Inhibitory effect of thymoquinone from Nigella sativa against SARS-CoV-2 main protease. An in-silico study

Efeito inibitório da timoquinona de Nigella sativa contra a principal protease do SARS-CoV-2. Um estudo in silico

M. T. Khan A. Ali X. Wei T. Nadeem S. Muhammad A. G. Al-Sehemi Dongqing Wei About the authors

Abstract

Nigella sativa is known for the safety profile, containing a wealth of useful antiviral compounds. The main protease (Mpro, 3CLpro) of severe acute respiratory syndrome 2 (SARS-CoV-2) is being considered as one of the most attractive viral target, processing the polyproteins during viral pathogenesis and replication. In the current investigation we analyzed the potency of active component, thymoquinone (TQ) of Nigella sativa against SARS-CoV-2 Mpro. The structures of TQ and Mpro was retrieved from PubChem (CID10281) and Protein Data Bank (PDB ID 6MO3) respectively. The Mpro and TQ were docked and the complex was subjected to molecular dynamic (MD) simulations for a period 50ns. Protein folding effect was analyzed using radius of gyration (Rg) while stability and flexibility was measured, using root means square deviations (RMSD) and root means square fluctuation (RMSF) respectively. The simulation results shows that TQ is exhibiting good binding activity against SARS-CoV-2 Mpro, interacting many residues, present in the active site (His41, Cys145) and also the Glu166, facilitating the pocket shape. Further, experimental approaches are needed to validate the role of TQ against virus infection. The TQ is interfering with pocket maintaining residues as well as active site of virus Mpro which may be used as a potential inhibitor against SARS-CoV-2 for better management of COVID-19.

Keywords:
Nigella sativa; main protease; thymoquinone; SARS-CoV-2

Resumo

Nigella sativa é conhecida pelo perfil de segurança, contendo uma grande variedade de compostos antivirais úteis. A principal protease (Mpro, 3CLpro) da síndrome respiratória aguda grave 2 (SARS-CoV-2) está sendo considerada como um dos alvos virais mais atraentes, processando as poliproteínas durante a patogênese e replicação viral. Na presente investigação analisamos a potência do componente ativo, timoquinona (TQ) de Nigella sativa contra SARS-CoV-2 Mpro. As estruturas de TQ e Mpro foram recuperadas de PubChem (CID10281) e Protein Data Bank (PDB ID 6MO3), respectivamente. O Mpro e o TQ foram acoplados e o complexo foi submetido a simulações de dinâmica molecular (MD) por um período de 50ns. O efeito de dobramento de proteínas foi analisado usando o raio de giração (Rg) enquanto a estabilidade e a flexibilidade foram medidas usando a raiz quadrada média dos desvios (RMSD) e a raiz média quadrada da flutuação (RMSF), respectivamente. Os resultados da simulação mostram que o TQ está exibindo boa atividade de ligação contra o SARS-CoV-2 Mpro, interagindo em muitos resíduos presentes no sítio ativo (His41, Cys145) e também o Glu166, facilitando o formato da bolsa. Além disso, são necessárias abordagens experimentais para validar o papel do TQ contra a infecção pelo vírus. O TQ está interferindo nos resíduos de manutenção do bolso, bem como no sítio ativo do vírus Mpro, que pode ser usado como um potencial inibidor contra o SARS-CoV-2 para um melhor gerenciamento da Covid-19.

Palavras-chave:
Nigella sativa; protease principal; timoquinona; SARS-CoV-2

1. Introduction

The main protease (Mpro, 3CLpro) is an important drug targets of sever acute respiratory syndrome 2 (SARS-CoV-2) proteome, processing the polyproteins. A number of studies has well characterized the Mpro (Anand et al., 2005ANAND, K., YANG, H., BARTLAM, M., RAO, Z. and HILGENFELD, R., 2005. Coronavirus main proteinase: target for antiviral drug therapy. In: A. SCHMIDT, O. WEBER and M.H. WOLFF, eds. Coronaviruses with special emphasis on first insights concerning SARS. Basel: Birkhäuser Basel, pp. 173-199. http://dx.doi.org/10.1007/3-7643-7339-3_9.
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) and its potential role. This viral protein acts on 11 positions along polyproteins. Human proteases do not share the cleavage specificity with NSP5 of SARS-COV-2 (Lee et al., 2020LEE, J., WORRALL, L.J., VUCKOVIC, M., ROSELL, F.I., GENTILE, F., TON, A.T., CAVENEY, N.A., BAN, F., CHERKASOV, A., PAETZEL, M. and STRYNADKA, N.C.J., 2020. Crystallographic structure of wild-type SARS-CoV-2 main protease acyl-enzyme intermediate with physiological C-terminal autoprocessing site. Nature Communications, vol. 11, no. 1, p. 5877. http://dx.doi.org/10.1038/s41467-020-19662-4. PMid:33208735.
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, p. 2).

The substrate-binding sites residues 10-99 (Domains I) and 100-182 (Domain II) in picornavirus, are six-stranded antiparallel β-barrels while domain III (198-303) forming five helices, regulating the dimerization of Mpro (Shi and Song, 2006SHI, J. and SONG, J., 2006. The catalysis of the SARS 3C-like protease is under extensive regulation by its extra domain. The FEBS Journal, vol. 273, no. 5, pp. 1035-1045. http://dx.doi.org/10.1111/j.1742-4658.2006.05130.x. PMid:16478476.
http://dx.doi.org/10.1111/j.1742-4658.20...
). Residues Cys145 and His41 form the catalytic site. The catalytic activity depends on the dimerization of the enzyme, as the N-finger interacts with Glu166 to facilitate the S1 pocket shape of the substrate-binding site (Anand et al., 2002ANAND, K., PALM, G.J., MESTERS, J.R., SIDDELL, S.G., ZIEBUHR, J. and HILGENFELD, R., 2002. Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain. The EMBO Journal, vol. 21, no. 13, pp. 3213-3224. http://dx.doi.org/10.1093/emboj/cdf327. PMid:12093723.
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). The residue T285 and I286in CoV-2 Mpro is substituted by A285 and L286 respectively (Zhang et al., 2020ZHANG, L., LIN, D., SUN, X., CURTH, U., DROSTEN, C., SAUERHERING, L., BECKER, S., ROX, K. and HILGENFELD, R., 2020. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science, vol. 368, no. 6489, pp. 409-412. http://dx.doi.org/10.1126/science.abb3405. PMid:32198291.
http://dx.doi.org/10.1126/science.abb340...
) leading to a threefold upsurge Mpro (Lim et al., 2014LIM, L., SHI, J., MU, Y. and SONG, J., 2014. Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain. PLoS One, vol. 9, no. 7, p. e101941. http://dx.doi.org/10.1371/journal.pone.0101941. PMid:25036652.
http://dx.doi.org/10.1371/journal.pone.0...
). Inhibitors may be useful to reduce the catalytic degree against these locations.

Phytocompounds have been found, affective against many viral targets (Raj and Varadwaj, 2016RAJ, U. and VARADWAJ, P.K., 2016. Flavonoids as multi-target inhibitors for proteins associated with ebola virus: in silico discovery using virtual screening and molecular docking studies. Interdisciplinary Sciences, Computational Life Sciences, vol. 8, no. 2, pp. 132-141. http://dx.doi.org/10.1007/s12539-015-0109-8. PMid:26286008.
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; Setlur et al., 2017SETLUR, A.S., NAIK, S.Y. and SKARIYACHAN, S., 2017. Herbal lead as ideal bioactive compounds against probable drug targets of ebola virus in comparison with known chemical analogue: a computational drug discovery perspective. Interdisciplinary Sciences, Computational Life Sciences, vol. 9, no. 2, pp. 254-277. http://dx.doi.org/10.1007/s12539-016-0149-8. PMid:26857866.
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; Ismail and Jusoh, 2017ISMAIL, N.A. and JUSOH, S.A., 2017. Molecular docking and molecular dynamics simulation studies to predict flavonoid binding on the surface of DENV2 E protein. Interdisciplinary Sciences, Computational Life Sciences, vol. 9, no. 4, pp. 499-511. http://dx.doi.org/10.1007/s12539-016-0157-8. PMid:26969331.
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; Li et al., 2020LI, C., WANG, L. and REN, L., 2020. Antiviral mechanisms of candidate chemical medicines and traditional Chinese medicines for SARS-CoV-2 infection. Virus Research, vol. 286, p. 198073. http://dx.doi.org/10.1016/j.virusres.2020.198073. PMid:32592817.
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; Khare et al., 2020KHARE, P., SAHU, U., PANDEY, S.C. and SAMANT, M., 2020. Current approaches for target-specific drug discovery using natural compounds against SARS-CoV-2 infection. Virus Research, vol. 290, p. 198169. http://dx.doi.org/10.1016/j.virusres.2020.198169. PMid:32979476.
http://dx.doi.org/10.1016/j.virusres.202...
). Among the plants, Nigella sativa (Black cumin) is an annual flowering plant under the family Ranunculaceae (Amin and Hosseinzadeh, 2016AMIN, B. and HOSSEINZADEH, H., 2016. Black cumin (Nigella sativa) and its active constituent, thymoquinone: an overview on the analgesic and anti-inflammatory effects. Planta Medica, vol. 82, no. 1-2, pp. 8-16. http://dx.doi.org/10.1055/s-0035-1557838. PMid:26366755.
http://dx.doi.org/10.1055/s-0035-1557838...
). Its fruit is in inflated capsule of seeds, native to North Africa, Southeast Asia, Southern Europe, Mediterranean and Middle Eastern region (Ahlatci et al., 2014AHLATCI, A., KUZHAN, A., TAYSI, S., DEMIRTAS, O.C., ALKIS, H.E., TARAKCIOGLU, M., DEMIRCI, A., CAGLAYAN, D., SARICICEK, E. and CINAR, K., 2014. Radiation-modifying abilities of Nigella sativa and thymoquinone on radiation-induced nitrosative stress in the brain tissue. Phytomedicine, vol. 21, no. 5, pp. 740-744. http://dx.doi.org/10.1016/j.phymed.2013.10.023. PMid:24268807.
http://dx.doi.org/10.1016/j.phymed.2013....
). N. Sativa seed is composed of some major components including 35.6-41.5% of fatty oil, fat (28.5%), proteins (26.7%), carbohydrates (24.9%) and several vitamins (A, B1, B2, B3, C) and minerals (Ca, K, Se, Cu, P, Zn, Fe) (Ahlatci et al., 2014AHLATCI, A., KUZHAN, A., TAYSI, S., DEMIRTAS, O.C., ALKIS, H.E., TARAKCIOGLU, M., DEMIRCI, A., CAGLAYAN, D., SARICICEK, E. and CINAR, K., 2014. Radiation-modifying abilities of Nigella sativa and thymoquinone on radiation-induced nitrosative stress in the brain tissue. Phytomedicine, vol. 21, no. 5, pp. 740-744. http://dx.doi.org/10.1016/j.phymed.2013.10.023. PMid:24268807.
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; Islam, 2016ISLAM, M.T., 2016. Biological activities and therapeutic promises of Nigella sativa L. International Journal of Pharma Sciences and Scientific Research, vol. 2, no. 6, pp. 237-252. http://dx.doi.org/10.25141/2471-6782-2016-6.0237.
http://dx.doi.org/10.25141/2471-6782-201...
). The volatile oil of N. sativa seeds has saturated fatty acids including thymohydroquinnone (THQ), dithymohydroquinone, carvacrol, thymoquinone (TQ), nigellone, thymol, α and β-pinene, d-citronellol, d-limonene, p-cymene volatile oil, t-anethole, longifoline and 4-terpineol (Enomoto et al., 2001ENOMOTO, S., ASANO, R., IWAHORI, Y., NARUI, T., OKADA, Y., SINGAB, A.N.B. and OKUYAMA, T., 2001. Hematological studies on black cumin oil from the seeds of Nigella sativa L. Biological & Pharmaceutical Bulletin, vol. 24, no. 3, pp. 307-310. http://dx.doi.org/10.1248/bpb.24.307. PMid:11256491.
http://dx.doi.org/10.1248/bpb.24.307...
). The medicinal characteristics focusing on various pharmacological efficacies of N. sativa seeds like: gastroprotective (El-Abhar et al., 2003EL-ABHAR, H.S., ABDALLAH, D.M. and SALEH, S., 2003. Gastroprotective activity of Nigella sativa oil and its constituent, thymoquinone, against gastric mucosal injury induced by ischaemia/reperfusion in rats. Journal of Ethnopharmacology, vol. 84, no. 2-3, pp. 251-258. http://dx.doi.org/10.1016/S0378-8741(02)00324-0. PMid:12648823.
http://dx.doi.org/10.1016/S0378-8741(02)...
), anti-oxidant (Hosseinzadeh et al., 2013HOSSEINZADEH, H., TAFAGHODI, M., MOSAVI, M.J. and TAGHIABADI, E., 2013. Effect of aqueous and ethanolic extracts of Nigella sativa seeds on milk production in rats. Journal of Acupuncture and Meridian Studies, vol. 6, no. 1, pp. 18-23. http://dx.doi.org/10.1016/j.jams.2012.07.019. PMid:23433051.
http://dx.doi.org/10.1016/j.jams.2012.07...
), anti-cancer (Khan et al., 2011KHAN, A., CHEN, H.-C., TANIA, M. and ZHANG, D.-Z., 2011. Anticancer activities of Nigella sativa (black cumin). African Journal of Traditional, Complementary, and Alternative Medicines, vol. 8, no. 5S, pp. 226-232. http://dx.doi.org/10.4314/ajtcam.v8i5S.10. PMid:22754079.
http://dx.doi.org/10.4314/ajtcam.v8i5S.1...
), anti-viral activity against cytomegalovirus have been reported in recent years. In some studies, the TQ was effective against avian influenza virus (H9N2 AIV) and cytomegalovirus infection in murine model l (Salem and Hossain, 2000SALEM, M.L. and HOSSAIN, M.S., 2000. Protective effect of black seed oil from Nigella sativa against murine cytomegalovirus infection. International Journal of Immunopharmacology, vol. 22, no. 9, pp. 729-740. http://dx.doi.org/10.1016/S0192-0561(00)00036-9. PMid:10884593.
http://dx.doi.org/10.1016/S0192-0561(00)...
; Umar et al., 2016UMAR, S., MUNIR, M.T., SUBHAN, S., AZAM, T., NISA, Q., KHAN, M.I., UMAR, W., REHMAN, Z., SAQIB, A.S. and SHAH, M.A., 2016. Protective and antiviral activities of Nigella sativa against avian influenza (H9N2) in turkeys. Journal of the Saudi Society of Agricultural Sciences. http://dx.doi.org/10.1016/j.jssas.2016.09.004. In press.
http://dx.doi.org/10.1016/j.jssas.2016.0...
). Nigella sativa extract prior decreases the coronavirus replication and significant reduction in coronavirus survival virus load inside cells. Recently, a am insilico study also proposed that thymoquinone (TQ) may interfere with ACE2 binding receptors, preventing virus entry.

In the drug discovery and their mechanism of action are important for better understanding the insight of the molecules. The molecular biologist desire to know that how a protein and small molecules works. An atomic level information is typically generating significant insight information of biomolecular interactions. The intermolecular interactions could be explored through the dynamic’s studies. Unfortunately, such kind of information is difficult to obtained through experimental approaches. An alternative to such approaches is computational molecular dynamics simulation (MD) of proteins and natural compounds to understand the atomic level mechanism underlined. The MD simulations are time efficient and may accurately predict how interact (Liu et al., 2018LIU, X., SHI, D., ZHOU, S., LIU, H., LIU, H. and YAO, X., 2018. Molecular dynamics simulations and novel drug discovery. Expert Opinion on Drug Discovery, vol. 13, no. 1, pp. 23-37. http://dx.doi.org/10.1080/17460441.2018.1403419. PMid:29139324.
http://dx.doi.org/10.1080/17460441.2018....
; Hollingsworth and Dror, 2018HOLLINGSWORTH, S.A. and DROR, R.O., 2018. Molecular dynamics simulation for all. Neuron, vol. 99, no. 6, pp. 1129-1143. http://dx.doi.org/10.1016/j.neuron.2018.08.011. PMid:30236283.
http://dx.doi.org/10.1016/j.neuron.2018....
). These MD studies are useful to capture a variety of biomolecular interactions, including ligand binding, protein folding, and changes in proteins behavior over time.

Knowing the strength of MD simulations and the importance of pharmacological characteristics of N. sativa, we performed the current study on thymoquinone (TQ) against Mpro to analyze the behavior of target protein at molecular level and their molecular effect on the SARS-CoV-2 target proteins.

2. Methods

2.1. Protein preparation

The worldwide biomolecular structural information is being archived at Brookhaven National Laboratories, called Protein Data Bank (PDB) (Bernstein et al., 1977BERNSTEIN, F.C., KOETZLE, T.F., WILLIAMS, G.J., MEYER JUNIOR, E.F., BRICE, M.D., RODGERS, J.R., KENNARD, O., SHIMANOUCHI, T. and TASUMI, M., 1977. The Protein Data Bank: a computer-based archival file for macromolecular structures. Journal of Molecular Biology, vol. 112, no. 3, pp. 535-542. http://dx.doi.org/10.1016/S0022-2836(77)80200-3. PMid:875032.
http://dx.doi.org/10.1016/S0022-2836(77)...
; Berman et al., 2000BERMAN, H.M., WESTBROOK, J., FENG, Z., GILLILAND, G., BHAT, T.N., WEISSIG, H., SHINDYALOV, I.N. and BOURNE, P.E., 2000. The Protein Data Bank. Nucleic Acids Research, vol. 28, no. 1, pp. 235-242. http://dx.doi.org/10.1093/nar/28.1.235. PMid:10592235.
http://dx.doi.org/10.1093/nar/28.1.235...
). The researchers around the world can easily retrieve the crystal structures of biomolecules. The crystal structure of COVID-19 Mpro (PDB ID: 6M03) was retrieved from PDB. Prior to further analysis, the structure was subjected to Preparation, using MOE (molecular operating environment) (Vilar et al., 2008VILAR, S., COZZA, G. and MORO, S., 2008. Medicinal chemistry and the Molecular Operating Environment (MOE): application of QSAR and molecular docking to drug discovery. Current Topics in Medicinal Chemistry, vol. 8, no. 18, pp. 1555-1572. http://dx.doi.org/10.2174/156802608786786624. PMid:19075767.
http://dx.doi.org/10.2174/15680260878678...
). The partial charges and missing hydrogen were assigned. The thymoquinone (CID:10281) was also prepared. Protein and ligand were docked and the complex was subjected to dynamics study.

2.2. Molecular Dynamics (MD) simulation

The molecular dynamics (MD) simulations in drug discovery capture the behavior of proteins in full molecular and atomic detail to an extent of fine temporal resolution (Liu et al., 2018LIU, X., SHI, D., ZHOU, S., LIU, H., LIU, H. and YAO, X., 2018. Molecular dynamics simulations and novel drug discovery. Expert Opinion on Drug Discovery, vol. 13, no. 1, pp. 23-37. http://dx.doi.org/10.1080/17460441.2018.1403419. PMid:29139324.
http://dx.doi.org/10.1080/17460441.2018....
; Hollingsworth and Dror, 2018HOLLINGSWORTH, S.A. and DROR, R.O., 2018. Molecular dynamics simulation for all. Neuron, vol. 99, no. 6, pp. 1129-1143. http://dx.doi.org/10.1016/j.neuron.2018.08.011. PMid:30236283.
http://dx.doi.org/10.1016/j.neuron.2018....
). The Mpro and TQ docking complex were subjected to molecular dynamics (MD) simulation as described in our previous study using Amber package (Berendsen et al., 1995BERENDSEN, H.J.C., VAN DER SPOEL, D. and VAN DRUNEN, R., 1995. GROMACS: a message-passing parallel molecular dynamics implementation. Computer Physics Communications, vol. 91, no. 1-3, pp. 43-56. http://dx.doi.org/10.1016/0010-4655(95)00042-E.
http://dx.doi.org/10.1016/0010-4655(95)0...
; Khan et al., 2020KHAN, M.T., ALI, S., ZEB, M.T., KAUSHIK, A.C., MALIK, S.I. and WEI, D.Q., 2020. Gibbs free energy calculation of mutation in PncA and RpsA associated with pyrazinamide resistance. Frontiers in Molecular Biosciences, vol. 7, p. 52. http://dx.doi.org/10.3389/fmolb.2020.00052. PMid:32328498.
http://dx.doi.org/10.3389/fmolb.2020.000...
). Briefly, MD simulation was performed on docking complex with the ff14SB force field through Amber14 package (Salomon‐Ferrer et al., 2013SALOMON‐FERRER, R., CASE, D.A. and WALKER, R.C., 2013. An overview of the Amber biomolecular simulation package. WIREs Computational Molecular Science, vol. 3, no. 2, pp. 198-210. http://dx.doi.org/10.1002/wcms.1121.
http://dx.doi.org/10.1002/wcms.1121...
; Sun et al., 2014bSUN, H., LI, Y., TIAN, S., XU, L. and HOU, T., 2014b. Assessing the performance of MM/PBSA and MM/GBSA methods. 4. Accuracies of MM/PBSA and MM/GBSA methodologies evaluated by various simulation protocols using PDBbind data set. Physical Chemistry Chemical Physics, vol. 16, no. 31, pp. 16719-16729. http://dx.doi.org/10.1039/C4CP01388C. PMid:24999761.
http://dx.doi.org/10.1039/C4CP01388C...
,aSUN, H., LI, Y., SHEN, M., TIAN, S., XU, L., PAN, P., GUAN, Y. and HOU, T., 2014a. Assessing the performance of MM/PBSA and MM/GBSA methods. 5. Improved docking performance using high solute dielectric constant MM/GBSA and MM/PBSA rescoring. Physical Chemistry Chemical Physics, vol. 16, no. 40, pp. 22035-22045. http://dx.doi.org/10.1039/C4CP03179B. PMid:25205360.
http://dx.doi.org/10.1039/C4CP03179B...
). To solvate each system the TIP3P water model was applied while system was neutralized with counterions (Jorgensen et al., 1983JORGENSEN, W.L., CHANDRASEKHAR, J., MADURA, J.D., IMPEY, R.W. and KLEIN, M.L., 1983. Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics, vol. 79, no. 2, pp. 926-935. http://dx.doi.org/10.1063/1.445869.
http://dx.doi.org/10.1063/1.445869...
). The system was energy minimized and conjugate gradient followed by heating upto 300K and 1atm pressure to equilibrate the system. Temperature regulations was achieved with the Langevin thermostat while Particle Mesh Ewald algorithm was applied for long-range electrostatic interactions (Essmann et al., 1995ESSMANN, U., PERERA, L., BERKOWITZ, M.L., DARDEN, T., LEE, H. and PEDERSEN, L.G., 1995. A smooth particle mesh Ewald method. The Journal of Chemical Physics, vol. 103, no. 19, pp. 8577-8593. http://dx.doi.org/10.1063/1.470117.
http://dx.doi.org/10.1063/1.470117...
; Darden et al., 1993DARDEN, T., YORK, D. and PEDERSEN, L., 1993. Particle mesh Ewald: an N⋅log(N) method for Ewald sums in large systems. The Journal of Chemical Physics, vol. 98, no. 12, pp. 10089-10092. http://dx.doi.org/10.1063/1.464397.
http://dx.doi.org/10.1063/1.464397...
). The MD simulation production step was carried with pmemd code 30 (Götz et al., 2012GÖTZ, A.W., WILLIAMSON, M.J., XU, D., POOLE, D., GRAND, S.L. and WALKER, R.C., 2012. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. Generalized born. Journal of Chemical Theory and Computation, vol. 8, no. 5, pp. 1542-1555. http://dx.doi.org/10.1021/ct200909j. PMid:22582031.
http://dx.doi.org/10.1021/ct200909j...
).

3. Results and Discussion

The current study shows that TQ may be effective against Mpro of SARS-CoV-2. The calculation of drug-likeness may help to understand the pharmacokinetic of a novel compound as well the pharmaceutical properties before its clinical application. The TQ drug likeness properties, calculated through Swiss ADME, is also in accordance the drug likeness rule (Walters and Murcko, 2002WALTERS, W.P. and MURCKO, M.A., 2002. Prediction of ‘drug-likeness’. Advanced Drug Delivery Reviews, vol. 54, no. 3, pp. 255-271. http://dx.doi.org/10.1016/S0169-409X(02)00003-0. PMid:11922947.
http://dx.doi.org/10.1016/S0169-409X(02)...
; Daina et al., 2017DAINA, A., MICHIELIN, O. and ZOETE, V., 2017. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific Reports, vol. 7, no. 1, p. 42717. http://dx.doi.org/10.1038/srep42717. PMid:28256516.
http://dx.doi.org/10.1038/srep42717...
). The drug likeness properties and absorption has been given in Table 1. The pharmacokinetics of TQ seems in accordance with desire drug like compound. Similarly, drug likeness properties also favor its clinical application (Brüstle et al., 2002BRÜSTLE, M., BECK, B., SCHINDLER, T., KING, W., MITCHELL, T. and CLARK, T., 2002. Descriptors, physical properties, and drug-likeness. Journal of Medicinal Chemistry, vol. 45, no. 16, pp. 3345-3355. http://dx.doi.org/10.1021/jm011027b. PMid:12139446.
http://dx.doi.org/10.1021/jm011027b...
; Vistoli et al., 2008VISTOLI, G., PEDRETTI, A. and TESTA, B., 2008. Assessing drug-likeness – what are we missing? Drug Discovery Today, vol. 13, no. 7-8, pp. 285-294. http://dx.doi.org/10.1016/j.drudis.2007.11.007. PMid:18405840.
http://dx.doi.org/10.1016/j.drudis.2007....
; Ursu et al., 2011URSU, O., RAYAN, A., GOLDBLUM, A. and OPREA, T.I., 2011. Understanding drug-likeness. WIREs Computational Molecular Science, vol. 1, no. 5, pp. 760-781. http://dx.doi.org/10.1002/wcms.52.
http://dx.doi.org/10.1002/wcms.52...
). The TQ docked against SARS-CoV-2 Mpro seems potent, altering the stability of protein. The RMSD of TQ and Mpro in Figure 1 shows stability. The Mpro is not stable in the whole simulation period which might be useful for effective inhibition of viral activity.

Table 1
Chemical properties of thymoquinone.
Figure 1
RMSD of TQ and Mpro during the 50ns MD simulation period. Stability seems fluctuating from 1.0612Å at 2ns and 2.94 Å at 18ns. Target stability seems unstable even at the end of simulation period.

The RMSD graph during the 50ns simulation period shows that the Mpro exhibited an unstable fluctuation. The RMSD at the start (1.06Å) is rising to 2.9Å at 18ns simulations. A downfall fall in fluctuation was again observed to 1.4Å at the end of 50ns MD simulation. The RMSD of Mpro seems highly unstable due to TQ which may alter the stability of target, assisting in the inhibition of viral proteins. Flexibility is also one of the important thermodynamic properties, maintaining the optimal functions of proteins (Nagasundaram et al., 2015NAGASUNDARAM, N., ZHU, H., LIU, J., KARTHICK, V., C, G.P.D., CHAKRABORTY, C. and CHEN, L., 2015. Analysing the effect of mutation on protein function and discovering potential inhibitors of CDK4: molecular modelling and dynamics studies. PLoS One, vol. 10, no. 8, p. e0133969. http://dx.doi.org/10.1371/journal.pone.0133969. PMid:26252490.
http://dx.doi.org/10.1371/journal.pone.0...
). A large change in this property may alter the biomolecules optimal function. The TQ may cause an increase in the flexibility of Mpro (Figure 2) which might be useful for better management of SARS-CoV-2 infections. The Mpro exhibited the RMSF among 5Å and 25Å at residues position 48 and 310 respectively. Residues at location 145 to 160 also attained a high RMSF (20.4Å) which contains the active site residue Cys145. MD simulations may explore the insight mechanisms of changes at the molecular level (Liu and Yao, 2010LIU, H. and YAO, X., 2010. Molecular basis of the interaction for an essential subunit PA-PB1 in influenza virus RNA polymerase: insights from molecular dynamics simulation and free energy calculation. Molecular Pharmaceutics, vol. 7, no. 1, pp. 75-85. http://dx.doi.org/10.1021/mp900131p. PMid:19883112.
http://dx.doi.org/10.1021/mp900131p...
; Liu et al., 2018LIU, X., SHI, D., ZHOU, S., LIU, H., LIU, H. and YAO, X., 2018. Molecular dynamics simulations and novel drug discovery. Expert Opinion on Drug Discovery, vol. 13, no. 1, pp. 23-37. http://dx.doi.org/10.1080/17460441.2018.1403419. PMid:29139324.
http://dx.doi.org/10.1080/17460441.2018....
; He et al., 2018HE, M., LI, W., ZHENG, Q. and ZHANG, H., 2018. A molecular dynamics investigation into the mechanisms of alectinib resistance of three ALK mutants. Journal of Cellular Biochemistry, vol. 119, no. 7, pp. 5332-5342. http://dx.doi.org/10.1002/jcb.26666. PMid:29323742.
http://dx.doi.org/10.1002/jcb.26666...
) which might be difficult through experimental work. Several studies reported that any change in protein function might be due the change in RMSF (Berhanu and Masunov, 2011BERHANU, W.M. and MASUNOV, A.E., 2011. Molecular dynamic simulation of wild type and mutants of the polymorphic amyloid NNQNTF segments of elk prion: structural stability and thermodynamic of association. Biopolymers, vol. 95, no. 9, pp. 573-590. http://dx.doi.org/10.1002/bip.21611. PMid:21384336.
http://dx.doi.org/10.1002/bip.21611...
; Chong et al., 2011CHONG, S.-H., LEE, C., KANG, G., PARK, M. and HAM, S., 2011. Structural and thermodynamic investigations on the aggregation and folding of acylphosphatase by molecular dynamics simulations and solvation free energy analysis. Journal of the American Chemical Society, vol. 133, no. 18, pp. 7075-7083. http://dx.doi.org/10.1021/ja1116233. PMid:21500781.
http://dx.doi.org/10.1021/ja1116233...
; Bavi et al., 2016BAVI, R., KUMAR, R., CHOI, L. and LEE, K.W., 2016. Exploration of novel inhibitors for Bruton’s tyrosine kinase by 3D QSAR modeling and molecular dynamics simulation. PLoS One, vol. 11, no. 1, p. e0147190. http://dx.doi.org/10.1371/journal.pone.0147190. PMid:26784025.
http://dx.doi.org/10.1371/journal.pone.0...
).

Figure 2
Residue’s flexibility of TQ and Mpro complex during simulation. Flexibility is very high in last amino acid residues. This high flexibility may change the protein function, required for virus activity.

The degree of folding stability could be measured through Rg. Fluctuations in Rg with respect to time shows unstable folding while a straight value reveals stability in folding (Lobanov et al., 2008LOBANOV, M.Y., BOGATYREVA, N.S. and GALZITSKAYA, O.V., 2008. Radius of gyration as an indicator of protein structure compactness. Molecular Biology, vol. 42, pp. 623-628. http://dx.doi.org/10.1134/S0026893308040195. PMid:18856071.
http://dx.doi.org/10.1134/S0026893308040...
; Smilgies and Folta-Stogniew, 2015SMILGIES, D.-M. and FOLTA-STOGNIEW, E., 2015. Molecular weight–gyration radius relation of globular proteins: a comparison of light scattering, small-angle X-ray scattering and structure-based data. Journal of Applied Crystallography, vol. 48, pp. 1604-1606. http://dx.doi.org/10.1107/S1600576715015551. PMid:26500468.
http://dx.doi.org/10.1107/S1600576715015...
; Khan et al., 2019,KHAN, M.T., KHAN, A., REHMAN, A.U., WANG, Y., AKHTAR, K., MALIK, S.I. and WEI, D.Q., 2019. Structural and free energy landscape of novel mutations in ribosomal protein S1 (rpsA) associated with pyrazinamide resistance. Scientific Reports, vol. 9, no. 1, p. 7482. http://dx.doi.org/10.1038/s41598-019-44013-9. PMid:31097767.
http://dx.doi.org/10.1038/s41598-019-440...
2021). A protein with misfolding shows variations in Rg over time (Figure 3). The plot shows large variations between 22Å and 22.8Å. Majority of the variations have been found from 811ps to 2026ps. The lowest Rg was detected at 811ps (22Å) while the highest at 1216ps (22.8Å). This shows that the TQ may affect the folding of Mpro which might be important to inhibit the protein activity. The Rg plot of Mpro is not stable during the simulation peried which shows the potential activity of TQ. Change in protein stability may be due to the alteration of thermodynamic property (Chen and Shen, 2009CHEN, J. and SHEN, B., 2009. Computational analysis of amino acid mutation: a proteome wide perspective. Current Proteomics, vol. 6, no. 4, pp. 228-234. http://dx.doi.org/10.2174/157016409789973734.
http://dx.doi.org/10.2174/15701640978997...
). This includes protein RMSD, fluctuations and also the protein folding. Destabilization in folding and thermodynamic stability may affect biomolecules function.

Figure 3
Radius of gyration TQ and SARS-CoV-2 Mpro complex. A constant Rg value is a measure of correct folding. Fluctuations in Rg shows that protein folding is not stable.

The TQ is fitting in the pocket, interacting with active site (C145, H41) (Figure 4), altering the catalytic activity of viral protein. The residues located in the binding pocket and its surrounding (T24, L27, H41, F140, C145, H163, M165, P168, and His172) are imported for a natural compound to interact with. The phytocompound TQ form a hydrogen bond with Glu166, facilitating the pocket shape of the substrate-binding site (Anand et al., 2002ANAND, K., PALM, G.J., MESTERS, J.R., SIDDELL, S.G., ZIEBUHR, J. and HILGENFELD, R., 2002. Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain. The EMBO Journal, vol. 21, no. 13, pp. 3213-3224. http://dx.doi.org/10.1093/emboj/cdf327. PMid:12093723.
http://dx.doi.org/10.1093/emboj/cdf327...
) and many hydrophobic interactions with active site (His41, Cys145) and its surrounding residues (Figure 5).

Figure 4
Thymoquinone and SARS-CoV-2 main protease interaction. (A) Docked thymoquinone. (B) Thymoquinone in binding Pocket. (C) Residues in the surrounding thymoquinone.
Figure 5
Mpro of SARS-CoV-2. (A) Domain organization. Active site residues have been shown. (B) Dimerization of two Mpro monomers and location of E166. (C) Impact of E166A mutation on the dynamics of Mpro. The mutant gain flexibility and show destabilizing effect (Rodrigues et al., 2018RODRIGUES, C.H., PIRES, D.E. and ASCHER, D.B., 2018. DynaMut: predicting the impact of mutations on protein conformation, flexibility and stability. Nucleic Acids Research, vol. 46, no. W1, pp. W350-W355. http://dx.doi.org/10.1093/nar/gky300. PMid:29718330.
http://dx.doi.org/10.1093/nar/gky300...
).

4. Conclusion

TQ shows good binding affinity with SARS-CoV-2 NSP5, interacting with active site residues and also with Glue166, maintaining the pocket shape for viral enzymatic activity. This phytomedicine alters the overall thermodynamics properties of SARS-CoV-2 Mpro which may useful for better management of COVID-19 in future. Further experimental validation is required to observe the TQ effect in vivo. The TQ may be used as therapeutic compound against SARS-CoV-2 after experimental confirmation.

Abbreviations

AIV: Avian influenza virus

COVID-19: Coronavirus disease-19

MOE: Molecular operating environment

Mpro: Main protease

MD: Molecular dynamics

NSP5: Non-structural protein 5

NPT: Number of moles, pressure, temperature

NVT: Number of moles, volume, temperature

RMSD: Root means square deviation

RMSF: Root means square fluctuation

Rg: Radius of gyration

SARS-CoV-2: Severe acute respiratory syndrome 2

TQ: Thymoquinone

PDB: Protein data bank

SPC: Simple point charge

Acknowledgements

The authors are thankful to the Institute of Research and Consulting Studies at King Khalid University for funding this research through grant number 2-N-20/22 and the support of the Research Center for Advanced Materials Science is highly acknowledged. Dong-Qing Wei is supported by grants from the National Science Foundation of China (Grant No. 32070662, 61832019, 32030063) the Key Research Area Grant 2016YFA0501703 of the Ministry of Science and Technology of China, the Science and Technology Commission of Shanghai Municipality (Grant No.: 19430750600), as well as SJTU JiRLMDS Joint Research Fund and Joint Research Funds for Medical and Engineering and Scientific Research at Shanghai Jiao Tong University (YG2021ZD02). The computations were partially performed at the Pengcheng Lab. and the Center for High-Performance Computing, Shanghai Jiao Tong University.

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Publication Dates

  • Publication in this collection
    15 Apr 2022
  • Date of issue
    2024

History

  • Received
    04 Apr 2021
  • Accepted
    24 Jan 2022
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