phytochemicals, and acid (DHA) as a Novel Inhibitors of IL-6 and ACE2 receptors, a Therapeutic Strategy for targeting COVID-19 Cell Entry and Cytokine Storm. An insilico Approach

Natural Phenformin, Docosahexaenoic ABSTRACT Cytokine storm syndrome (CSS) is a life-threatening consequence of inflammatory immunological illnesses; it can also occur with COVID-19 infection . CSS is characterized by a disruption in cytokine synthesis, including regulatory , pro-inflammatory and anti-inflammatory cytokines, resulting in pathologic stimulation of innate in addition to adaptive (Th17 and Th1 mediated) response. In the pathophysiology of CSS, interleukin-6 could play a key role. The significant role of IL-6 in COVID-19 pathogenesis was established in a wide variety of researches , which reported that the plasma concentration of IL-6 was raised in COVID-19 patients with severe symptoms . COVID-19 spike protein binding to angiotensin-converting enzyme 2 (ACE2), the virus's cellular receptor, causes a cascade of molecular processes that could result in hyperinflammation which may lead to cytokine storm . Therefore, the development of new natural therapies and repurposing some drugs such as Phenformin and Docosahexaenoic acid that could compete with COVID-19 for ACE2 binding or inhibit IL-6 activity may possibly help COVID-19 patients avoid a cytokine storm and save their lives through inhibiting IL-6 and preventing SARS-CoV-2 RBD attachment to

ACE2. Herein we made a docking based screening for some natural phytochemicals and drugs that could be repurposed according to our findings to counter COVID-19 cell entry and inhibit the hyper activation of IL-6. Our results revealed that a five phytochemicals including Epigallocatechin gallate (EGCG) , bromelain , luteolin , vitexin and isovitexin) showed a high binding affinities with best interactions with the active sites of IL-6 . The binding affinities of these phytochemicals including, EGCG , bromelain , luteolin , vitexin and isovitexin with IL-6 were (-7.7, -6.7, -7.4, -7.2 and -7.3 ), respectively. In addition to , phenformin showed a high binding affinity with best interactions with the active sites of IL-6 and ACE2 . The binding affinity of phenformin with IL-6 was (-7.4) and with ACE 2 ( -7.2). Docosahexaenoic acid (DHA) had a moderate binding affinity and moderate interactions with the active sites of IL-6 and had a high binding affinity with best interactions with ACE2 active sites. The binding affinity of Docosahexaenoic acid(DHA) with IL-6 was (-5.3) and with AC2 (-6.3).

Conclusion
Proposing possible IL-6 inhibitors with less adverse effects has been suggested as a way to aid COVID-19 patients who are suffering from severe cytokine storms. This study has been designed to elucidate the potential of potent antiviral phytocompounds as well as phenformin and Docosahexaenoic acid (DHA) as a potent ACE2 and IL-6 inhibitors. The compounds interact with different active sites of IL6 and ACE2 which are involved in direct or indirect contacts with the ACE2 and IL-6 receptors which might act as potential blockers of functional ACE2 and IL-6 receptor complex. It worth mentioning that phenformin which showed high binding affinity with both

Introduction
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) an enveloped RNA virus that causes the recent pandemic of COVID-19 principally affects the human respiratory system but can also affect the nervous system , urogenital system, circulatory system and could also COVID-19 infection has been linked to an increased response of the immune system in some covid-19 patients, which is governed by an excessive release of circulating cytokines known as cytokine release syndrome (CRS). 7 One of the key causes of COVID-19 patients' significant deterioration, which leads to multiorgan failure, has been identified as cytokine release syndrome. 8 Increased levels of interleukin (IL)-6, interferon (IFN)-, and tumour necrosis factor (TNF)-, are the most common features of a cytokine storm, which is known as a "cytokine storm." 9 IL-6 is a kind of interleukin that is produced by the Significant proinflammatory qualities play a critical role in ARDS, systemic inflammation, pneumonia associated with respiratory failure 10 Interleukin-6 levels have been associated to the severity of COVID-19 infection and have been found to be high in individuals with respiratory dysfunction.. 11 The quantity of interleukin-6 is linked to a higher risk of death, more than three times higher in individuals with complex COVID-19 than in those with simple disease. 12 As a result, IL-6 blocking drugs, as well as techniques aimed at decreasing this cytokine, have been successfully used in the treatment of persons with hyper-inflammatory conditions.. 13 In addition, we suggest that medication that could compete with covid-19 for ACE2 may prevent COVID-19 entry and hyper inflammation and cytokine storm cascade . According to our findings many drugs such as, phenformin and active ingredient found in natural supplements like Docosahexaenoic acid (DHA) could be repurposed and screened for investigating their efficacy for competing with COVID-19 for ACE2 receptors a critical point that could prevent COVID-19 cell entry or inhibiting cytokine storm via binding directly to IL-6. Phenformin is a biguanide anti-diabetic drug that may be made in a single step chemical synthesis. It is an oral diabetes medication that aids in the regulation of blood sugar levels 14 .Phenformin has been shown to lower influenza mortality in mice. Because of the modest inhaled dose, buformin or phenformin inhalation for coronavirus could be a viable new treatment that reduces the danger of systemic side effects associated with biguanides 15 .
As part of normal metabolism, certain algae create long chain omega-3 fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic (DHA).
These fatty acids are critical nutrients for the health of many species, including humans, when they enter the food chain in nature 16 .Docosahexaenoic acid (DHA) is an omega-3 (n-3) fatty acid with a long chain. It has a structure that provides it with distinct physical and functional characteristics 17 . Docosahexaenoic acid (DHA) is crucial for the growth and functional development of the brain in babies and is biologically connected to other n-3 fatty acids. DHA is also essential for adults to maintain optimal brain function 17 .According to our findings phenformin and Docosahexaenoic acid (DHA) may be used as a therapeutic agents against COVID-19 because of their inhibitory activity against IL-6 and ACE2 receptors of COVID-19.In addition , many natural components generated from plants, such as phytochemicals, may provide preliminary evidence for the use of plant extracts to suppress IL-6. Phytochemicals can provide a wide range of chemical diversity as well as anti-inflammatory properties, making them potentially useful as COVID-19 treatment agents.. Plants may be able to supply a number of low-cost medications that can standardize IL-6 levels.. 18 The use of phytochemicals as anti-IL-6 agents could thus be a helpful technique for decreasing COVID-19's negative effects. 19 In this study, EGCG , bromelain , luteolin , vitexin and isovitexin are found to be potential phytocompounds to inhibit the IL-6 along with phenformin and Docosahexaenoic acid (DHA

Interaction studies
The protein-ligand interactions and binding conformational behaviour within the active pocket of the targeted protein IL6 were studied using molecular docking.. The active sites of IL6 were found in the literature prior to online tool using Galaxy binding site server ad docking trials. 22 . 23 The active site residues of a protein were surrounded by a grid. using HDOCK server was used to perform docking experiment. 24

Molecular docking of proteins and ligands
The

Binding free energy calculation
The MMGB/PBSA approach was used to calculate the binding affinities of the optimal docking conformations acquired as a result of molecular docking. 25 The computations were carried out using the AMBER MMPBSA.py model. 25 The following formulas were used to determine binding free energy using Prime v3.5 and four protein-inhibitor complexes and their binding modes:

4-ΔGsol=ΔGPB/GB+ΔGSA
The gas-phase MM energy, the solvation free energy, and the conformational entropy are represented by E MM, G sol, and TS, respectively. In EMM, all bond, angle, and dihedral energies were addressed, as well as electrostatic internal energies (Eele) and the van der Waal's (Evdw). The polar contribution was calculated using the GB or PB models, while G was the sum of electrostatic solvation energies.

Interaction analysis
The  Table 1, Table2 and Table 3 Table1: The binding affinities between phytochemicals and Il-6

Active bonds with IL-6
The interactive residues of each compound with IL-6 are showed in (Fig 1).
Docking of bromelain with interleukin 6, the two-dimensional interaction

Conclusion
Our findings demonstrated the possible efficacy of the investigated compounds including Epigallocatechin gallate (EGCG) , bromelain , luteolin , vitexin and isovitexin as well as phenformin and Docosahexaenoic acid (DHA) as a potent inhibitor of Il-6 and ACE 2. Therefore, theses compounds could be used as a novel treatments for inhibiting COVID-19 cell entry and preventing its inflammatory complication

Conflict of Interest Statement
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest