Active-site Molecular docking of Nigellidine to nucleocapsid/Nsp2/Nsp3/M Pro of COVID-19 and to human IL1R and TNFR1/2 may stop viral-growth/cytokine-ood, and the drug source Nigella sativa (black cumin) seeds show potent antioxidant role in experimental rats.

The recent outbreak of SARS CoV-2 has changed the global scenario of human lives and economy. In this pandemic-outbreak the ratio of infected person is much higher than the death encountered. Most of the dead patients were observed with dysfunction/failure of cardiac and renal systems. Beside this a ‘cytokine storm’ namely TNF-α/IL1 receptors i.e. TNFR1/TNFR2/IL1R over-functioning was reported in the infected-persons. Here, nigellidine, an indazole-alkaloid and key-component of Nigella Sativa L. (NS); black-cumin-seed, has been analyzed for COVID-19 different protein and TNFα receptors TNFR1/TNFR2 and IL1R inhibition through molecular-docking study and biochemical-study of cumin-seed extract exposure to experimental-rat. The NMR, X-ray-crystallographic or Electron-microscopic structures of COVID-19 Main-protease(6LU7), Spike-glycoprotein(6vsb), NSP2(QHD43415_2), N-terminus-protenase (QHD43415_3), Nucleocapsid(QHD43423) and Human IL1R (1itb), TNFR1 (1ncf), TNFR2 (3alq) from PDB were retrieved/analyzed for receptor-ligand interaction in normal condition. Then those structures were docked with nigellidine using Autodock-software and Patchdock-server. Where nigellidine showed highest binding-energy of -7.61 (kcal/mol) and ligand-eciency value of (-0.35) forming bonds with amino acids THR943/LYS945/MET1556/ALA1557/PRO1558/ILE1559. Highest ACE-value of -356.72 was also observed for nigellidine N-terminal-protease interaction. Nigellidine also showed strong interaction with NSP2 (-6.28) and Mpro/3CLpro_Q (-6.38s). Nigellidine showed anity to TNFR1 (-6.81), IL1R (-6.23) and TNFR2 (-5.16). In rat experiment 2-groups (vehicle and NS treated) of female Wistar-rats were taken for experiments. The NS treated tissue showed marked decline in ALP/SGPT/ SGOT/MDA level then the basal-levels. From the Western-blot or activity analysis it was observed that Nigellidine, the sulfuryl-group containing drug showed no impact on Phenol-catalyzing ASTIV or Steroid-catalyzing EST expressions/activities and thus have no inuence in sulfation-mediated adverse metabolic-processes. Current-results concluded that Nigellidine has hepato/reno-protective; immunomodulatory/anti-inammatory and antioxidant activities as well as it inhibits important proteins of COVID-19. With steps to further validation/checking nigellidine can be used in COVID-19 infection.


Introduction
The pandemic outbreak from SARS CoV 2 has claimed a large number of lives from millions of infections globally. A horri ed and lockdown situation has generated a post infection and post remedial traumatized situation that is creating a long term health hazards also. When the number of deaths alone is counted it is found to be no doubt huge but when it is calculated with respect to the number of infections it is found approximately one of twentieth. The question arise how these nineteen people get escape or what type of physiological measures they possess. In other words, what condition makes that one unfortunate person to be more morbid? Is it not the point that co-morbid status might have increased the risk of death? When it is noticed from the statistics that elder persons and males are more affected then it can be clari ed that these two factors can increase the risk. One of the adverse physiological conditions in the elder persons is their weaker immunological status than the younger one. Nevertheless, over ow of the in ammatory Page 3/21 responses make the situations more unfavorable in these patients. In this conditions speci c new drug targeting, drug repurposing for the speci c proteins on the CoV 2 structure and vaccination strategies are the demand of the time. Our laboratory is screening from a large number of drug target for the purpose.
Some traditional molecules as phytochemical sources have been tested here and found that nigellidine from Nigella Sativa L.; black cumin seed may be of great importance. It (Ranunculaceae) is an annual herb extensively used in the Middle-East, India and other countries. Its traditional use is reported in ancient texts and historical documents [1]. The rst indazole alkaloids nigellidine and nigellicine which can remain in sulfated-form are accumulated in this seed coat B.
Sulfated form increases its polarity and bioavailability. Nigellicine, nigellidine, thymoquinone, dithymoquinone from this extract has shown strong as anti-oxidant, anti-bacterial, anti-hypertensive, antiinflammatory and immunomodulatory effects [2,3].Toxicity studies from our lab and other have suggested the safety of N. sativa extract in different form i.e. fat soluble or water soluble [4].
Antioxidant role of NS extract has been shown by lower serum levels of MDA in the experimental rats suggest that it can be used in oxidative stress condition. No signi cant impact was found on TNF-α, IL-10 and hs-CRP levels of the exposed group [5]. But the eventual in ammatory signaling has not been tested here. Or their receptor binding capacity has not been screened. Nigellidine has been shown to be immunomodulatory and anti-in ammatory. Beside immunological protection, NS serves as metabolic protector.
Administration of Nigella sativa might improve lipid and blood sugar pro le in postmenopausal women with different types of metabolic syndrome [6,7], and it also act as hepato-and reno-protective compound [8]. NS seed proteins has potential as therapeutic agent for cancer [9]. Most importantly, in SARS CoV-2 infection ACE-2 mediated impairment of aldosterone system may be repaired by NS. Vasorelaxant and anti-hypertensive function of NS helps in the modulation of renin angiotensin system (RAS) or the diuretic activity [10], which is one of the major targets of COVID. It might have great protective role during post infective secondary disorder of the peripheral vasculature namely cardiac and renal systems. In most of the instances patients die due to this organ dysfunction/failure in COVID-19 infection.
Nigella sativa was shown to signi cantly improve laboratory parameters of hyperglycemia and diabetes control, glycated hemoglobin, and insulin resistance, and a rise in serum insulin [7].
In this background, present study tested the possible active site docking of nigellidine to several important SARS CoV 2 proteins and the human in ammatory molecules which are reported to create a 'cytokine storm' namely TNF-α receptors i.e. TNFR1, TNFR2 and IL1R. In the experimental rat model the source of this drug Nigella sativa; black cumin seed extracts were tested for its role on antioxidant, hepatic and renal status. This work will help in the urgent therapeutic intervention against COVID-19 global pandemic.

Preparation of both Receptor and Ligand Molecules
Receptor molecules both from human coronavirus and human receptor and other molecules were found with numerous water molecules, ions and in some their respective ligands. The water and ions were removed and rest of the structures was saved in .pdb format. For ligand bound receptors initially the interaction patterns were analyzed using Pymol molecular graphics visualize and documented. Then the ligand molecules were removed and receptor molecules were prepared for molecular docking with nigellidine and the modi ed les were saved in .pdb format. The .sdf format of chemical ligand was converted to .pdb also using Pymol.

Molecular Docking
The molecular docking of nigellidine with all the selected proteins was performed through AutoDock 4.2 [11] o ine software and Patchdock online server for interactive docking. Autodock calculates the global minimum energy in the interaction between the substrate and the target protein, calculating all the degrees of freedom (DOF) available for the system. During docking parameter le generation, all nonpolar hydrogens were removed except polar and charged one. The receptors were added with Kolman Charges and ligand was calculated for Gasteiger charges and saved in PDBQT format then these formats were used for molecular docking. PatchDock is developed as geometry-based molecular docking algorithm. It calculates the docking transformation between two molecules to get the best molecular interface complementarity which nds out the ligand posture in receptor with maximum interface area covered and minimum steric hindrance [12]. It also calculates the Atomic Contact Energy (ACE) Value of each docking positions to indicate the amount of required desolvation free energies to transfer the ligand molecule from water to protein (Receptor) interior. The docked structures were visualized and represented using Pymol and AutoDock tool. The drug-protein interactions were represented through LIGPLOT software.
Biochemical impact of black-cumin seed-extract exposure to experimental rat Female Wistar rats were purchased from a small-animal rm house (govt. registered) that follows all ethical norms and maintain requisite regulatory affairs. The rm house is a Government accredited Rats of ageing 3 to 4 weeks were acclimatized for 10 days at 12-hour light-dark cycle, 25ºC ± 2ºC temperature, 50%-70% humidity in the institutional animal resource facility. Those were fed with a standard pellet diet (Hindustan Lever Ltd, Mumbai, India) and water ad libitum. Studies were carried out in accordance with the National Institutes of Health, USA guidelines and the institutional ethical concerns were maintained throughout the investigation. Rats were randomly distributed in 2 groups (vehicle and NS treated) having 6 in each.
On the day of sacri ce, animals were experienced cervical dislocation and initially their blood was collected using a disposable syringe (21-gauge needle), serum was separated from the collected blood samples. The liver tissue was carefully collected and stored at -20ºC for experimental purposes.
Evaluation of General Toxicity.
Serum glutamate pyruvate transaminase (SGPT), SGOT, alkaline phosphatase (ALP), urea, and creatinine were measured from the rats by standard protocol with the assay kits (Ranbaxy, India or other reputed company).

Estimation of Malondialdehyde (MDA) Levelsin liver tissue
Liver tissue was homogenized (10 % w/v) in the ice-cold phosphate buffer (0.1 mol /L, pH 7.4) and the homogenate was centrifuged at 10,000 rpm at 4ºC for 10 min. The MDA assay was conducted using the supernatant following the protocol of Buege and Aust, 1978 [13,14]. To chelate iron and reduce its interference in peroxidation reaction of unsaturated fatty acid, 1 mM EDTA was used in the reaction mixture. To reduce the interference caused by a yellow-orange colour produced by some carbohydrates, the reaction mixture was heated at 80oC instead of 100ºC. Finally, the MDA was measured and calculated utilizing the molar extinction coe cient of MDA (1.56 x 105 cm2/ mmol).

Estimation of Non Protein Soluble Thiol (NPSH) in liver tissue
The NPSH in serum and liver tissue homogenates (prepared in 0.1 M phosphate buffer, pH 7.4) were determined by the standard DTNB (5, 5'-dithiobis-2-nitrobenzoic acid) method with a slight modi cation [26]. In brief, the protein was precipitated by trichloroacetic acid and clear cytosol was added to 0.1 M sodium phosphate buffer containing 5 µM DTNB. The level of NPSH was determined against a GSH standard curve [15].
ASTIV activity (PNPS assay method) in liver tissue β-Naphthol sulfation activity from liver cytosols was determined as previously described [16,17]. This assay determines phenol sulfation activities of different isoforms of phenol sulfating SULTs. Brie y, sulfation activity was determined in a reaction mixture containing 50mM Tris buffer, pH 6.2, 5mM PNPS, 20 μM PAPS, and 0.1mM β-naphthol. Rat liver cytosols (50 μg protein) were used as the enzyme source in a total reaction volume of 250 μl. After 30 min incubation at 37 º C in a shaking water bath, the reaction

Results And Discussion
Viral protein used in this experiment; https://zhanglab.ccmb.med.umich.edu/COVID-19/ Nigellidine binding to N terminus protease nsp3_QHD43415_3 (Fig 1) This is known as Papain-like proteinase. It is responsible for the cleavages located at the N-terminus of the replicase polyprotein. It participates together with nsp4 in the assembly of virally-induced cytoplasmic double-membrane vesicles necessary for viral replication. Nigellidine showed highest binding energy value of -7.61 and its ligand e ciency was also high (-0.35). According to the AutoDock result the position A having binding energy -7.61, forms some unstable bonds with amino acids THR943, LYS945, MET 1556, ALA1557, PRO1558, ILE1559 (Fig  1). Binding energy range of -7.59 to -4.56 (total posture 4) were found at position B. It showed an entry/exit pocket where nigellidine binds to TRP1632 through an N-O bond and other hydrogen bond with HIS1630, TRP1632, ALA1878 and THR1774. The ACE value was also good at this site (-284.28) and nigellidine a nity was also found. At site C binding energy (-6.18 & -5.05) was also high and the highest ACE value (-356.72) was observed. Here also nigellidine formed stable bond with GLY251 (C-C & O-C) and ASN 244 (N-O) and some hydrogen bond with other mentioned amino acids. From this observation it could be concluded that a nity of nigellidine at site was very high but then it may transferred to site B and C and may hamper the normal function of N-terminal protease of coronavirus.
This protein cleaves the C-terminus of replicase polyprotein at 11 sites. Recognizes substrates containing the core sequence [ILMVF]-Q-|- [SGACN]. Also able to bind an ADP-ribose-1''-phosphate (ADRP). It is noticed from both Patchdock and Autodock results nigellide binds with the high e ciency with the important amino acids in the active sites of the enzyme which could have altered its activity and viral metabolism signi cantly. The ACE value was found to be -265. 82. Active site biding energy was found to be -6.38 and -4.19 suggesting potential impairment due to drug binding effect. One previous report suggests Mpro protease structure in COVID-19 may bind nigellide molecule at it active site [19].

Nigellidine binding to Non-structural protein 2 nsp2_QHD43415_2; Fig 3
This protein plays a role in the modulation of host cell survival signaling pathway by interacting with host PHB and PHB2, which helps to maintain the functional integrity of the mitochondria and protecting cells from various stresses. According to Angeletti et.al., 2020 [20], NSP2 protein of COVID 19 has an entry pocket (Fig 3). The spike glycoprotein interact with the angiotensin convertage enzyme 2 (ACE 2) receptor present at human cell surface and initiate the viral entry into the host cell. No satisfactory attachment of nigellidine was observed at the active site. The highest binding energy value was -6.11, whereas, presence of nigellidien at the central core was observed with -4.77 binding energy. The ligand e ciency ranged from -0.28 to -0.22. Though the highest ACE value was -340.50 and strong a nity was observed .
Human receptors, in ammatory signal molecules and other proteins Nigellidine binding to IL1R, Fig 5 The highest binding energy for IL1R and nigellidine was -6.23 but that location was not at the active site. Although the ACE value of TNFR2 -Nigellidine binding was moderate and the highest value was -258.71. But, the normal function could be hampered if nigellidine concentration will increase. The highest binding energy value for this interaction was -5.16 and the ligand e ciency value was ranged from -0.23 -0.19.

Results on rat experimental rat model
Present results suggest in the gure 8 that ALP, SGPT and SGOT and MDA remarkably lower in the NS treated tissues compared to that of their basal level. Serum urea and creatinine levels were also found to decline from that of vehicle treated group (data not shown). Nigellidine is the rst indazole alkaloid which has been found with a sulfuryl group. The sulfated compound is possibly favorable for higher rate of solubility and increased bioavailability. So, this compound might have role in the induction of sulfotranseferase; SULTs that catalyzes sulfuryl group transfer. These enzymes may modulate phase II drug metabolism, drug-drug interactions. In the current study, we found no impact of nigellidine on Phenol catalyzing ASTIV or Steroid catalyzing EST expressions/activities. So this drug does not in uence at least sulfation mediated adverse metabolic processes. This is demonstrated in the gure 8.
Our present nding has two parts. In the molecular docking strategies with both PatchDock and Autodock were run to verify the binding of nigellidine and its locations on different proteins of SARS CoV 2. It is noticed that the drug very effectively binds with the N terminus protease, nucleocapsid and Main protease which are absolutely important for viral maturations in proteins structures, RNA packaging and other functions. Another point is that during post infection period this virus has been reported to greatly impair the human immune system by the extravagant activities of different cytokines namely TNFα, IL1, IL6 and others. These signaling molecules perform after binding with their speci c receptors like TNFR1/2, IL1R and IL6R respectively. So blocking of these cytokines or their receptors may help to break the cascade of cytokine signaling. This could be one of the steps to decrease the severity of the SARS CoV 2 infection. Other than nigellidine there are several compounds like thymoquinone groups of drugs, α-hederin and these compounds are reported to have signi cant therapeutic activities against different types of pathogen infections. The docking results revealed promising inhibitory potential of thymoquinone against Cag A and Vac A, H. pylori oncoproteins with comparison to the standard drug, metronidazole [22].
Complex glycan molecules are the frequent components on the viral and bacterial surfaces; sometimes lipopolysaccharides or glycated proteins generate signi cant and unwanted immunological reactions which instead potentiating, exhaust the host immune system. As for example, in the present case, SARS CoV 2 spike which is covered with a large number of NAG disfavors the proper presentation of the epitopic part at the time of MHC presentations. This glycan molecule also restricts proper drug targeting to the spike proteins. This has been demonstrated in our previous ndings on some suitable epitopescreening from SARS CoV 2 spike [23]. Our earlier study also demonstrated that Epigallocatechin gallate (EGCG) and Thea avin gallate (TDG) are the potent binder to the CoV 2 spike channel [24]. Increased cytokines and other hematopoetic parameters (oxidative stress markers) by LPS-administration have been shown to terminate by Nigella sativa (NS) extract [25]. Cardio-respiratory and endothelial dysfunction is one of the major symptoms of SARS CoV 2 infection. And this dysfunction may be counteracted by NS and its component thymoquinone by restraining interleukin-1β, TNF-α and NF-κB signaling [26]. Promising anxiolytic and anti-in ammatory activities of NS has been demonstrated [27]. Hypertensive renin-angiotensin system is an obvious co-morbid condition in the elderly persons and that has been targeted in the SARS CoV 2 infection. Nevertheless, hypertension associated diabetic immunesuppressive status is the major target of this viral infection. So drug prohibition of these secondary disorders is of great importance in decreasing mortality rate. The NS component, nigellidine has been decisively shown to bind to the active sites of the IL1 and TNF-α receptors ( g 6 and 7). The immune response and pathogenicity of the H9N2 avian in uenza virus has been restricted by the Nigella sativa [28]. The use of N. sativa seed xed oil can inhibit the in ammation of sinuses and respiratory airways, microbial infections, such as coryza, nasal congestion [29]. Report revealed from some prospective study that, killed and re-assorted in uenza virus supplemented with natural adjuvant like Nigella sativa has been more responsive than NS alone to generate IgG and IgM responses via augmented CD4+-and CD8+ signaling [30]. A signi cant decline in the number of peripheral lymphocyte counts, mainly CD4 T and CD8 T cells in COVID-19 patients is shown to relate disease severity and further opportunistic infection. Several peripheral; tissues like spleen, lymph nodes, and lymphoid tissue and T lymphocytes have been shown to carry COVID-19 RNA. It is suggested that enhanced CD4+ mediated responses generated by NS in the decrease of HIV-RNA load in the patients. [31] In the current study, we have decisively shown by molecular modeling that nigellidine can bind in the active sites of several important proteins of SARS CoV 2, several host receptors speci c for SARS CoV 2 induced in ammatory markers IL1, IL6, TNF-α. Moreover, the extract from black cumin seed has been shown in experimental rat to be highly antioxidative, hepato-and reno-protective. Further studies are necessary to verify the potential effects of nigellidine in in vivo laboratory experimental animal model.  Interaction of nigellidine with IL1R. Common TYR127 and TYR261 are involved to both interactions IL1 and the drug nigellidine.

Figure 6
Interaction of nigellidine with TNFR1. Site A and B representing the amino acid interactions within TNFR1 and also nigellidine binding at the same site.

Figure 8
Effects of NS treatment on hepatic functions and antioxidant status in experimental rat model. Drug metabolizing enzymes ASTIV and EST/SULT1E1 activity/expression has been checked in the rat liver in response to NS extract exposure.