Identification of Compounds from Nigella Sativa as New Potential Inhibitors of 2019 Novel Coronasvirus (Covid-19): Molecular Docking Study.

The spread of the global COVID-19 pandemic, the lack of specific treatment and the urgent situation requires use of all resources to remedy this scourge. In the present study, using molecular docking, we identify new probable inhibitors of COVID-19 by molecules from Nigella sativa L , which is highly reputed healing herb in North African societies and both Islamic and Christian traditions. The discovery of the M pro protease structure in COVID-19 provides a great opportunity to identify potential drug candidates for treatment. Focusing on the main proteases in CoVs (3CL pro /M pro ) (PDB ID 6LU7 and 2GTB); docking of compounds from Nigella Sativa and drugs under clinical test was performed using Molecular Operating Environment software (MOE). Nigelledine docked into 6LU7 active site gives energy complex about -6.29734373 Kcal/mol which is close to the energy score given by chloroquine (-6.2930522 Kcal/mol) and better than energy score given by hydroxychloroquine (-5.57386112 Kcal/mol) and favipiravir (-4.23310471 kcal/mol). Docking into 2GTB active site showed that α- Hederin gives energy score about-6.50204802 kcal/mol whcih is better energy score given by chloroquine (-6.20844936 kcal/mol), hydroxychloroquine (-5.51465893 kcal/mol)) and favipiravir (-4.12183571kcal/mol). Nigellidine and α- Hederin appeared to have the best potential to act as COVID-19 treatment. Further, researches are necessary to testify medicinal use of identified and to encourage preventive use of Nigella Sativa against coronavirus infection.


Introduction
During December 2019 a novel coronavirus  has been reported from Hubei province in China i . The virus associated with human to human transmission is causing several human infections and disorder not only in the respiratory apparatus but also in the digestive tract and systemically ii iii iv . On March 11, 2020, world health organization characterizes COVID-19 as a pandemic which caused until 30, March, 2020 30,105 death and 638,146 confirmed cases over the world v . Due to gravity of the situation, urgent and complementary efforts from researchers are necessary to find therapeutic agents and new preventive methods. Description of COVID-19 virus shown three important proteins know as papain-like protease (PL pro ), 3C-like protease (3CL pro ) and spike protein to be attractive target for drug development vi . Viral polypeptide onto functional proteins is processed by Coronavirus PL pro which is also a deubiquitinating enzyme that can dampen host anti-viral response by hijacking the ubiquitin (Ub) system vii viii . It has been shown also that SARS-3CL pro is a cysteine protease indispensable to the viral life cycle ix . Angiotensinconverting enzyme 2 (ACE2) is used by Coronavirus spike protein as a receptor to help the virus enter cells x .The potential target (M pro )/chymotrypsin-like protease (3CL pro ) from COVID-19 (6LU7) have been successfully crystallized by Liu et al (2020) and repositioned in Protein Data bank (PDB) xi . Medicinal chemists are focusing also on the main protease of SARS-Coronavirus (2GTB) to develop antiviral treatments of the virus causing COVID-19 xii because it shares 96 % similarity xiii . Some in silico preliminary studies have been conducted to find small molecules from herbal plants with the potential to inhibit 2019 novel coronavirus xiv xv xvi .
Contagious disease treatment and control is widely demonstrated by effectiveness of medicinal herbs xviixviii xix xx xxi . Absence of specific therapy for COVID-19 leads population over many regions in the world to use medicinal herbs knows in ethnophamacologie as antiviral. In our present study and inspired by recent molecular docking studies xxii xxiii we illustrate interactions between small molecules from North African medicinal herb; Nigella sativa L in order to identify the favorable molecules for COVID-19 treatment and compare them to proposed drugs such as chloroquine hydroxychloroquine,azithromycin, arbidol, remdesivir, and favipiravir xxiv xxv . The in silico study was done using Molecular Operating Environment software (MOE) xxvi . The present study will provide other researchers with important investigation way to identify new COVID-19 treatment and use of natural products.

Medicinal herb choice
Based on local survey we reported that Nigella sativa L. commonly known as black seed or black cumin (Haba sawda) is widely recommended in society during the COVID-19 crisis for their probable antiviral effects. The large traditional use of black cumin as panacea (universal healer) in North African societies came from Islamic belief and also Bible xxvii . Nigella sativa is cited by many research papers for its multiple benefits as antiviral, anti-inflammatory, anti-cancer, analgesic…etc xxviii xxix xxx xxxi .

Preparation of both enzymes and ligands
Download of 3cl pro /M pro COVID-19 and 3cl pro /M pro SARS-coronavirus three dimensional structures were done from Protein Data Bank xxxii under PDB ID 6LU7 and 2GTB respectively xxxiii xxxiv . Crystallographic properties of 6LU7 and 2GTB are reported in table 1. Table 2 reports major chemical compounds of Nigella sativa L collected from literature xxxv xxxvi xxxvii xxxviii xxxix xl . The 3-dimensional (3D) structures of main chemical compounds from Nigella sativa were downloaded in .sdf format from PubChem xli . Lipinski's physicochemical parameters rule xlii xliii xliv were also studied for each ligand and reported in table 3. Chemical structures of main drugs under clinical tests for treatment of COVID-19 are reported in table 4 xlv xlvi xlvii . Identification of the preferred region of the receptor that interacts with ligands is known by active site prediction and isolation protocol xlviii . Using Hamiltonian AM1 (Austin model 1) implanted in MOE and field strengths in the MMFF94x (Merck molecular force field) energy of the protein was minimized. In addition, water molecules were removed from the protein surface so that the interaction region will not be hidden while docking. By use of site-finder module implanted in MOE, active sites of 6LU7 and 2GTB were identified and shown in figure 1 and 2 respectively. Also both natural ligands (compounds from Nigella sativa L ) and proposed drugs were submitted to energy minimizing under default conditions of temperature = 300°K and pH = 7.

Docking and Building Complexes
Docking using Dock module implanted in MOE, consists of positioning ligands into active site of 6LU7 and 2GTB with most of default tools to predict how molecules interacts with the binding site of the receptor xlix l li . First docked molecules series were proposed drugs and respective reference inhibitors (PRD_002214 of 6LU7 and AZP for 2GTB) in order to compare obtained score with score from chosen ligands of Nigella sativa L. Table 5 gives obtained scores by drugs under clinical test and inhibitor ligands (PRD_002214 and AZP). Table 6 shows scores of second docked ligand series from compounds from Nigella Sativa.

Results and discussion
Obtained results showed that Nigellidine gives the lowest energy (-6.29734373 Kcal/mol) in complex with 6LU7, which is the best score when compared to other docked compounds. Nigellidine gives score close to the one given by chloroquine (-6.2930522 Kcal/mol) and better score than hydroxychloroquine (-5.57386112 Kcal/mol) and favipiravir (-4.23310471 kcal/mol). Nigellidine in complex with 6LU7 ( Figure  3A and 3B) shows two hydrogen possible interactions with amino acid MET49 (H-donor) with a distance about 4.25Å and energy of -0.7Kcal/mol and π-H interaction with amino acid THR190 with a distance about 4.24Å and energy of -1.3Kcal/mol. Interactions between the rest of compounds from Nigella sativa and 6LU7 are reported in table 7. Docking results with 2GTB show that α-Hederin gives better score (-6.50204802 kcal/mol) than chloroquine (-6.20844936 kcal/mol), hydroxychloroquine (-5.51465893 kcal/mol)) and favipiravir (-4.12183571kcal/mol). Alpha-hedrin in complex with 2GTB (figure 4A and 4B) show that only one hydrogen interaction (H-acceptor) with amino acid Gly 143 is possible with distance about 2.92 Å and energy of -2.2 Kcal/mol.. Interactions between the rest of compounds from Nigella sativa and 2GTB are reported in table 9.

Nigellimine
Only one hydrogen interaction (π-π) is possible with amino acid HIS 41 with distance about 3.95 Å.

Carvacrol
There are non-perceptible interactions, only electrostatics (Van der Waals) interactions are perceptible.

Thymol
There are non-perceptible interactions, only electrostatics (Van der Waals) interactions are perceptible.

Thymoquinone
There are non-perceptible interactions, only electrostatics (Van der Waals) interactions are perceptible.

Dithymoquinone
There are non-perceptible interactions, only electrostatics (Van der Waals) interactions are perceptible.

Thymohydroquinone
Only one hydrogen interaction (Hacceptor) is possible with amino acid GLY143 with distance about 3.20 Å and energy of -0.7 Kcal/mol.

Conclusion
The aim of the present study is to identify molecules from natural products which may inhibit COVID-19 by acting on the main protease (M pro ). Obtained results by molecular docking showed that Nigellidine and αhederin are main compounds from Nigella sativa which may inhibit COVID-19 giving the same or better energy score compared to drugs under clinical tests. Those results encourage further in vitro and in vivo investigations and also encourage traditional use of Nigella sativa preventively.