Repositioning of ligands that target spike glycoprotein as potential drugs against SARS-CoV-2

The worldwide health emergency of the SARS-CoV-2 pandemic and the absence of a specic treatment for this new coronavirus have led to the use of computational strategies (drug repositioning) to search for treatments. The aim of this work is to identify FDA-approved drugs with the potential for binding to the spike structural glycoprotein at the hinge site, receptor binding motif (RBM), and fusion peptide (FP) using molecular docking simulations. It was identied drugs that binding to amino acids crucial for down to up conformational change, receptor recognition, and fusion of the viral membrane with the cell membrane. Results show some drugs that bind to hinge site amino acids (Varenicline, or even steroids as Betamethasone) while other drugs bind to crucial amino acids for RBM (Naldemedine, Atovaquone, Cefotetan) or FP (Edarbi, Maraviroc, Diuprednate); and highlights the Saquinavir that binds both the RBM and the FP. Therefore, these drugs could inhibit spike glycoprotein and prevent viral entry (possible anti-COVID-19 drugs). Several drugs are in clinical studies; focused on another pharmacological target (candesartan, Atovaquone, Losartan, Maviroc and Ritonavir) in this work we propose an additional target, the spike glycoprotein. These results can impact in the proposal of treatments that can inhibit the rst steps virus replication cycle.


Introduction
The COVID-19 disease is produced by the virus called SARS-COV-2 and emerged in China at the end of 2019. Currently, it has spread worldwide, so the World Health Organization (WHO) has declared a pandemic for which there is no treatment or vaccine [24].
The process to generate and test a vaccine will take at least a year [8]. However, by computational tools such as repositioning, already known drugs (known information on pharmacokinetics, pharmacodynamics and toxicity) and used for other pathologies can be proposed.
Repositioning drugs shows several advantages, for example, the time and the production costs of the drugs are dismissed because the drugs are already available in the market [17]. In the case of aspirin, this drug was initially used as analgesic and antipyretic and derived from the repositioning strategies; currently its new indication is as a treatment for colorectal cancer.
Another example is Hydroxychloroquine an antiparasitic and it is now used in the treatment of anti-arthritic systemic lupus erythematosus [33].
Conformationally, spike glycoprotein is organized on the viral surface in homotrimers [29]. When the RBM are hidden, the conformation is called down (receptor-inaccessible) (see Fig.1C). However, the homotrimer is asymmetric, because they constantly undergo structural rearrangement (up conformation) to fuse the viral membrane with the host cell membrane [13]. When two RBD domains are hidden (receptor-inaccessible) one RBD domain is exposed (receptor-accessible), named up conformation (see Fig.1D). This is due to the fact that the RBD of S1 undergoes hinge-like movements [32]. In SARS-CoV two hinge sites were characterized (hinge 1 site (354-361) and hinge 2 site (552-563), which are responsible for the switch in the up and down conformation [25]. The RBD in up position recognizes the receptor, through the receptor-binding motif (RBM), which binds to the outer surface of the claw-like structure of angiotensin-converting enzyme 2 (ACE2). The importance of RBM was demonstrated by comparing the sequences between SARS-CoV and SARS-CoV-2; and the (Leu455, Phe486, Gln493, Ser494 and Asn501) ve amino acid mutation improves a nity for the receptor. Therefore, it is believed that SARS-CoV-2 is more infectious than SARS-CoV [30].
After recognition and binding of the RBM by the ACE2, the spike glycoprotein needs to be proteolytically activated at the S1/S2 boundary, such that S1 dissociates and S2 undergoes a dramatic structural change. These SARS-CoV-2 entry-activating proteases include cell surface type II transmembrane serine proteases (TMPRSS2) and lysosomal proteases cathepsins. The structural changes that S2 shows when dissociated is the exposure of the fusion peptide (FP) located at the amino acid position 816 to 855.
Subsequently triggering the insertion of the FP into the host membrane and allowing the fusion of membranes (viral and cellular) to release the viral genome into the cytoplasm. [26].
As mentioned above, the spike glycoprotein is in rst contact with the host cell and it is crucial for various processes such as attachment, receptor binding, membrane fusion via conformational changes, internalization of the virus, host tissue tropism [7,30]. Therefore, it is one of the main targets for the development of vaccines and antivirals.
In this work, we repositioned drugs that have been previously approved by the FDA for other therapeutic use, which could be used as a treatment against COVID-19 disease, targeting the spike glycoprotein involved in three speci c events (conformational change down to up, recognition of the ACE2 enzyme and membrane fusion).

Methodology
Molecular modeling of spike glycoprotein SARS-CoV-2 Two crystal structures which represent down and up conformational states of the spike glycoprotein were used. The ID PDB: 6VYB represents the up conformation whereas ID PDB: 6VXX constitutes the down conformation. Both crystals showed missing fragments and two disul de bridges (480-488 and 840-851) which are important in the RBM and FP region respectively, thus, these were completed using the Program Modeller 9.23 (scripts: loop.py, segment.py and model-disul de.py) [31]. After adding the missing residues and two disul de bridges, the resultant tridimensional (3D) models were denominated as 6VXX-ll and 6VYB-ll down and up conformation correspondingly. In the model 6VYB-ll the chain B the site RBD is exposed. The crystals glycosylation's were preserved and added by the Chimera program [20] to the models 6VXX-ll and 6VYB-ll.

Selection of drugs to be repositioned
The 3D structures of the drugs were obtained from the database Zinc (http://zinc.docking.org/), accessed March 2020, download the subset fda, drugs which are approved by the FDA, per DrugBank selecting those that comply with the rules of Lipinski and have the properties ADMET (Absorption, Distribution, Metabolism, Elimination and Toxicity), binding to albumin, and solubility in water and in DMSO [14]. Also, the subset fda was submitted to Data Warrior program to predict drugs that were mutagenic, tumorigenic, reproductive or irritants effect. The drugs that presented those effects were eliminated and selecting for docking studies only those drugs that were safe were docked. In total were analyzed 1372 drugs; according to the criteria, they were nally chosen 1322 drugs were selected to be coupled to spike glycoprotein by docking studies.

Molecular docking
Molecular docking studies were carried out using the AutoDock Vina program [28]. The spike glycoprotein was rigid and the ligands exible. The drugs with the highest a nity and those that reach key amino acids that in uences the spike glycoprotein function were selected. The dockings simulation were focused to the hinge sites, the RBM and FP, for the 3 chains of the spike glycoprotein trimmer in the up and down conformation. In total, 1322 dockings were achieved for the A, B and C chain for the down and up conformation.

Drugs selection
Once nishing the docking simulations, Discovery Studio Visualizer (https://www.3dsbiovia.com/products/collaborativescience/biovia-discovery-studio/) and Chimera [20] programs were used to visualize the docking results. Drugs with the highest a nity (more negative) and that interact with key amino acids for function at the interest sites (hinge, RBM and FP) were selected.

Results
Molecular modeling of spike glycoprotein Fig. 2 shows the conformational structure of the models obtained by molecular modeling. The 6VXX-ll model is seen in the down conformation, and the up conformation is seen in the 6VYB-ll model.

Molecular docking and drug selection
Once the molecular docking was performed, the drugs with the highest a nity for the spike protein (mainly with amino acids belonging to the hinge site, the RBM and the FP) were selected. The drugs are listed in tables.The tables show the drug name, database Zinc identi cation number, use according to currently described pharmacological action, FDA approval number, inclusion in clinical studies related to COVID-19, a nity in kcal/mol (from highest to lowest a nity) and amino acid interactions from spike glycoprotein are indicated. The general scheme shows the possible sites of interaction (Fig.1).

Docking simulations on the hinge site
Docking simulations were performed on hinge sites (1 and 2), however only drug interactions were found at hinge site 1 (see table   1 and table 2). The a nity binding scores and molecular interactions of the drugs for down conformation are detailed in the Table1. Fig. 3A shows Varenicline which binds to chain B and C in the hinge site 1, RBM and NTD with an a nity of -7. 6 kcal/mol in down conformation, interacting by Van der Waals forces (Pro230C, Lys458B and Arg466B), π-interactions (Ile231C and Tyr200C), alkyl Interactions (Trp353B and Arg355B), π-cation interactions (Asp467B and Arg355B) and nally forming hydrogen bonds (Gly232C, Glu465B, Asp198C and Gly199C).In addition Fig. 3B shows that Docosahexaenoic acid interacts by Van der Waals forces with the amino acids Pro230B, Val227B, His207B, Asp228B, Leu229B, Phe168B, Tyr170B, Ile128B, Ile203B, Phe194B, Ile119B, Phe192B, Trp104B and Asn121B, by an electrostatic interaction and a hydrogen bonds with Arg357B, and nally alkl interactions with Leu179B and Val126B. Other interesting drug was sulbactam (Fig. 3C) which interacts with in the hinge 1 site, the RBM and the NTD by Van der Waals forces with the amino acid Asp198C, Pro426B, Leu518B, Glu516B, Phe515B, Tyr396B and Ser514B, and two hydrogen bonds with the amino acids Arg466B and Arg355B.
Docking simulations on the RBM Table 3 and 4 show the drugs selected for the RBM in the down and up conformation, respectively.
Regarding the drug Cefotetan, although it did not show one of the highest a nities, this drug is the one that interacts with the most crucial amino acids for the formation of the spike-ACE2 complex. The drug Cefotetan (Fig. 6C) is bound in the up conformation by Van der Waals forces (Ser349B, Tyr489B, Leu455B, Pro491B, Gly476B, Ser477B, Phe490B, Gln493B, Leu492B, Tyr351B, Tyr449B and Asp467B), forms hydrogen bonds (Tyr449B, Asn450B, Arg454B and Thr478B), alkyl interactions (Pro479B and Leu452B) and π-sulfur interactions (Phe456B and Tyr449B).

Docking simulations on the FP
The drugs which displayed the highest a nity for the fusion peptide in both the down and up conformation are summarized in tables 5 and 6 respectively. Results from the docking analyses are ordered from highest to lowest a nity. Some representative drug interactions are described below.

Discussion
Spike glycoprotein is the structural protein of the SARS-CoV-2 virus that allows adhesion and binding to the receptor, making it crucial in the rst step of infection [34]. Currently it has been considered as one of the main targets for vaccine design. However, this protein evades the immune response due to the dynamic structure, for example, when is found in the down conformation (receptor-inaccessible) where it hides RBD (hidden RBD) and prevents the binding of neutralizing antibodies. Additionally, when is found in the up conformation (accessible receptor) shows the RBD which has high a nity for the receptor (higher a nity than SARS-CoV). In addition to the above, the virus can enter through two routes, by receptor-mediated endocytosis and by direct fusion to the cell membrane because the fusion peptide can be exposed by cell surface proteases (depends on cell type). So can be developed vaccines with target the membrane fusion S2 subunit, however S2 subunit is less immunogenic than S1 subunit [6,23]. Therefore, in this work, we have employed an in silico strategy called drug repositioning, in which we looked for drugs available and could target the spike glycoprotein (hinge sites, RBM and FP of interest). From this analysis, it was possible to identify 43 drugs, of which 8 are included in reported clinical trials (https://clinicaltrials.gov/).
Regarding the hinge region in the down conformation, 3 drugs were found with the possibility of binding and preventing the change to the up conformation and therefore avoiding exposure of the receptor binding site. The drug with highest a nity is Varenicline (-7.6 kcal/mol), however it is not found in any clinical study, unlike Docosahexaenoic acid (DHA) and Sulbactam, which are being studied as a complementary part of a main treatment. In the DHA clinical trial, the main treatment with Fenretinide (LAU-7b) was a synthetic retinoid derivative and has been used in the treatment of some types of cancer and cystic brosis. DHA is an omega-3 fatty acid and can be used as part of the diet. Another fatty acid that has been reported in 5 clinical trials is Icosapent Ethyl (VascepaTM) but not as part of a dietary food supplement. The effect is found on In ammatory Biomarkers in Individuals with COVID-19. Arachidonic acid (AA) and other unsaturated fatty acids (especially eicosapentaenoic acd, EPA and DHA) are known to inactivate enveloped viruses and inhibit proliferation so they can serve as endogenous antivirals [5].
Furthermore, Sulbactam is an antibiotic inhibitor of the beta lactamase enzyme and was used in conjunction with intravenous ampicillin [9] to avoid secondary infection in patients with Cytokine Adsorption in Severe COVID-19 Pneumonia Requiring Extracorporeal Membrane Oxygenation (CYCOV).
As mentioned before, spike glycoprotein has been shown to be very dynamic due to the hinge sites, preventing switch up to down conformation can help to avoid hiding the receptor binding site and in therapy together (with neutralizing antibodies) can prevent the viral attachment. The best evaluated drugs to bind at the hinge site in the up position were 5 drugs (Accolate, Tigecycline, Betamethasone, Triamcinolone acetonide, Atazanavir), which two are in clinical trials (Betamethasone and Triamcinolone acetonide; both corticosteroids). Betamehasone is a steroid from the group of corticosteroids, it was used in pregnancies complicated by SARS-CoV-2 for its immunosuppressive and anti-in ammatory properties. However, a potential diabetogenic effect was found, in addition to that SARS-CoV-2 and other coronavirus may have effects on glycemia [10].
Therefore, its use in clinical trials is not recommended and has not been reported. In contrast, the use of Dexamethasone for the short-term in severe, intubated, COVID-19 patients has recently been recommended due to the anti-in ammatory effect that would limit the production of and damaging effect of the cytokines, but will also inhibit the protective function of T cells and block B cells from making antibodies [27]. The use of Betamehasone and Dexamehasone effect on hinge site blockage not been explored, the two corticosteroids have the same molecular weight and high structural similarity (the only difference is the orientation of the methyl group on carbon 16) so which could have similar effects. Use of inhaled corticosteroids had been associated with lower expression of ACE2 and TMPRSS2, but treatment with Triamcinolone Acetonide did not decrease expression of either gene [19]. On the other hand, the use of Atazanavir (protease inhibitor) has been reported in computer studies with inhibitory potency activity with Kd of 94.94 nM against the SARS-CoV-2 3C-like protease inhibitor [2].
On the other hand, we have found 10 better evaluated drugs from the docking studies at the RBM site in the down conformation, which although it would not prevent the change to the up conformation, would prevent receptor binding. Nine of the drugs show interaction with two amino acids (Leu455 and Gln493), of the 5 reported (Leu455, Phe486, Gln493, Ser494 and Asn501) as crucial for the RBM site in the formation of a stable complex between spike viral glycoprotein and human ACE2 [15,30]. Only Saquinavir drug shows interaction with a crucial amino acid Leu455. Despite these interactions, the a nity for the glycoprotein is different, with the highest a nity being Naldemedine (-12.7 kcal/mol) and the lowest a nity Suvorexant, Riociguat, Diabeta and Candesartan drugs (-10.6 kcal/mol). Candesartan is included in three clinical studies due to the selective antagonist function of angiotensin II AT1 receptors; therefore, the present study reports an additional mechanism of this drug to avoid SARS-CoV-2 infection.
When the conformation of the spike glycoprotein in the S1 domain changes to the up position, it shows high a nity for the receptor, which can be blocked by drugs that bind to the RBM in up position. 6 drugs were found to show a nity for the RBM site in up position. However, the a nity values are lower compared to the a nity shown in down position (-9.7 kcal/mol in up vs -12.7 kcal/mol in down position). The drugs with the highest a nity were Tedizolid Phosphate and Atovaquone with (-9.7 kcal/mol).
Atovaquone is in a clinical trial (in combination with Azithromycin) to treat COVID-19, due to its ability to treat or prevent pneumonia caused by Pneumocystis carinii (clinical trials). On the other hand, the drug with the lowest a nity was Rosiglitazone (-8.3 kcal/mol) and was used to regulate glucose in type 2 diabetes, however its use is discontinued [21].
Considering another point in the viral replication cycle, we looked for drugs capable of binding to FP and taking into account that it is a conserved sequence with SARS-CoV that shows two FP sites. FP1 (amino acid 798 to 818) and FP2 (amino acid 816 to 835) which have characteristics of an active fusion region working, furthermore, they postulate that the regions function cooperatively as an extended FP (FP1-2) [12,16]. For the case of SARS-CoV-2 spike glycoprotein, Tang and collaborators recently determined that FP is found in the region located in amino acids 816 to 855, which corresponds to the region 798-835 for SARS-CoV spike glycoprotein. The amino acids Leu803, Leu804, Phe823, Cys822, Cys833, Asp830 and Leu831 in SARS-CoV (corresponding to the amino acids Leu821, Leu822, Phe823, Cys840, Asp848, Leu849, and Cys851 in SARS-CoV-2) have been shown by biophysical, site-directed mutagenicity functional assays to be essential for membrane fusion function [12] (Lai et al, 2017). In addition, these amino acids are conserved in SARS-CoV, MERS-CoV and SARS-CoV-2 [26].
Of worthy interest is that nine drugs were found to be bound in the down conformation, within the FP and interact mainly with two highly conserved amino acids (Cys851A and Leu849A) which are crucial for the process of membrane fusion in the family of the coronavirus [26]. However, no drug has been reported in clinical studies. The drug with the highest a nity was Saquinavir (-11.1 kcal/mol) and the lowest a nity was Di uprednate (-8.9 kcal/mol). However Di uprednate interacts with an additional amino acid to modulate the membrane fusion process (Cys840A) in total three critical amino acids (Cys840A, Cys851A and Leu849A). In order to be able to use these drugs in future clinical studies (in addition to the interactions found), the commercial presentation must be considered. Such is the case of Terconazole which can be administered as a cream and suppository vaginal presentation (https://www.accessdata.fda.gov), so it could not be used for treatment of COVID-19 in that presentation.
On the other hand, when the protein is in the up position, the best evaluated drugs to bind FP are 10, of which 5 (Maraviroc, Ritonavir, Bosentan, Fosinopril sodium, Ceftazidime) are found in clinical trials. The Maraviroc, a C-C Chemokine Receptor 5 (CCR5) antagonist, is well-tolerated without signi cant side effects in its current use in patients with HIV; in patients with  has been used as a drug against the main protease (Mpro) of SARS-CoV-2 (determined by computer methods) [22]. The drug found in the largest number of clinical trials is Ritonavir, most of it is used in combination with Lopinavir both are protease inhibitors to treat HIV and had been used against other coronaviruses (SARS-CoV and MERS-CoV) [4]. The use in SARS-CoV-2 also seeks to inhibit cellular proteases to prevent completion of the viral replication cycle; however some clinical trials have not been concluded and others have concluded that there is no difference in using ritonavir in hospitalized adult patients with severe COVID-19 and standard medical care treatment [3]. Another class of drugs that have been used for the treatment of COVID-19 are ACE inhibitors such as Fosinopril sodium, which is being studied in a clinical trial (https://clinicaltrials.gov/), which seeks to block the virus receptor. Therefore, these 3 drugs, in addition to the effect described in the clinical trial, could have an effect in preventing the fusion of the viral membrane with the cell membrane. Interesting, Saquinavir has been found to be the most a nity for the FP for both conformations (up and down), and showing an a nity of -11 kcal/mol and in addition it binds to four crucial amino acids in the membrane fusion process (Cys851C, Cys840C, Leu849C and Asp848C). Furthermore it can bind to the RBM in down position. Therefore, this drug is a potential candidate to inhibit the FP and RBM sites of the spike glycoprotein. It has also been reported by computational methods effect on 3C-like protease (3CL pro) also called the main protease (Mpro) [1,11], so it could be considered a multi-target drug, which confers a greater probability of success.
Considering the data reported, the use of multi-target drugs can be proposed, and by interfering at different points in the replication cycle, it could have a better effect. It is even possible to propose combined therapies that prevent both viral entry (spike target) and replication cycle events inside the host cell to be used as a preventive and therapeutic treatment.

Conclusion
By using in silico tools, a subgroup of drugs previously approved by the FDA for use in various human diseases has been analyzed. From this group of drugs, we were able to identify potential drugs that could inhibit the function of spike glycoprotein interfering, the entry of SARS-CoV-2 on host cell inhibiting the viral infection. Among these potential drugs some are reported in clinical trials as therapy in conjunction with other drugs (DHA, Sulbactam and Atovaquone); others are part of the main therapy due to its effect on the ACE2 receptor (Fosinopril sodium and Candesartan), anti-in ammatory effect (Betamethasone, Triamcinolone acetonide); effect on proteases (Atazanavir and Ritonavir). Additionally, Maraviroc is used as a CCR5 antagonist and determined by computer methods as a possible protease inhibitor. On the other hand, even though Saquinavir has not been reported in clinical trials it has been suggested from theoretical and in silico studies, a 3C-like protease effect was determined.
Therefore, in this work, we propose other therapeutic targets that could bind the spike protein and which may have a summative effect with the other reported targets/uses.