Discovering of GPCRSs and GnRHRs as SARS-CoV-2 binding receptors, the Scientic Breakthrough that could explain the observed Hypogonadism, Hypothyroidism, Anosmia, Retinol deciency, Neurological and Menstrual disturbance among SARS-COV-2 patients.

A common symptoms of COVID-19 is a change or disorder in hormonal balance and olfactory function which may persist after recovery including COVID-19-related anosmia and hypogonadism. Hormonal problems including Hypogonadism and Hypothyrodism are being observed in with Covid-19. of imbalance post recovery nervous

binding 2 -CoV -as SARS GnRHRs and GPCRSs Discovering of that could explain the he Scientific Breakthrough receptors, t mystery of its common symptoms with unknown aetiology.In . silico research Background A common symptoms of COVID-19 is a change or disorder in hormonal balance and olfactory function which may persist after recovery including COVID-19-related anosmia and hypogonadism. Hormonal problems including Hypogonadism and Hypothyrodism are being observed in patients with . Rise in cases of hormonal imbalance post COVID recovery is a cause for concern. Moreover, anosmia is a well-tolerated symptom of COVID-19, but their aetiology isn't understood. The studies demonstrated that the new coronavirus could affect the central nervous system through the olfactory bulb or blood circulation. Furthermore, in addition to anosmia or hyposmia induction, as well as taste disorders, the virus may cause hormonal imbalance ,retinol deficiency, eye-ache, earache, dizziness and hallucination. It was showed that G-protein coupled receptors (GPCRS) and Gonadotropin-releasing hormone receptors (GnRHRs, a subtype of GPCRS), were expressed sufficiently in olfactory region and hypothalamus as well as the lung Herein by using molecular docking and stimulation analysis , we succeeded to elucidate the direct neuroinvasive route of COVID-19 into the nasal epithelium and human brain cells which may lead to anosmia and hormonal imbalance mainly through the olfactory route by direct binding to G-protein coupled receptors (GPCRS). Furthermore, we strongly suspect that binding of COVID-19 to the expressed GPCRS in the lung is a main cause of ion changing disruption leading to pulmonary edema and failure . Moreover, we confirmed our results by investigating Gonadotropin-releasing hormone receptors (GnRHRs) as a novel binding receptor of COVID-19.

Methodology
In the current study, we used PatchDock server to conduct a docking study of the SARS-CoV-2 Spike protein with both of GnRHRs and GPCRSs protein. The structure of the crystal structure of the proteins were retrieved from RSCP (https://www.rcsb.org/ ) with accessions numbers (PDB ID 7BR3 and 6P9X respectively. we obtained the crystal structure of spike with accession number (PDB ID: 6VYB). The proteins are downloaded in the pdb format. The spike -receptor protein was investigated to determine the conservative residues of binding of Spike protein with the GnRHRs and GPCRS proteins in order to discover the ability of Spike to interact with GnRHR and GPCR receptors. We performed Molecular Dynamics (MD) Simulation to investigate the positional and conformational changes of the included proteins in relation to the binding site that provides insight into the binding stability. MD simulation of the complex was carried out with the GROMACS 4.5.4 package using the GROMOS96 43a1 force field . Introduction s) are well known to be GPCRS protein coupled receptors (  -G  expressed throughout the body, and they represent the genome's  ,  )  1  (  largest superfamily of signalling proteins  .  are the largest and  nd  a They the most diverse group of membrane receptors in eukaryotes. ligands, including light energy, lipids, are activated by a wide range of which transport information , (2,3,4) sugars, peptides, and proteins from the outside environment into the cell in order to mediate the s bind to ligands, GPCRS When . corresponding functional responses conformational changes trigger a cascade of biochemical their reactions within the cell. These intracellular reactions control sensory functions like smell, taste, and vision, as well as a wide range of physiological processes like secretion, neurotransmission, metabolism, cellular differentiation, inflammation, and immune patients infected 19 -Almost 80% of COVID . ) (5,6,7,8 responses experience significant symptoms which is neurological origin such as anosmia , unconsciousness ,dizziness, headaches, muscle tiredness and irritability. (9) A study showed that COVID-19-related anosmia has been linked to inflammation and viral persistence in the olfactory epithelium region , as well as infection of the brain in hamsters models (10). Although, previous research showed that COVID-19 enters the brain via an olfactory path from the nose toward entry receptor, angiotensin 19 -COVID he But, t . 11,12,13,14) ( brain olfactory of receptor converting enzyme 2 (ACE2), is not expressed in is limited to a minor fraction of these generation neurons, or its s GPCRS Expression profiling of 100 . (15,16,17,18)

Dataset of the proteins
In the current study we used PatchDock server to conduct a docking study of the SARS-CoV-2 Spike protein with both of GnRHRs and GPCRSsprotein.. The structure of the crystal structure of the proteins are retrieved from RSCP (https://www.rcsb.org/ ) with accessions numbers (PDB ID 7BR3 and 6P9X respectively. we obtained the crystal structure of spike (PDB ID: 6VYB). The proteins are downloaded in the pdb format. The spike receptor protein was investigated to determine the conservative residues of binding of Spike protein with the GnRHRs and GPCRS proteins in order to discover the ability of Spike to interact with GnRHRs and GPCRSs and to explain the loss of smelling and taste as well as retinol deficiency , hormonal imbalance and lung edema if the spike protein of the virus are bind with GNRHRS and GPCRSs in a good binding affinity that declare the mechanism of the interaction which lead to lose of the smelling of the human . protein Docking study of each Spike -GNRHRS GPCRSs protein were carried out using PatchDock server, this uses molecular docking algorithm based on structure geometry. Firstly, we put the proteins (spike , GNRHRS and GPCRS) after we download it from RSCP we submit it on SAMSON software For pre-docking, all water molecules and ligands were removed while hydrogen atoms were added to the target proteins. In addition, the affinity minimization was performed. For Spike with all of them GPCRS and GNRHRS proteins to make docking between them to get know are the spike will bind to anther receptor GPCRS or GNRHRS .Secondly we submited the data into the server , spike as receptor (spike -receptor) and the ligand (GPCRS),at the first then (spike as a receptor) and the ligand (GNRHRS), in which both amino acid sequences and PDB structures are supported. Then, submitted into the server PatchDock program.

PatchDock program
We performed docking analysis using PatchDock program. This uses molecular docking algorithm based on structure geometry. The PatchDock algorithm divides the Connolly dot surface representation of the protein molecules into three classes, namely, convex, concave, and flat patches (64,65). Then, complementary patches were matched to generate the candidate transformations. Each of the candidate transformation is additionally evaluated by a scoring function which considers both the atomic desolvation energy and geometric fit (66). Next, root mean square deviation (RMSD) clustering is applied to the candidate solutions to discard redundant solutions. The input parameters for the docking are the PDB coordinate file of the protein and ligand molecule. Three major steps are followed in the PatchDock analysis: (i) surface patch matching, (ii) molecular shape representation, and (iii) filtering and scoring.

Molecular Dynamics Simulation
MD simulation of the complex was carried out with the GROMACS 4.5.4 package using the GROMOS96 43a1 force field. The lowest binding energy (most negative) docking conformation generated by AutoDock was taken as initial conformation for MD simulation. The topology parameters of proteins were created by using the Gromacs program. The complexes (spike -GNRHRS ) and (spike -GPCRS )was immersed in an octahedron box of simple point charge (SPC) water molecules. ( 336 -267 ) Na+ counter-ions were added by replacing water molecules to ensure the overall charge neutrality of the simulated system. (spike -GNRHRS ) and (spike -GPCRS ) complexes were energy-minimized initially by steepest descent 10,000 steps, followed by conjugate gradient method 10,000 steps. In order to equilibrate the system, the solute was subjected to positionrestrained dynamics simulation ( NPT) at 300 K for 300 ps. Finally, the full system was subjected to MD production run at 300 K temperature and 1 bar pressure for 20 000 ps. MD simulations were repeated thrice in order to verify the reproducibility of our study.

Analysis of Molecular Dynamics Trajectory
The trajectory files were analyzed by using g_rms, g_rmsf, and g_sas GROMACS utilities in order to obtain the root-mean-square deviation (RMSD), root-mean square fluctuation (RMSF), and solvent accessibility surface area (SASA). Numbers of distinct intermolecular hydrogen bonds formed during the simulation were calculated using g_h bond utility. The trajectory files of PCA were analyzed through the use of g_covar and g_anaeig of GROMACS utilities in order. The analysis of the secondary structure elements of the protein was performed using the program "do_dssp," which utilizes the DSSP program [54].

Docking Analysis
To investigate the binding of the spike with GPCRS and GNRHRS proteins, docking analysis was carried out with a specific GPCRS and GNRHRS proteins .  Figure 1, it is clear that in the native model of the spike and its binding to GNRHRS are take an alternative way to bind to spike protein like ACE2 .also and GPCRS are bind in deferent chain to get the effect of losing the smelling .  Results were described before to optimize the Docking score of the native model of spike with GNRHRS and GPCRS. the calculated energy are done by using Patch Dock ( Table 1). The obtained results confirmed that the native model binds to GPCRS with the highest docking score of -1451.8 when compared to the other GnRHRs complexes, which have the lowest binding affinity, as evidenced by the docking score of -1424.9.. These results signifies better conjugation of GNRHRS to the binding pocket of the spike receptor in the RDB of the spike protein . Comparing the binding free energy of GPCRS to GNRHRS showed that the GNRHRS protein was found to bind to the vital residues in the RBD of the spike protein. But GPCRSs protein were found to bind to RDB in other place in chain B of the spike.

Simulation Study of (spike -GNRHRS ) and (spike -GPCRS )Complexes
The results obtained from the above docking analysis provoked us to explore the dynamic behavior of (spike -GNRHRS ) and (spike -GPCRS )Complexes. We analyzed the root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), number of hydrogen bonds (NH) The main purpose of the MD simulation studies was to investigate the positional and conformational changes of GNRHRS and GPCRS proteins in relation to the binding site of spike protein RDB that provides insight into the binding stability. MD revealed that GNHR could efficiently activate the biological pathway with changing the conformation in c terminal and N terminal but its effects on the middle of the protein in between 1000 : 1500 residues in RMSF plot in the binding site of spike protein as in figure 4 (spike -GNHR ) in addition to the MD simulation of GPCR are revealed that its efficiently activate the biological pathway with changing in the conformation in c terminal and the middle of the protein in between 500 : 1000 residues in the C terminal and from 1300 : 1500 in RMSF plot in the binding site of spike protein as in figure 4 (spike -GPCR).
To evaluate the stabilities of (spike -GNHR ) and (spike -GPCR )Complexes, during the MD simulations, root mean square deviation (RMSD) where it is used to measure the average change in displacement of a selection of atoms for a particular frame with respect to a reference frame. It was calculated for all frames in the trajectory. The RMSD for frame x is for (spike -GNHR ) and (spike -GPCR )Complexes was calculated with respect to the initial structures along (the 50 (ns)) trajectories (Fig. 4). The trajectories indicated for (spike -GNHR ) and (spike -GPCR )the binding of the receptor on the active site after 50 ns in system with a mean RMSD value of (7.5 ns ,4.9 ns) representatively . In addition, the confirmation change of the (spike -GNHR ) protein between 1000 : 1500 in the system also proved the credibility of the docking results. Total energy of the most active conformation of the molecule was −1424.9 K.cal / mol . Also the confirmation change of the (spike -GPCR) protein are affected in the C terminal and the middle so it is between 500 : 1000 residues in the C terminal and from 1300 : 1500 in the middle in the system also proved the credibility of the docking results and the binding between (spike -GPCR explore new binding site in spike protein .Total energy of the most active conformation of the molecule was −1424.9 K.cal / mol , -1451.8 K.cal / mol for (spike -GNHR ) and (spike -GPCR) representatively The temperature and pressure do not have any effect on the conformation of the structure. The hydrogen bonds formed between the protein and inhibitor after simulation were mostly concentrated in the activation loop region of the protein which is responsible for the catalytic machinery and substrate binding. This is explicitly understood from the above observation.

Figure6. The plot above reports SSE distribution by residue index throughout
the spike -GnHR and spike -GPCR structure. The plot below summarizes the SSE composition for each trajectory frame over the course of the simulation where it done for 50 nsec , and the plot at the bottom monitors each residue and its SSE assignment where its 30% over time.
Binding of COVID-19 to GNRHRS, a subtypes of GPCRSS could lead to smell losing and hypogonadism.
The new coronavirus was found to have the ability to affect the According to our findings, COVID-19 could bind to GnRHRs leading to blocking the binding of GnRH to this receptor and disrupts its signal resulting in hypogonadism and anosmia. It was found that congenital anosmia (inability to smell) is frequently associated with GnRH deficiency in humans, leading to the widely held belief that GnRH neurons rely on olfactory structures to reach the brain, but this hypothesis has yet to be proven. (21). The olfactory bulb (OB) is a conserved region found in brain that its main function is receiving sensory neurons direct synaptic input in the nasal epithelium part and conveys that instructions to the rest of the brain (22). It gets instructions from the brain regarding odours recognized by cells in In 257 (89.8%) of the hospitalized patients, testosterone levels suggestive of hypogonadism were detected (29).The hypothalamus is a critical region located in the brain that produces, integrates, and regulates several processes including the blood pressure, hormonal balance, body temperature, circadian rhythm, basal homeostasis, emotion and sexual behavior (30,31) .

COVID-19 could block thyroxin GPCRSs leading to hypothyroidism and anosmia.
There is an increasing body of literature on the impact of COVID-19 on the pituitary-thyroid axis. Currently, we know that SARS-CoV-2 could lead to short-term and reversible thyroid dysfunction. According to our findings, COVID-19 could bind to thyroxin GPCRSs leading to blocking the binding of thyroxin to this receptor and disrupt its signal resulting in hypothyroidism . The thyroid-stimulating hormone (TSH) or thyrotropin (34) receptor (TSHR) (35,36) is a member of the class A G-protein-coupled receptors (GPCRSs) (37). It was revealed that a significant proportion of hypothyroidism associated with COVID-19 and altered thyroid hormones was significantly more in COVID-19 patients as compared to control groups (38) . It was showed that Hypothyroidism is associated with prolonged COVID-19-induced anosmia (39) COVID-19 could block GPCRSs leading to disorder in tastes .

COVID-19 could bind to GPCRSs leading to blocking of GPCRS signaling and pulmonary edema
COVID-19 mortality is primarily driven by abnormal alveolar fluid metabolism of the lung, leading to fluid accumulation in the alveolar airspace. This condition is generally referred to as pulmonary edema and is a direct consequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection(43). GPCRSs are primarily responsible for signal transduction/propagation cascades (44,45) . GPCRSs located on the cell surface transduce exogenous signals that activate GTP-binding "G" proteins, which in turn activate effector proteins (such as adenylyl cyclase and phospholipases) and second messengers (such as calcium or cAMP) (44,45) . The cAMP/PKA pathway (46) regulates CFTR activity and is typically induced by Gs-coupled GPCRSs that stimulate adenylyl cyclase (AC), raising cAMP levels and stimulating PKA (47). Invading pathogens, on the other hand, frequently exploit these endogenous signalling pathways (48).‫ال‬The A2B adenosine receptors and the 2 adrenergic receptors are two specific GPCRSs that are primarily involved in CFTR regulation and are abundantly expressed in the airways (47, 49). Under physiological conditions, the adenosine-CFTR regulation system is essential for mucosal airway surface protection (50) and alveolar surface layer (ASL) regulation (51). Viruses, on the other hand, are well known for their ability to not only use GPCRSs to enter host cells, but also to use their intracellular signalling pathways for survival and replication (48). Based on this general concept, it is possible that SARS-CoV-2 may also compromise GPCRS signalling, and this effect may contribute to the pathophysiology of pulmonary edema.

COVID-9 could disrupt progesterone signaling via binding to progesterone receptors (GPCR)
The progesterone prepares the body for pregnancy in the event that the released egg is fertilized. If the egg is not fertilized, the corpus luteum breaks down, the production of progesterone falls and a new menstrual cycle begins. Membrane progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ, and mPRε) were identified as putative G protein-coupled receptors (GPCRs) for progesterone (69). It was showed that female patients with COVID-19 had various extents of transient menstrual changes, mainly manifesting as prolonged cycles and decreased volume. A few patients also showed shortened or disordered menstrual cycles and increased volume, which were rarely observed in the control group (70). Therefore, COVID-19 could disrupt progesterone signaling leading to menstrual cycle disturbance.
COVID-9 could disrupt FSH signaling leading to low expression of Rbp4 and . retinol deficiency ovaries in vivo and in granulosa cells cultured in vitro, both at the mRNA and protein levels (76). Therefore,COVID-9 could inhibit Rbp4 and retinol enzyme activation leading to retinol deficiency via disrupting FSH signaling .

Conflicts of interest
The authors have no conflicts of interest to declare that are relevant to the content of this article

Acknowledgments
Contains gratitude and appreciation for Egypt for providing the funds for this project and the parties involved in the research but not included in authorship.