Discovering of GPCR and GnRHR as SARS-CoV-2 binding receptors, the Scientic Breakthrough that could explain the mystery of its common symptoms with unknown aetiology. In silico research.

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 Covid-19. 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 etiology 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 ,headache, eye-ache, earache, dizziness and hallucination. It was showed that G-protein coupled receptors (GPCR) and Gonadotropin-releasing hormone receptors (GnRHR), a subtype of GPCR were expressed suciently 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 (GPCR). Furthermore, we strongly suspect that binding of COVID-19 to the expressed GPCR in the lung is a main cause of ion changing disruption leading to pulmonary edema and failure. Moreover, we conrmed our results by investigating Gonadotropin-releasing hormone receptors (GnRHR) as a novel binding receptor of COVID-19. In the current study, we used PatchDock server to conduct a docking study of the SARS-CoV-2 Spike protein with both of GnHR and GPCR receptor 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 performed to positional into simulation out with the using analysis of simulations molecular dynamics molecular high between and both GPCR and GnRHR. Results indicated obtained results model compared results better vital The COVID-19 entry receptor, angiotensin-converting enzyme 2 (ACE2), is not expressed in the receptor of olfactory neurons, or its generation is limited to a minor fraction of these neurons. A change or disorder in hormonal balance and olfactory function is a common symptom of COVID-19, but its aetiology is unknown. SARS-CoV-2 was found to bind strongly and directly to both GPCR and GnRHR which expressed suciently in olfactory neurons. As a result, we conrm that COVID-19 could use these receptors as a direct neuroinvasive route into human brain cells, potentially leading to long-term neurological complications and hormonal imbalance via the olfactory route. Our ndings may also shed a new light on the mechanism of pulmonary edema in COVID-19 patients. Therefore ,we propose that GPCR and is involved in COVID-19 pathophysiology and can be exploited as a potential therapeutic target for COVID-19.

Introduction G-protein coupled receptors (GPCRs) are well known to be expressed throughout the body, and they represent the genome's largest superfamily of signalling proteins (1),.and are the largest and the most diverse group of membrane receptors in eukaryotes. They are activated by a wide range of ligands, including light energy, lipids, sugars, peptides, and proteins (2,3,4), which transport information from the outside environment into the cell in order to mediate the corresponding functional responses. When GPCRs bind to ligands, their conformational changes trigger a cascade of biochemical 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, in ammation, and immune responses (5,6,7,8). Almost 80% of COVID-19 infected patients experience signi cant 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 in ammation and viral persistence in the olfactory epithelium region , as well as infection of the brain in hamsters models (10). Although, previous research showed that  enters the brain via an olfactory path from the nose toward brain (11,12,13,14). But, the COVID-19 entry receptor, angiotensin-converting enzyme 2 (ACE2), is not expressed in receptor of olfactory neurons, or its generation is limited to a minor fraction of these neurons(15, 16,17,18). Expression pro ling of 100 GPCRs demonstrates that most are expressed in multiple tissues and that individual tissues express multiple GPCRs. Over 90% of GPCRs are expressed in the brain(52). GPCRs expressed su ciently in olfactory bulb (OB) and act as olfactory receptors(53) GPCRs are signal receptors that respond to a wide range of stimuli. Chemosensory GPCRs (csGPCRs) are receptors for sensory signals from outside the body that are detected as odors, pheromones, or tastes (54,55). Peptides, lipids, neurotransmitters, and nucleotides are all examples of endogenous signals that GPCRs respond to (56,57). These GPCRs are involved in a variety of physiological processes, including neuronal excitability regulation, metabolism, reproduction, development, hormonal homeostasis, and behaviour. Two speci c GPCRs, A2B adenosine receptors and 2 adrenergic receptors, are primarily involved in CFTR regulation and are abundantly expressed in airways (58,59). Under physiological conditions, the adenosine-CFTR regulation system is critical for mucosal airway surface protection (60) and alveolar surface layer(ASL) regulation (61). Viruses, on the other hand, are well known for their ability to not only exploit GPCRs to enter host cells, but also to use their intracellularsignalling pathways for survival and replication (62).

Methodology Molecular Docking
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 GnRHR and GPCR receptor protein.. The structure of the crystal structure of the ) with accessions numbers (PDB ID 7BR3 andhttps://www.rcsb.org/proteins are retrieved from RSCP ( 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 GnRHR and GPCR proteins in order to discover the ability of Spike to interact with GnRHR and GPCR receptor and to explain the loss of smelling and taste as well as hormonal imbalance and lung edema if the spike protein of the virus are bind with GNRHR and GPCR receptor in a good binding a nity that declare the mechanism of the interaction which lead to lose of the smelling of the human . protein Docking study of each Spike -GNRHR GPCR receptor protein were carried out using PatchDock server, this uses molecular docking algorithm based on structure geometry.
Firstly, we put the proteins (spike , GNRHR and GPCR) 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 a nity minimization was performed. For Spike with all of them GPCR and GNRHR proteins to make docking between them to get know are the spike will bind to anther receptor GPCR or GNRHR .Secondly we submited the data into the server , spike as receptor (spike -receptor) and the ligand (GPCR),at the rst then (spike as a receptor) and the ligand (GNRHR), in which both amino acid sequences and PDB structures are supported. Then, submitted into the server 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 at 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 t (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 le 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) ltering 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 eld. 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 -GNRHR ) and (spike -GPCR )was immersed in an octahedron box of simple point charge (SPC) water molecules. ( 336 -267 ) Na+ counterions were added by replacing water molecules to ensure the overall charge neutrality of the simulated system. (spike -GNRHR ) and (spike -GPCR ) 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 position-restrained 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. The gromacs topology le for ligands was generated using the PRODRG2 server (http://davapc1.bioch.dundee.ac.uk/prodrg). The time constant for berendsen temperature coupling and berendsen pressure coupling was both set at 0.1. The environment was set to 300 K and 1 bar. All of the complexes were energy minimized using steepest descent method. Further, a 20 ps position restraining simulation was carried out to restrict the movement of the protein in the simulation. For the long range electrostatic interactions, Particle Mesh Ewald (PME) electrostatic was used. The cut-off for coulomb interaction and Vander Waal interaction was set to 1.0 nm and 1.4 nm, respectively. The LINCS algorithm was used for all bond constraints.

Docking Analysis
To investigate the binding of the spike with GPCR and GNRHR proteins, docking analysis was carried out with a speci c GPCR and GNRHR proteins . Results indicated that the spike are bind to GNRHR in (PHE 456-GLN 493 -GLY 496 -THR 500 -GLY 502 -LEU 455 -TYR 449 -LYS 417) which are the vital residues in the RDB of the Spike proteins which bind to GNRHR by binding energy -1424.7 k.cal/mol GPCR Results indicated that the spike are not bind to RDB of which are the vital residues in the RDB of the Spike proteins which bind to GPCR by binding energy -1451.8 k.cal/mol to Spike proteins as showed in table 1.  Figure 1, it is clear that in the native model of the spike and its binding to GNRHR are take an alternative way to bind to spike protein like ACE2 .also and GPCR 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 GNRHR and GPCR. the calculated energy are done by using Patch Dock ( Table 1). The obtained results con rmed that the native model binds to GPCR with the highest docking score of -1451.8 when compared to the other GnHR complexes, which have the lowest binding a nity, as evidenced by the docking score of -1424.9.. These results signi es better conjugation of GNHR to the binding pocket of the spike receptor in the RDB of the spike protein . Comparing the binding free energy of GPCR to GNRHR showed that the GNRHR protein was found to bind to the vital residues in the RBD of the spike protein. But GPCRs protein were found to bind to RDB in other place in chain B of the spike.

Analysis of Molecular Dynamics Trajectory
The trajectory les 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 uctuation (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 les 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].
Simulation Study of (spike -GNRHR ) and (spike -GPCR )Complexes The results obtained from the above docking analysis provoked us to explore the dynamic behavior of (spike -GNRHR ) and (spike -GPCR )Complexes. We analyzed the root mean square deviation (RMSD), root mean square uctuation (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 GNRHR and GPCR proteins in relation to the binding site of spike protein RDB that provides insight into the binding stability. MD revealed that GNRHR could e ciently activate the biological pathway without changing the conformation in the binding site of spike protein .
To evaluate the stabilities of (spike -GNRHR ) and (spike -GPCR )Complexes during the MD simulations, root mean square deviation (RMSD) was calculated with respect to the initial structures along (the 3.0 (ns)) trajectories (Fig. 4). The trajectories indicated the stabilization of the receptor on the binding of (spike -GNRHR ) and (spike -GPCR )Complexes in the active site after 1.0 ns in system with a mean RMSD value of 2.5 nm. In addition, the stability of the system also proved the credibility of the docking results. Total energy of the most active conformation of the molecule was −8.79801e+06 kJ/mol. 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.
Binding of COVID-19 to GNRHR, a subtypes of GPCRS could lead to smell losing and hypogonadism.
The new coronavirus was found to have the ability to affect the central nervous system via the olfactory bulb or blood circulation in many studies. In addition to causing anosmia or hyposmia, as well as taste disorders, the virus can also cause headaches, eye pain, ear pain, dizziness, and hallucination (63). The gonadotropin-releasing hormone receptor (GnRHR), also known as the luteinizing hormone releasing hormone receptor (LHRHR), is a member of the seven-transmembrane, G-protein coupled receptor (GPCR) family(20). According to our ndings, COVID-19 could bind to GnRHR 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 de ciency 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 the nasal cavity. Axons of the olfactory sensory neurons extends to the region of the olfactory bulb, which is dedicated to process odour-related instructions (23).The nervus terminalis, or zeroeth cranial nerve, contains speci c neurons that produce gonadotropin-releasing hormone (GnRH). All vertebrate animals without sharks have a nervus terminalis, a chain of neurons implanted within vomeronasal or olfactory nerves in the region of the nasal canal, where it is considered a distinct nerve.
The main role of the gonadotropin-releasing hormone ( GnRH) constituent of the nervus terminalis is supposed to have neuromodulatory properties . (24). Numerous studies suggested that the role of the intranasal gonadotropin-releasing hormone ( GnRH) system is to adapt and modify olfactory information, maybe at opportune times for reproduction.(24) 30 to 40 percent of neurons located in the region of the nervus terminalis genetically express gonadotropin-releasing hormone (GnRH), and a small dozen of these neurons may produce gonadotropin-releasing hormone (GnRH) directly into blood veins underlying the olfactory epithelium (OE). (25). During prenatal GnRH neurons emerge from the nasal placode and travel into the brain (26). These neurons become critical ingredients of the hypothalamicpituitary-gonadal axis, which is required for activity of reproduction, after they enter the brain. Hypogonadotropic hypogonadism (HH) is caused when this mechanism is disrupted (HH). Kallman syndrome is a clinical term for HH that is accompanied by anosmia (KS) (5). A. Maestre de San Juan in 1856, he was the rst to describe a loss of smell and hypogonadism (a disorder in which the body produces insu cient amounts of a hormone). followed by F. J. Kallmann in 1944. (26,27)Kallmann discovered a co-segregation of hypogonadism and anosmia (loss of smell) in 3 families and hypothesised that the disease, now known as Kallmann syndrome, was hereditary (KS) (27). Changes in sense of smell are potentially connected with Covid-19, speci cally in patients with fever symptoms and women , according to 17 research articles found in databases; these changes rise Covid-19, degree of suspicion, and they warrant the implementation of surveillance and isolation measures as soon as possible(28). Although no current research has looked at its function in host contagion, but dysosmia (disordered smell perception) which has been found in cases with covid-19 infection might recall disorders related to the sense of smell typical of the Kallmann syndrome(KS), where the terminal nerve may play an important role in disruption of hormones . Furthermore, a study showed that COVID-19 patients had considerably lower levels of total Testosterone (tT) and luteinizing hormone ( LH) than control (p 0.0001), while controls had lower levels of circulating Estradiol(E2). 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) . Through the circulating amounts of gonadal sex steroids and stress hormones, the hypothalamus is functionally connected to the pituitary gland, gonads and adrenal gland (32) The primary regulator of mammalian function of reproduction in both men and womens is gonadotropin-releasing hormone (GnRH). It acts via distinct receptors, G-protein coupled receptors (GnRH) found in gonadotropes to induce production of the gonadotropin hormones, follicle (LH) (33).,and luteinizing -stimulating hormones (FSH) .COVID-19 could block thyroxin GPCR receptor 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 ndings, COVID-19 could bind to thyroxin GPCR receptor leading to blocking the binding of thyroxin to this receptor and disrupt its signal resulting in hypothyroidism . The thyroidstimulating hormone (TSH) or thyrotropin (34) receptor (TSHR) (35,36) is a member of the class A Gprotein-coupled receptors (GPCRs) (37). It was revealed that a signi cant proportion of hypothyroidism associated with COVID-19 and altered thyroid hormones was signi cantly 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 GPCR receptor leading to disorder in tastes .
According to the report of Moein et al., the publications included case reports or self-report surveys among different countries, and researchers proved the loss of smell and taste as a predictor of COVID-19 (40). Taste is one of the most important sensations for human life, enabling us to perceive different tastes from the diverse range of food available in nature and is a major determinant of our ingestion decisions (41). The anatomical units of taste detection are taste receptor cells (TRCs) that are assembled into taste buds distributed across different papillae of the tongue and palate epithelium. Taste processing is rst achieved at the level of TRCs that are activated by speci c tastants. They transmit information via sensory afferent bers to the gustatory cortex in the brain for taste perception . Three different morphologic subtypes of TRCs in taste buds sense the different tastes we perceive. Type I glial-like cells detect salty taste while type II cells expressing GPCRs detect sweet, umami, and bitter tastes. Type III cells sense sour stimuli (41,42). Therefore, according to our ndings COVID-19 could target GPCR receptor in Type II cells leading to taste disorder COVID-19 could bind to GPCR receptor leading to blocking of GPCR signaling and pulmonary edema COVID-19 mortality is primarily driven by abnormal alveolar uid metabolism of the lung, leading to uid 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).
GPCRs are primarily responsible for signal transduction/propagation cascades (44,45) . GPCRs 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 GPCRs 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). The A2B adenosine receptors and the 2 adrenergic receptors are two speci c GPCRs 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 GPCRs 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 GPCR signalling, and this effect may contribute to the pathophysiology of pulmonary edema..

Con icts of interest
The authors have no con icts of interest to declare that are relevant to the content of this article Funding None Figures Figure 1 shows the spike protein with GNRHR in the left side as see in the ger its bind like an ACE2 in the same binding energy in the right side spike protein binding with GPCR Figure 2 shows the binding of the spike protein which is in blue ,green and rose which is the tree chains of the protein chain A,B and C which bind to the white part which is GPCR as show its bind in alternative way to get the effect of losing smelling Figure 3 the binding of the spike protein which is in blue ,green and rose which is the tree chains of the protein chain A,B and C which bind to the white part which is GNRHR as show it binds in the RDB of spike protein so it is an alternative way to get the effect of losing smelling Page 20/21