1.1 Phosphoprotein

Human proteins that interacted with the proteins of covid-19 in 68 of these proteins can be applied as targets for suitable drugs (Table 1) [¹²]. SARS-COV-2 strain is about 75% similar to SARS-COV strain and 35% close to the MERSCOV strain [¹³].

Effects of covid-19 can be occurring within 3 days or sometimes up to two weeks after exposure and there is an equal genomic system known as of β-coronaviruses containing ORF1(ab), NSP1, S- protein from 5'-UTR (untranslated region) and E- protein, M-protein from 3'-UTR and also ORF6(a), ORF7(a), ORF(8), N-protein, ORF(10), and several other non-structural items.

1.2. Neutrophil & Hemoglobin Specification:

By this disease, the neutrophil and hemoglobin amounts have reduced while the indexes of erythrocyte sedimentation rates, serum ferritins, C-reactive proteins, albumins, and lactate dehydrogenases increase significantly. This phenomenon indicates that by decreasing hemoglobin, HEM increases and too much harmful iron will accumulate in the body that will cause smart pain in the human and increase the albumin. Consequently cells produce, large scale of serum ferritin for reducing injury. Hemoglobin contains 4 subunits, 2-alpha and 2-beta, in which each subunit has iron-hemoglobin bonded [¹⁴]. Due to Fe2+ the CO2 gases might be separated from hemoglobin and consequently capture O atoms and consequently Fe2+ is oxidized to Fe3+. Hemoglobin can release oxygen atoms and capture CO2 and then Fe3+ is reduced to Fe2+. Because of no clinical trial-based vaccine present, vaccinations are a reliable effective way.

So availability of genomic information and biological algorithms including immunological information might be help researchers for identifying the effective epitopes that can be used for developing vaccines [¹⁵,¹⁶]. The subunit vaccine consists of several segments of antigen which can be simulated the presence of the natural pathogen towards an immune situation of pathogen [¹⁷,¹⁸].

Basically, vaccinations designing against MERS, SARS, Ebola, ZIKA and Chikungunya viruses have produced promising results [¹⁹,²⁰]. The modeling and simulation methods with genetic algorithms decrease the number of experimental activities and save expenditure and also increase the potential of a successful vaccination. A large amount of peptides involved in the multi-epitopes vaccine in which induce the activation of AIR (Adaptive Immune Response) are suitable and fast method for the viral-infections therapy and covid-19 [²¹,²²]. By this work, SARS-CoV-2 proteomes were explored for determining the antigenic proteins and various B&T-cells epitopes were foreteller with their main histocompatibility complexes characteristic. In addition conserved domain, homologies simulation and molecular docking methods were applied for analyzing the structure of virus-depended proteins. This investigation exhibited that ORF9b and surfaces glycoproteins had a segment for combining with porphyrin to make the complexes structures, while ORF1(ab), ORF10, ORF(3a) coordinately attack the hemoglobin on the 1-β chain for dissociating the iron to form the porphyrin. Moreover the amino acid sequences of target proteins (ORF1, S-(protein), ORF3 (a), E-protein, M-protein, ORF6, ORF(7a), ORF8, ORF10 and ORF9b of SARS- CoV-2 were extracted from the Gen-bank. This phenomenon of the coronavirus inhibited the usual metabolic pathways of hemoglobin, and made human show symptoms of these problems.

2.1. Simulation & Docking analysis

Auto dock software (iGEM-DOCK) has been used and via this tool, the suitable receptors can be distinguished in whole COVID-19 component structures for forming a complex. “iGEMDOCK” is suitable to define the binding site quickly. Molecular docking simulation is a way of finding the best matching pattern among molecules via geometric and energies matching. Following items have been done in docking modeling and simulation : (A), Guess for a place of binding site on the ORF series for separation the iron for forming the porphyrin. (b) Browsing and selecting the protein file and in the initial step, the sequence of polypeptides were extracted from gen-BANK [²³]. finally, whole of the sequences can be saved as FASTA format for any further evaluation. (c) Designing the binding sites as a bonded ligand. (d) Designing and preparing the center of the active site via proper ligands. (e) Setting the binding and active sites in view point of size and atoms by the extended radius from the choices ligands - proteins complex. These sequences were extracted from NCBI and also all covid-19 proteins. (f) Hemoglobin -binding proteins, Hemoglobin- oxidase and peptide sequences were used for analyzing conserved chain. It is notable that all polypeptides of Wuhan novel covid-19 were applied for constructing 3-D structures via homology simulation.

IGEMDOCK produces an analysis environment including visualization with analysis tools for applicants that can be utilized the docked situation and related category via the system. The energies poses of each items would be outputted based on the position of “best: Pose”. That information is premeditated with analyzing of these poses. Through looking at the peptide bonded of various ligands, they might be selected through the checked box of ligands. If the cocrystallization ligands are saved on the active site structures, it will be predicted poses. Cluster analyses are the partitioning of the information set into subsets. This information in each subset will share some general aspects. Interaction specifications can be extracted via the protein-ligand intercalation and are accounted atomic kinds in several functional categories. The data in each subset will share some usual properties. Finally, the PDB files were obtained from the bioinformatics sites such as PDB database web sites. By this work, the main items which have been done via molecular docking by “Discovery-Studio” that can be summarized as: Preparation of a ligand perspective, Opening the ligands file, clicking “Prepare Ligands” in the “Dock Ligands” sub-menu of the “Receptor-Ligand Interactions” menu for generating the heme ligands. For the preparing our protein receptors, the protein's pdb file has been generated via homology modeling by the “Dock Ligands” submenu of the “Receptor-Ligand Interactions” menu for generating the protein receptor models for our docking model. We set our docking data via the generating protein receptor system from the “Define and Edit Binding Site” submenus in the “Receptor-Ligand Interactions” menu. The binding energies were calculated via choosing the pose with the largest binding energies.

The Expassy-Protparam tools were applied for determining the chemical & physical properties of molecules [²⁴]. For checking antigenicity of proteins, the related software (Vaxijen 2.0) was also applied [²⁵] and for the predication of secondary polypeptides “Alignment self-optimized prediction method” (SOPMA) tools has been used [²⁶]. In addition several additional online software or tools such as Swiss model [²⁷], “Phyre2”, and “RaptorX” was applied for the tertiary polypeptide segment [²⁷,²⁸]. Model recovered was then refined via “Galaxy Refine Server” and accredited also via Ramachandran plots.

2.2. Genetic and Sequence Analysis

Conserved of viral proteins can be analyzed through MEME website [²⁹–³¹]. These models are applied for predicting differences between the viral system and human proteins. The 3-D structures of viral proteins were structured through homology modeling of Swiss-model [³²,³³]. Sequence length extension and homology modeling should be adopted via applying molecular docking, and also the ligand-receptor of viral proteins with porphyrins might be simulated in real situation.

Consequently, a life cycle system of the virus were structured and configured within related proteins of the covid-19 that was proposed. Molecular docking simulation can be built on homology or 3-D molecular modeling. For the analysis of mentioned domains “MEME” suite websites where integrates several keys of predicting is suitable. By the MEME, the polypeptides sequences can be merged into a text file and then select the number of motifs which is needed before any clicking of the "Go" button. For the homology modeling SWISS-MODELs are completely automatic, which can be accessed via a web system through a few running steps such as entering into the Swiss-model, writing the sequence, and clicking "Search Template" for performing a simple template search. Second step is choosing a template for modeling and third is Building Model command that a template model is selected automatically.

2.3. Molecular Dynamic Modeling

Molecular dynamics modeling for polypeptide-ligand complexes were accomplished using the Desmond software [³⁴]. The OPLS and charm force fields were applied for modeling the protein-small molecules interactions. Long-range electrostatic forces were estimated using the Particle-mesh Ewald (PME) software with a grid spacing of 0.75 Å. Nose-Hoover thermometry and Martyna-Tobias-Klein method were applied for maintaining the temperature and constant pressure, respectively. The formula of motion was considered using the multi run RESPA by 3.0 fs time step for bonded and non-bonded interactions within a low cutoff. An outer time step of 5.0 fs was used for non-bonded forces beyond the cutoff.

Although, accurate structures of the phosphor lipid's bilayers which are in biochemistry pertaining fluid medium are not possible for getting experimental data, fluctuations of these types of dynamic bilayers demonstrate correct structures. Quantum mechanics and Molecular dynamics simulation (QM/MM) are strong tools for tabulating and guiding the interpretation of these experimental parts. The validity of modeling may be measured in contrast existing experimental results. There are several and various techniques such as Deuterium NMR quadrupol splitting which can give certain results of physical & chemical properties. Membrane electrostatics area per lipid and membrane thickness and acyl parameters are also important to study for any further simulation. The absence of experimental data and results are reversed in molecular modeling of membrane protein (M) due to several force fields parameterization. Tight level AB-initio estimations are needed for parameterization of these types' force fields and presently allow evaluation of the heavy atoms for gaining accurate results. Moreover, there are some limitations in weak QM calculations due to London's dispersion of non-bonded interactions for such molecules. We simulated our model based on our previous works (Scheme 2) [³⁵].

Scheme 2 Membrane simulation including 180 molecules of DPPC phospholipids. 

2.4. Isotropy & Anisotropy of NMR Analysis

The anisotropy data of shielding and non-shielding spaces of the hetero rings in all antibiotics, (σ₁₁, σ₂₂, σ₃₃), are labeled based on IUPAC instruction. Moreover, σ₃₃ indicates the direction of minimum shielding, with the highest frequency, while σ₁₁ exhibited a direction of maximum shielding, with the lowest one. In addition, the orientations of the asymmetry tensors are given by (k=3aΩ) and the skew is κ=3(σ1so−σ22)Ω(13) (−1≤κ≤+1) . In our calculations of various halogenated antibiotic's rings, (κ) is basically positive, and the negative values are related to some critical or boundary points. The stabilities of the isotropies are tightly affected on suitable places in the shielding area spaces and are dependent on the configuration of aromatic rings. Therefore, using this method of isotropy can calculated as an aromaticity criterion for any further simulation of covid-19 due to hydrogen bonds between Vidarabine″, “Cytarabine”, “Gemcitabine” and “Matrine” and covid-19 proteins. Obviously that structural function cause changes in the magnetic field experienced through the nuclei and change the resonant frequencies. So, the chemical shielding and other properties such as hydrogen bonding and magnetic anisotropy of π-systems might be changed due to the electrons around the proton which produce a magnetic field, countering the applied fields. Consequently reduces the field experienced at the nucleus. In other words, electrons are said to shield the proton, an effect that is exactly dependent on the distance of the center. By this simulation we have calculated those parameters for any binding of those natural products to the Covid-19 components (Fig. 1 and Tables 2 & 3).

Figure 1 ¹³C NMR and Chemical shifts of Cytarabine and Vidarabine.