Severe acute respiratory syndrome corona-virus-2 (SARS-COV 2) pandemic is the infectious cause whose exceptional character has resulted in a serious global attempt for its diagnosis and treatment [1, 2]. The SARS-COV 2 is a member of the coronavirus family and contains the enveloped positive-sense single-stranded RNA (+ssRNA) . Sore throat, fever, shortness of breath, multi-organ failure, or even death are some symptoms and outcome of SARS-COV 2 which is now called coronavirus disease 2019 (COVID-19) [1, 2]. The metabolic pathway and mechanism of action of existing drugs targeting the key proteins of SARS-COV 2 have been evaluated and ranked in combat against this disease . Among them, hydroxychloroquine (HCQ) [5-7] and Remdesivir [8-10], respective drugs of malaria  and Ebola , have been repurposed for COVID-19 therapy. Despite the approval of Remdesivir by the food and drug administration (FDA) of the United States , no reliable drug has been yet identified for SARS-COV 2.
Immunoinformatics can collaborate with the structural-based drug design approaches for the COVID-19 treatment . The molecular docking approach is one of the collaborations with epitope-based immunoinformatics which has revealed the applicability of the new vaccine [14-16]. This approach was employed to the structural virology and antibody-antigen recognition , structural validation against crystal structures of carbohydrate ligands to antibodies , an epitope-based peptide vaccine against DENV-NS3 protein , characterization of the common epitope-based peptide vaccine for Nipah and Hendra viruses , and a multi-epitope based subunit vaccine against the dengue infection . Also, the molecular docking study of antibody-antigen complexes was applied for pitfalls illustrated by influenza hemagglutinin  and investigation of the protein-protein interactions in serine protease inhibitor and antibody-antigen complexes . Moreover, the molecular docking and molecular dynamics (MD) simulation have been carried out for an invitro identification of human anti-complement factor H (CFH) antibody Ab42 and CFH polypeptide .
The application of natural drugs and vaccines for COVID-19 therapy is one of the popular trends in controlling the pandemic. It was seen, the classical plant cannabinoids Dronabinol and Epidiolex have shown various biological activities against SARS-COV2 . The virtual screening of 40 natural compounds particularly employed in traditional Iranian medicine for blocking the enzymatic activity of SARS-CoV-2 3CLpro has revealed the inhibition potential of kappa-carrageenan conformer, beta-D-galactopyranosyl and calycosin 7-O-glucoside phytochemicals .
Nanobody refers to a group of the single-domain antibody (sdAb) that were first engineered from heavy-chain antibodies naturally found in the camelids (called VHH fragments) . These sdAbs overcome many application problems of the monoclonal antibodies in molecular imaging, diagnostic kits, and therapeutic medicines [27-29]. The nanometer-scale antibody with a molecular weight of 12–15 (kDa) and a light chain can effectively penetrate the tissues . It has been confirmed that some natural [30-33] and synthetic nanobodies [34-38] have the ability to neutralize the binding of the trimeric spike glycoprotein receptor-binding domain (RBD) of SARS-COV 2 against the host receptor human angiotensin-converting enzyme 2 (ACE2) .
These identifications promise to evaluate the blocking ability of some nanobodies against RBD of SARS-COV 2. This ability of the nanobodies was experimentally explored in human immune systems. Hence, a molecular docking strategy was employed for evaluation using CoDockPP Server and COVID-19 Docking Server.
MOLECULAR DOCKING STUDY
Firstly, crystallography structures of studied nanobodies and RBD-ACE2 of SARS-COV 2 (6LZG) were taken from a protein data bank (PDB) . The structural and antigenic related data were checked in the structural antibody database (SAbDab)  which is an online resource of all publicly available antibody structures annotated and presented in a consistent manner. The data are annotated with several properties such as experimental information, gene details, correct heavy, light chain pairings, and antigen details. The candidate nanobodies were blocking the Nef  and capsid protein p24 [43, 44] of human immunodeficiency viruses 1 (HIV-1) or capsid protein VP3 of poliovirus [45, 46]. Moreover, different conformations of nanobodies that are experimentally involved in the RBD of SARS-COV 2 were adapted from PDB [30-33, 35]. The nanobodies name, their viral types and macromolecules, and PDB ID are listed in Table 1. The heavy chain name in the PDB ID may be different due to several binding sites on the target protein of the virus that are experimentally involved in human immune systems. Regardless of the synthetic nanobodies blocking of spike glycoproteins RBD of SARS-COV 2, some other nanobodies such as natural VHH (occurring in Lama glama, Vicugna pacos or Camelus dromedaries) were also addressed. In the next step, nanobodies, and ACE2 structures and affinity were checked using a multistage protein-protein docking program (CoDockPP Server)  while RBD (6LZG (B)) offered the target protein. This server operates based on shape complementarity using knowledge-based scoring function and site constraint. Then, nanobodies and ACE2 were presented to COVID-19 Docking Server  for determining the most stable complexes of RBD-nanobody and RBD-ACE2 where ACE2 (6LZG (A)) was defined as ligand proteins.
RESULT AND DISCUSSION
The neutralization ability of candidate VHH toward RBD of SARS-COV 2 was repurposed using CoDockPP Server and COVID-19 Docking Server. The predicted binding energies of nanobodies and ACE2 to RBD are listed in Table 1. The binding energy values obtained by the two servers well coincided for all complexes.
Seven nanobodies contain Ty1 , H11-D4 , H11-H4 , SR4, MR17 and MR17-K99Y , Sb23  were experimentally involved in human immune systems against RBD of SARS-COV 2. The results of molecular docking of these nanobodies showed that the binding energy values varied from -235.55 kcal/mol for Ty1 derived of Vicugna pacos (6ZXN (F))  to -342.56 kcal/mol for synthetic construct SR4 (7C8V (A)) . The other synthetic nanobodies have the binding energy of -262.63 and -283.52 for sybodies of MR17 and MR17-K99Y , -295.34 (7A29 (F)), -295.82 (7A29 (D)). and -298.74 (7A29 (E)) for Sb23 , and -342.56 for SR4 (7C8V (A)) , respectively. Moreover, the estimated binding energy for other Ty1 was -238.59 (6ZXN (E)) and -247.40 (6ZXN (D)) while these values were -285.87 (6YZ5 (F)), -292.91 (6Z43 (X)), -300.03 (6Z43 (Z)), and -302.56 (6Z43 (Y)) for Lama glama obtained H11-D4 , respectively. The binding energy was predicted as -328.62 for H11-H4 (6ZHD (E))  of Lama glama which can neutralize the binding of the RBD against the human ACE2. On the other hand, predicted binding energy of ACE2 with RBD receptor was -297.12 (kcal/mol). Accordingly, respective binding energies of Sb23 (7A29 (E)), SR4 (7C8V (A)), H11-D4 ((6Z43 (Z)) and (6Z43 (Y))) and H11-H4 (6ZHD (E)) (-298.74, -342.56, -300.03, -302.56, and -328.62) were greater than ACE2-RBD complex, so they are the top conformation of these nanobodies.
The predicted binding energies of all 15 reported nanobodies were greater than -235.55 (kcal/mol) for Ty1 (6ZXN (F)) while these values for 10 VHH were greater than RBD-ACE2 complex. VHH 7A, VHH PVSP29F, Cameld VHH 9, VHH PVSS8A, VHH 12B, VHH 59H10, VHH PVSP6A, VHH 10E, VHH 17B, and VHH 59H10 are proposed nanobodies for blocking the viral glycoprotein of SARS-COV 2. 5KU2 (7), 3JBC (7), 2XV6 (D), 3JBE (7), 5KTZ (7), 5O2U (D), 3JBD (7), 5KWL (7), 5KU0 (7), and 5O2U (B) are PDB ID (VHH heavy chain) of the selected nanobodies. The proposed VHH 17B and VHH 59H10 nanobodies have an affinity greater than -342.56 (kcal/mol) for synthetic construct SR4 (7C8V (A)) against trimeric spike glycoprotein RBD for SARS-COV 2.
The energy map for ACE2-RBD, VHH 17B-RBD and VHH 59H10-RBD was visualized using Molegro Molecular Viewer of MVD  software. The spherical space of mapping had a radius of 0.25 Å with the offset from the center for covering the binding site (X = -31.50, Y = 25 and Z = 13.62). According to the energy maps for ACE2-RBD (Fig. 1), hydrogen donor, steric, hydrogen acceptor and electrostatic interactions were significant in blocking of the host receptor human ACE2 to RBD of SARS-COV 2, respectively. It should be noted that for electrostatic interactions in the figures blue and red colored areas respectively correspond to the positive and negative charges. For example, the energy maps of VHH 17B-RBD and VHH 59H10-RBD are depicted in Figs. 2 and 3, respectively. As seen, in ACE2-RBD complexes, hydrogen donor, steric, hydrogen acceptor, and electrostatic interactions had the highest contributions in blocking RBD, respectively. This similarity in the energy maps of VHH 17B-RBD and VHH 59H10-RBD with ACE2-RBD confirmed that these nanobodies can be used for blocking RBD of SARS-COV 2. It should be noted that the different crystallography structures of VHH 59H10 (5O2U (B)) and (5O2U (D)) could be due to various experimental contact region of spike glycoprotein of SARS-COV 2 (Fig. 4). The top conformation and secondary structure view of VHH 59H10 nanobody in the active site of RBD for SARS-COV 2 are demonstrated in Figure 4.
The neutralization ability of some nanobodies was investigated against the trimeric spike glycoprotein RBD of SARS-COV 2. CoDockPP Server was applied for a multistage protein-protein molecular docking of nanobodies and ACE2 with RBD receptor. Moreover, COVID-19 Docking Server was employed to evaluate the blocking ability of nanobodies against RBD. Both servers predicted the equal values of binding energy for all studied nanobodies while the value of -297.12 (kcal/mol) was estimated for the ACE2-RBD complex. According to these values, all proposed nanobodies have values greater than -235.55 (kcal/mol) for Ty1 (6ZXN (F)). VHH 7A, VHH PVSP29F, Cameld VHH 9, VHH PVSS8A, VHH 12B, VHH 59H10, VHH PVSP6A, VHH 10E, VHH 17B, and VHH 59H10 are 10 nanobodies that can be proposed for blocking the RBD of SARS-COV 2. However, the two last nanobodies of VHH 17B and VHH 59H10 are more confident for neutralization due to their greater estimated binding energy against -342.56 (kcal/mol) which computed for synthetic construct SR4. According to the energy maps of all ACE2, VHH 17B and VHH 59H10, hydrogen donor, steric, hydrogen acceptor, and electrostatic interactions played the most significant role in blocking the RBD, respectively. This study offers structural insight for the blocking of pathogenic novel coronavirus with 15 reported nanobodies that had been proposed for the control of the SARS-COV 2 pandemic.
CONFLICTS OF INTEREST
The authors announce that there are no conflicts of interest.