RESEARCH ARTICLES Computational studies reveal piperine, the predominant oleoresin of black pepper (Piper nigrum) as a potential inhibitor of SARS-CoV-2 (COVID-19) Prassan Choudhary1, Hillol Chakdar1,*, Dikchha Singh1, Chandrabose Selvaraj2, Sanjeev Kumar Singh2, Sunil Kumar3 and Anil Kumar Saxena1 1ICAR-National Bureau of Agriculturally Important Microorganisms, Kushmaur, Mau 275 103, India 2Department of Bioinformatics, Alagappa University, Karaikudi 630 003, India 3Centre for Agricultural Bioinformatics, ICAR-Indian Agricultural Statistics Research Institute, New Delhi 110 002, India receptor binding conformations8. Inhibitors of protease In this study, we screened 26 bioactive compounds have the risk of causing severe side effects as they can in- present in various spices for activity against SARS- 9 CoV-2 using molecular docking. Results showed that hibit the cellular homologous proteases non-specifically . piperine, present in black pepper had a high binding Whole genome sequencing (WGS) has played a crucial affinity (–7.0 kCal/mol) than adenosine monophos- role in paving the way for exploration of novel drug phate (–6.4 kCal/mol) towards the RNA-binding pock- targets10. The GISAID database has undertaken a global et of the nucleocapsid. Molecular dynamics simulation initiative and currently holds WGS of approximately of the docked complexes confirmed the stability of 9300 different isolates of SARS-CoV-2, characterizing piperine docked to nucleocapsid protein as a potential the epidemiology and functional annotation of this virus inhibitor of the RNA-binding site. Therefore, piperine genome (https://www.gisaid.org/). seems to be potential candidate to inhibit the packag- Nucleocapsid (NC) is a highly conserved zinc finger ing of RNA in the nucleocapsid and thereby inhibiting structural protein which plays a crucial role in viral repli- the viral proliferation. This study suggests that 11,12 consumption of black pepper may also help to combat cation . This multimeric protein encapsulates the viral SARS-CoV-2 directly through possible antiviral genome while also facilitating entry into human cells 13 effects, besides its immunomodulatory functions. through the ACE2 receptors . NC along with Nsp3 plays a pivotal role in the coronavirus life cycle by controlling the replication–transcription complexes14. More im- Keywords: Binding affinity, black pepper, COVID-19, portantly, NC is necessary for viral RNA packaging in homology modelling, piperine. the early stages of viral infection15. These properties of NC make it a suitable drug target for a first generation of SARS-CoV-2 (COVID-19) is a novel human coronavirus anti-NC drugs. With therapeutic vaccines not available as belonging to Betacoronaviruses which originated from early as 2021 (ref. 16), there is an urgent requirement to Wuhan Province in China1,2. Since its outbreak around promote complementary and alternative medicine (CAM) November 2019, it has created havoc in more than 200 practices in order to combat this sudden outbreak till any countries infecting about two million people and leading concrete therapies/vaccines are available globally17. to 1.5 lakh deaths globally3. Throughout the world, scien- Moreover, alternative medicines are essential for deve- tists are engaged in the development of a vaccine in order loping countries which cannot bear the cost of vaccines18. to curb its viral action. According to Li et al.2, a total 73 Lack of enough testing kits and efficient outreach pro- vaccines are at preclinical or exploratory stages, while grammes for the promotion of such vaccines also greatly five candidate vaccines have entered phase-I clinical hinders the cause. trial4. Most of the lead candidates have structural spike The Ministry of Ayurveda, Yoga and Naturopathy, protein or the main protease (Mpro, 3CLpro) as their drug Unani, Siddha and Homoeopathy (AYUSH), Government target5–7. There are several limitations to these drug tar- of India (GoI) has issued an advisory on immunity-boost- gets as mutations in the S protein can help the virus elude ing measures which can help to fight SARS-CoV-2 infec- the therapeutic target and also lead to changes in host-cell tion. They have outlined in detail the use of spices like clove, ginger, cinnamon, black pepper, dalchini, cumin, *For correspondence. (e-mail: [email protected]) ajwain, etc. to boost immunity (https://www.mohfw. CURRENT SCIENCE, VOL. 119, NO. 8, 25 OCTOBER 2020 1333 RESEARCH ARTICLES gov.in/pdf/ImmunityBoostingAYUSHAdvisory.pdf). Spi- templates along with their percentage identity were: (i) ces and other Ayurvedic remedies are known to contain 6M3M (100%), (ii) 6YI3 (99.28%), (iii) 1SSK (92.03%), diverse bioactive compounds and well documented to (iv) 6VYO (100%), and (v) 2OFZ (92.06%). The best possess immunomodulatory properties19–21. It is also template (6YI3) was chosen based on high-resolution known that spices like ginger, turmeric, black pepper, etc. (1 Å), query coverage (69%) and percentage identity. On also have antimicrobial activities21. the basis of the lowest DOPE score, the final model was In this study we examined whether such spices can selected. RAMPAGE was used to carry out Ramachan- really be effective against SARS-CoV-2 apart from dran plot analysis and was represented using Discovery boosting immunity. Briefly, we predicted the 3D structure Studio module27,28. Expresso tool of T-COFFEE server of N-terminal RNA-binding domain of NC protein of was used for sequence alignment of the query sequence SARS-CoV-2 Wuhan-Hu-1 using homology modelling. with the templates using default parameters29. The mod- Molecular docking has been employed to screen predom- elled structure was superimposed onto the template 6YI3 inant bioactive compounds found in spices commonly using PYMOL software package30. used in households and as advised by the Ministry of AYUSH, GoI. We performed molecular dynamic simula- Molecular docking and interaction studies of tion which provided evidence to validate our findings. SARS-CoV-2 NC The study throws light on the significance of these natu- ral compounds in the fight against COVID-19. Autodock Vina module of UCSF Chimera was used for the docking studies on a Windows 10 platform23. The Methods prepared .mol2 files of receptor and ligands were imported and a search volume allotted to the receptor Data retrieval molecule for each docking study keeping all other param- eters constant31. The software uses Opal web service for The annotated sequence of NC protein sequence of docking and the files were allotted executable location on SARS-CoV-2 Wuhan-Hu-1 was obtained from the the local host computer. The best Autodock Vina score National Center for Biotechnology Information (NCBI; with the suitable energetically favoured conformations protein id: YP_009724397.2). BLASTp and CDD were was used for further analyses. The interaction of com- used to determine the N-terminal domain conserved site pounds with amino acid residues was analysed and repre- of the protein22. The sequence was curated to locate the sented using Discovery Studio Client. NTD, eliminating the rest of the amino acids from the study. The truncated protein (200 amino acids) was used Molecular dynamics simulation for further analysis. The 3D structures of 26 natural compounds from seven The apo protein of Npro (N protein) and the ligand com- different spices and few other drugs, including known plexes (Npro-AMP, Npro-piperine) were prepared, hy- synthetic anti-HIV analogues targeting NC used in the drogen bond-optimized, and the final complexes were study were directly imported from Pubchem database minimized till the root mean square deviation (RMSD) using UCSF Chimera v1.13.1 (Table 1)23. The structures value reached 0.30 Å (ref. 32). The prepared complex of adenosine monophosphate (AMP) and three synthetic was subjected to molecular dynamics simulation to un- analogues were also imported to examine their affinity to derstand the molecular stability of protein and protein– SARS-CoV-2 NC structure (Table 1). The imported ligand complex using the Desmond MD package, as ligand structures were prepared using Dock Prep tool of described earlier33,34. Autodock Vina, as reported previously24. Charges were computed using ANTECHAMBER with AMBER ff14SB charges allotted to standard residues and Gasteiger charg- Results and discussion es to other residue types, as reported in previous stud- ies25. The receptor protein was prepared following the Homology model of SARS-CoV-2 NC same protocol, barring the computation of charges step. All the prepared files were stored in .mol2 format for fur- The 3D structure of NC protein had a sequence identity ther evaluation and docking .analysis. of 99.28% with the template (PDB : 6YI3), with a query coverage of 69% (Figure 1 a). Two pairs of anti-parallel Homology modelling and Ramachandran plot -sheets (-hairpin) with a -core were found in the analysis structure. Ramachandran plot analysis showed 96% of the residues in the favoured region and 2% in the allowed Modeller v9.20 was used for homology modelling of the region, making it a robust structure (Figure 1 b). The protein sequence using Python script26. The PDB ID of modelled structure was used for docking and interaction 1334 CURRENT SCIENCE, VOL. 119, NO. 8, 25 OCTOBER 2020 RESEARCH ARTICLES Table 1. Bioactive natural compounds used in the study along with their sources Plant species Scientific name Common name Important chemical constituents Reference Syzygium aromaticum Clove Eugenol (~94.4% of essential