirmed cases so far as per World Health Organization data released till 17(th) July 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) is responsible for causing this pandemic across different continents. It is not only impacting the world economy but also quarantined millions of people in their homes or hospitals. PURPOSE: : At present, there is no Food and Drug Administration-approved drug or vaccine available to treat this disease. Still, people are trying various pre-existing medicines that are known to have anti-viral or anti-parasitic effects. In view of this, the present study aimed to study the binding potential of various phytochemicals present in multiple natural plant extract as a secondary metabolite to non-structural protein 15 (Nsp15) protein, a drug target known to play a crucial role in virulence of coronavirus. METHOD: : Nsp15 protein was selected because it shows 89% similarity to the other SARS-CoV, which caused the earlier outbreak. The assumption is that inhibition of Nsp15 slowdowns the viral replication. Phytochemicals are selected as these are present in various plant parts (seed, flower, roots, etc.), which are used in different food cuisines in different geographical regions across the globe. The molecular docking approach was performed using two different software, i.e., Autodock, and Swissdock, to study the interaction of various phytochemicals with Nsp15 protein. Hydroxychloroquine is used as a positive control as it is used by medical professionals showing some positive effects in dealing with coronavirus. RESULTS: : The present study demonstrated the binding potential of approximately 50 phytochemicals with Nsp15 and capable of inhibiting the viral replication, although in vitro and in vivo tests are required to confirm these findings. CONCLUSIONS: : In conclusion, the present study successfully demonstrated the binding of phytochemicals such as sarsasapogenin, ursonic acid, curcumin, ajmalicine, novobiocin, silymarin and aranotin, piperine, gingerol, rosmarinic acid, and alpha terpinyl acetate to Nsp15 viral protein and they might play a key role in inhibiting SARS-CoV-2 replication.

Phytochemicals; Non-structural protein 15 (Nsp15); Molecular Docking; Sarsasapogenin; SARS-CoV-2; Coronavirus disease 2019 (COVID-19).

Human coronavirus is an RNA virus that belongs to the family of coronaviruses that can infect both humans and animals, which causes severe acute respiratory syndrome coronavirus (SARS-CoV). It has created difficult pandemic situations across the globe by causing severe respiratory syndrome-associated with cough, sore throat, fever, headache, body ache, and Various plants and herbs are necessary for maintaining good health, nutrition, and have medicinal values as they are part of many home remedies for many common illnesses.

Phytochemicals present in these plants and herbs are generally non-toxic and have the potential for preventing chronic disorders. Therefore, the present study focusses on the role of these medicinally useful phytochemicals against Nsp15, the RNA-processing enzymes in Structural analysis (RMSD, RMSF, and radius of gyration) was performed and their 3D graphs were generated with xm grace (Graphing, Advanced Computation, and Exploration program).

The active site of SARS-CoV-2 is located in a shallow groove between two anti-parallel βsheets with six primary catalytic residues-His235, His250, Lys290, Ser294, Thr341, and Tyr343. Where His235, His250, and Lys290, which are proposed to form a catalytic triad of the protein. As evident from the table, the phytochemicals interacted with the main catalytic site residues with strong binding energies indicating irreversible protein inhibition. Table 1 shows the binding energies obtained with phytochemicals along with interacting residues and bond length of molecular interaction.

Amongst all studied phytochemicals, promising strong inhibitions could be observed with sarsasapogenin, ursonic acid, ajmalicine, novobiocin, silymarin, and aranotin ( Figure 1 ).

Sarsasapogenin interacted with catalytic triad residue Lys290 with a strong single hydrogen bond of such low binding energy of -8.5, which made sarsasapogenin a potential inhibitor with the highest binding energy of -8.5. 

ADMET studies of phytoconstituents showed that sarsasapogenin, ursonic acid, ajmalicine, silymarin, and aranotin are the phytochemicals with strong interactions and decent ADMET profile as they obey the Lipinski's rule of five (Table 2) . DruLito software was used to carry out ADMET analysis (www.niper website) for studying the ideal pharmacokinetics profile of the phytochemicals for drug designing. Lipinski's rule was used to screen the phytochemicals. According to the Lipinski rule, the weight of an ideal drug molecule should be below 500g/mol, groups donating hydrogen bond should be less than or equal to 5 and groups accepting hydrogen bond should be less than or equal to 10 along with a partition coefficient of 5 or less. Bioavailability was analyzed based on TPSA (total polar surface area)

of the drug molecule. Veber's rule states that the TPSA less than or equal to 140Å indicates good oral bioavailability Hydroxychloroquine and chloroquine were used as a standard drug against NSP-15 with deltaG value of -9.27 and 8.71kcal/mol, respectively. Chloroquine, a known anti-malaria, and the anti-inflammatory drug showed deltaG -7.43Kcal/mol and full fitness score of -3382.6494. In the case of hydroxychloroquine, a total of three hydrogen bonds are observed, two with Glu192 and one with Gln310, whereas, in the case of chloroquine, one hydrogen was observed with Met105 amino acid ( Figure 2 ). All the phytochemicals exhibited one hydrogen bond, whereas zingerone demonstrated two hydrogen bonds. Furthermore, piperine, gingerol, alpha terpinyl acetate, menthol, ajoene, and rosmarinic acid were found with a better FF score in comparison to hydroxychloroquine and chloroquine.

13 

Sarsasapogenin and ursonic acid protein complexes were solvated and made electro neutral by adding 12 sodium ions in each system using genion. The volume and density of both systems were observed with values 954.784 nm3 and 1007.86 (g/l), respectively. After NVT and NPT ensemble, proteins were at a dynamic state at 300K ( (Figures 3a and 4a ) and

constant pressure (Figures 3b and 4b) . The potential energy graph revealed a sudden drop in potential energy of the system in the first few ps but reached a constant value thereafter.

Potential energy minimization of the ursonic acid system achieved at 980 EM steps as compared to 1460 EM steps of sarsasapogenin, indicating that the structure ursonic acid equilibrated more quickly than sarsasapogenin (Figure 3c and 4c) . To study the degree of compactness of protein during simulation, the radius of gyration was calculated. Over a span of 1000ps, a variation of the only 0.05nm was observed, stating that proteins have folded in its stable form. Lesser value of Radius of gyration for ursonic acid system shows its compactness over sarsasapogenin (Figure 3d and 4d) . Further, RMSD value was calculated to determine the fluctuations in 3D structure of proteins with time. Changes in the order of 1-3

Cuminaldehyde Cinnamaldehyde Å are acceptable, and higher values indicate misbehaviour of protein during the simulation. It was seen that protein under simulation undergoes minute changes resulting in a slight increase of RMSD. The blackline with red dots represents the protein backbone during simulation, which remained under acceptable value with reference to crystal structure. Ursonic acid equilibrated system shows little more deviation to its crystal structure as compared to the sarsasapogenin system (Figure 3e and 4e) . The density of systems during the course varied from 1012 to 1020 kg/m^3, but the average was close to value 1017 kg/m^3.

Density of both the systems was found stable over time, indicating that systems were wellequilibrated with respect to pressure and density (Figures 3f and 4f) . 

In conclusion, the present study successfully demonstrated the binding of phytochemicals such as sarsasapogenin, ursonic acid, curcumin, ajmalicine, novobiocin, silymarin and aranotin, piperine, gingerol, rosmarinic acid, and alpha terpinyl acetate to Nsp15 viral protein and they might play a key role in inhibiting SARS-CoV-2 replication. Among the phytochemicals, sarsasapogenin and ursonic acid can be considered as the lead phytochemicals. Further, in vivo and in vitro assay are required in the BSL 3 category lab to confirm the above finding. 

Nil Table 1 . Phytochemicals with binding energy score and interacting residue wit Nsp15 protein target. Table 2 . Pharmacokinetics ADMET prediction by Drulito against Lipinski rule of five and blood-brain-barrier filter. 

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