key: cord-0743873-qeo4sm3k
authors: Kumar, Mohit; Sodhi, Kushneet Kaur; Singh, Dileep Kumar
title: Addressing the potential role of curcumin in the prevention of COVID-19 by targeting the Nsp9 replicase protein through molecular docking
date: 2021-01-18
journal: Arch Microbiol
DOI: 10.1007/s00203-020-02163-9
sha: 5eefc7a5b0315de30db90a02db159ceb61f5a2e8
doc_id: 743873
cord_uid: qeo4sm3k

The pandemics have always been a destructive carrier to living organisms. Humans are the ultimate victims, as now we are facing the SARS CoV-2 virus caused COVID-19 since its emergence in Dec 2019, at Wuhan (China). Due to the new coronavirus’ unexplored nature, we shed light on curcumin for its potential role against the disease. The Nsp9 replicase protein, which plays an essential role in virus replication, was extracted online, followed by 3D PDB model prediction with its validation. The in silico molecular docking of curcumin with the replicase enzyme gave insights into the preventive measures against the virus as curcumin showed multiple interactions with Nsp9 replicase. The current study showed the use of curcumin against the coronavirus and its possible role in developing medicine against it. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00203-020-02163-9.

The SARS CoV-2 is responsible for the severe acute respiratory syndrome in humans and is now popularly known as COVID-19 because of the 2019-20 pandemic of coronavirus disease-2019 (Wan et al. 2020 ). The existing virus is contagious to human health and originated in Wuhan, China, hence sometimes called the "Wuhan virus" (Ralph et al. 2020) . It mainly enters the human body through the nasal opening, mouth, and eyes, where it goes to the lungs and multiplies themselves using host cellular machinery. In response to virus attack, host cells start secreting the signaling molecules, which may be the critical markers of viral infection, e.g., difficult breathing (Fig. 1) . The coronavirus contains positive-sense single-stranded RNA as genetic material. Structurally, coronavirus contains three major surface proteins called, spike (S) protein (Gallagher and Buchmeier 2001) , membrane (M) protein (Neuman et al. 2011) , and envelope (E) protein (Ruch et al. 2012) . The S-protein makes the crown-like appearance of each virus particle and helps attach the virus to the host protein/glycoproteins present on the host cell surface. Finally, they help in the invasion of virus particles into host cells via the specific receptors called ACE2 receptors. The M-protein is a membrane protein and helps in the assembly of the virus membrane. The E-protein is also a structural protein and helps in the formation of the virion envelope.

Communicated by Erko Stackebrandt.

The online version contains supplementary material available at https ://doi.org/10.1007/s0020 3-020-02163 -9.

The coronavirus caused severe respiratory illness to the global human population (Su et al. 2016) ; hence, the WHO declared it a worldwide pandemic (WHO 2020a, b) . The virus is non-curable till now. The virus replicates and spreads at a high-speed rate in the human population, depending on their immunity response (The Novel Coronavirus Pneumonia Emergency Response Epidemiology Team 2020). The replicase proteins help the virus to replicate in its host (Littler et al. 2020 ). These proteins are majorly RNAdependent RNA polymerase, which binds to the RNA and helps in its replication (Kim et al. 2020) . The replicase protein used in the current study is a chain B, Nsp9 replicase from SARS CoV-2 under family Coronaviridae and genus Betacoronavirus.

However, the fact that coronavirus causes COVID-19 is almost non-curable. Still, some molecules/compounds may help prevent or slow down the infection rate by targeting the machinery of viral particles (sampangi et al. 2020). Curcuma longa produces turmeric (diferuloylmethane), named Indian saffron in Europe, with its medicinal uses, including antiviral and anti-inflammatory actions (Araujo et al. 2001) . It has shown that curcumin has its inhibitory effects on the virus, including HIV (Hergenhahn et al. 2002) , smallpox, measles, and chickenpox are being among its target. It inhibits the integrase and other replication activity needed for viral replication. Figure 1 described coronavirus's entry to the human body and its inhibition by curcumin at multiple steps. In the current study, we showed the possible use of curcumin in the prevention of COVID-19 by targeting the virus replicase protein Nsp9. Turmeric is the principle source of curcumin, and in India it is used as an essential daily ingredient in the food preparation while it has its own antiviral, antifungal, antiallergic properties. Hence, it is preferred over other medicinal compounds in the present study. Nsp9 (nonstructural protein 9) RNA binding protein of SARS CoV-2 encoded by ORF1a is supposed to be involved in the viral RNA synthesis (Sutton et al 2004) hence, this protein was targeted in the current study. As, curcumin also showed the antiviral properties, the interaction of curcumin and Nsp9 may be useful in understanding the novel SARS Cov-2.

The chain B, Nsp9 replicase protein, was found to be a sequence of 117 amino acids and was extracted from NCBI (https ://www.ncbi.nlm.nih.gov/prote in/6W4B_B) with PDB id; 6W4B. The 3D PDB model of the protein was formed by the SWISS-MODEL (https ://swiss model .expas y.org) and analyzed in PyMOL software (https ://pymol .org/2) (Schrodinger 2010). The quality of the predicted protein model was checked by the ProSA web server (https ://prosa .servi ces.came.sbg.ac.at/prosa .php) (Wiederstein and Sippl 2007) . The active amino acids of chain B, Nsp9 protein were found by the online CASTp server (http://sts.bioe.uic.edu/castp / calcu latio n.html) (Tian et al. 2018 ) with the default value Fig. 1 The common infection pathway of Coronavirus, causes COVID-19 in the human system, with the entry roots; oral, nasal, and mucus membranes from where they reach to the body system to replicate and amplify themselves. Their amplification caused the immune cells to secrete the cytokines (Velazquez et al. 2019), leads to the molecular signaling pathways, cause abnormal breath, hypoxia, etc. Curcumin has the potential to inhibit the virus operated pathways and can lower down the pathological consequences after the virus infection (Qin et al. 2014) parameter of 1.4 Å. The structure of curcumin was drawn by chem sketch (http://www.acdla bs.com). The molecular docking of different active amino acids of Nsp9 protein with curcumin was done by Autodock 4.2 software (http://autod ock.scrip ps.edu) (Morris et al. 2009 ), and the results were analyzed in UCSF chimera software (https ://www.cgl.ucsf. edu/chimr a) (Pettersen et al. 2004 ).

Bioinformatics is a successful initiator to explore the systems biology and chemistry at the molecular level while saving time at the critical global pandemic of COVID-19 viral disease. The Nsp9 protein is taking part in viral replication in the host (human) cells (Sutton et al. 2004 ). Miknis et al. (2009) showed that its dimerization is necessary for efficient viral growth. The 117 amino acid long Nsp9 we have used was extracted from NCBI for the study due to the pandemic of COVID-19. The Nsp9 protein was started from amino acid serine and ended with glutamine, and it contains the initial seven sheets region and one helix region at last. The predicted protein model of Nsp9 replicase was checked and found to be of good quality as more than 90% amino acids were in the favoured region Ramachandran plot (Fig. 2a) , and again the X-ray and NMR prediction by ProSA webserver (Fig. 2b) gave a z-score of − 4.2, confirmed the good quality of the protein model and allowed us to use it in the study. The 3D structure of Nsp9 was of good quality homo-dimer with the QMEAN value of − 0.66 (Fig. 2c ) and X-ray resolution of 2.95 Å. These quality checks suggest the protein model used by us is an acceptable model. Further, the CASTp server gave 11 active amino acids (MET 16, GLY 41, GLY 42, ARG 43, VAL 45, PHE 60, PRO 61, LYS 62, SER 63, ILE 69, THR 71) , which are docked with curcumin, with their confined coordinates. Docking of curcumin with Nsp9 results gave a ligand-binding pocket of the Nsp9 (Fig. 3) , and this was probably the confined site where the curcumin showed interaction with other amino acids. Out of 11 docking complexes, six showed direct interaction Fig. 2 a The Ramachandran plot for assessment of the overall quality of the protein model, indicating up to 90% of the total amino acids in the most favored region of Ramachandran plot (a, b and l). b The Z-score of the protein predicted model was -4.2 indicating the good quality for the study, based on the X-ray and NMR calculations through the ProSA web server. Both quality check program, allowed the predicted model to be used in the current study. c The QMEAN value along with other physical parameters by SWISS MODEL Fig. 3 The ligand-binding site of Nsp9 protein holding the curcumin in its pocket (Fig. 4) . They made eight hydrogen bonds with different docking coordinates assigned to them for different active amino acids. All the docking parameters shown in Table 1 and docking coordinates are shown in Suppl Table 1 . The hydrogen bonds formed with curcumin involved THR 113 (Fig. 4.1) , SER 17 ( Fig. 4.1) , GLY 41 (Fig. 4.3) , ARG 43 (× 3) (Fig. 4.3, 4.6, 4.7) , LYS 62 ( Fig. 4.10) , and VAL 45 (Fig. 4.11) with bond length 2.896, 3.047, 2.916, 3.046, 2.947, 2.912, 2.905 and 2.966 Å, respectively. The supplementary figure file 1 contains all the descriptive images of Fig. 4 (1-11) , obtained through molecular docking. Interestingly, ARG 43 was the most common; three times took part in bond formation with different docking coordinates. This suggested the critical role of these amino acids in the interaction with curcumin. Besides the direct interaction and bond formation, there were 21 more possibilities of bond formation between curcumin and amino acids of Nsp9 replicase protein. The six docked complexes showed both actual and possibilities of the bond formation while five complexes were involved in the possible interaction with curcumin. The molecular docking of GLY 41 and LYS 62 gave the same results as the involvement of the same amino acids; AGR 43 and SER63 with different bond length, again showed the importance of ARG 43 of Ns9.

As it is well known that in any kind of viral infection, the inflammatory cytokines, IL-1, IL-6, and TNF-α released more actively by immune cells (Velazquez et al. 2019) , and they are being the target of curcumin (also diagrammatically represents in Fig. 1 ). This supports the use of curcumin to reduce the pathological consequences that emerged due to coronavirus infection. So, by targeting the ssRNA of coronavirus at its initial replication stage, through curcumin, when it enters the human is a matter of immediate in-vivo research to possibly overcome the COVID-19 and explore the inhibitory pathways of curcumin to prevent the new coronavirus replication machinery in the human system. 

Biological activities of curcuma longa L. Memórias do

Coronavirus spike proteins in viral entry and pathogenesis

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Insufficient Sensitivity of RNA Dependent RNA Polymerase Gene of SARS-CoV-2 Viral Genome as Confirmatory Test using Korean COVID-19 Cases

Crystal structure of the SARS-CoV-2 non-structural protein 9

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AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility

A structural analysis of M protein in coronavirus assembly and morphology

UCSF Chimera-a visualization system for exploratory research and analysis

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Wuhan virus), a novel coronavirus: human-to-human transmission, travel-related cases, and vaccine readiness

The coronavirus E protein: assembly and beyond

Molecular docking analysis of selected natural products from plants for inhibition of SARS-CoV-2 main protease

The PyMOL molecular graphics system. Version

Epidemiology, genetic recombination, and pathogenesis of coronaviruses

The nsp9 replicase protein of SARS-coronavirus, structure and functional insights

The Novel Coronavirus Pneumonia Emergency Response Epidemiology Team (2020) The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19) in China

CASTp 3.0: computed atlas of surface topography of proteins

The role of interleukin 6 during viral infections

Receptor recognition by the novel coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS coronavirus

SWISS-model: homology modelling of protein structures and complexes

ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins

World Health Organization (2020a) Novel coronavirus (2019-nCoV): situation report, 3. World Health Organization (2020b) Coronavirus disease 2019 (COVID-19): situation report, 72

The author MK would like to thank Hindu col-