key: cord-1038875-pjd5r3r1
authors: Molavi, Zahra; Razi, Sara; Mirmotalebisohi, Seyed Amir; Adibi, Amirjafar; Sameni, Marzieh; Karami, Farshid; Niazi, Vahid; Niknam, Zahra; Aliashrafi, Morteza; Taheri, Mohammad; Ghafouri-Fard, Soudeh; jeibouei, Shabnam; Mahdian, Soodeh; Zali, Hakimeh; Ranjbar, Mohammad Mehdi; Yazdani, Mohsen
title: Identification of FDA Approved Drugs Against SARS-CoV-2 RNA Dependent RNA Polymerase (RdRp) and 3-chymotrypsin-like Protease (3CLpro), Drug Repurposing Approach
date: 2021-03-31
journal: Biomed Pharmacother
DOI: 10.1016/j.biopha.2021.111544
sha: d0ac7867da5a40d39d51b5dabca296222c47e960
doc_id: 1038875
cord_uid: pjd5r3r1

The RNA-dependent RNA polymerase (RdRp) and 3C-like protease (3CLpro) from SARS-CoV-2 play crucial roles in the viral life cycle and are considered the most promising targets for drug discovery against SARS-CoV-2. In this study, FDA-approved drugs were screened to identify the probable anti-RdRp and 3CLpro inhibitors by molecular docking approach. The number of ligands selected from the PubChem database of NCBI for screening was 1760. Ligands were energy minimized using Open Babel. The RdRp and 3CLpro protein sequences were retrieved from the NCBI database. For Homology Modeling predictions, we used the Swiss model server. Their structure was then energetically minimized using SPDB viewer software and visualized in the CHIMERA UCSF software. Molecular dockings were performed using AutoDock Vina, and candidate drugs were selected based on binding affinity (∆G). Hydrogen bonding and hydrophobic interactions between ligands and proteins were visualized using Ligplot and the Discovery Studio Visualizer v3.0 software. Our results showed 58 drugs against RdRp, which had binding energy of -8.5 or less, and 69 drugs to inhibit the 3CLpro enzyme with a binding energy of -8.1 or less. Six drugs based on binding energy and number of hydrogen bonds were chosen for the next step of molecular dynamics (MD) simulations to investigate drug-protein interactions (including Nilotinib, Imatinib and dihydroergotamine for 3clpro and Lapatinib, Dexasone and Relategravir for RdRp). Except for Lapatinib, other drugs-complexes were stable during MD simulation. Raltegravir, an anti-HIV drug, was observed to be the best compound against RdRp based on docking binding energy (-9.5 kcal/mole) and MD results. According to the MD results and binding energy, dihydroergotamine is a suitable candidate for 3clpro inhibition (−9.6 kcal/mol). These drugs were classified into several categories, including antiviral, antibacterial, anti-inflammatory, anti-allergic, cardiovascular, anticoagulant, BPH and impotence, antipsychotic, antimigraine, anticancer, and so on. The common prescription-indications for some of these medication categories appeared somewhat in line with manifestations of COVID-19. We hope that they can be beneficial for patients with certain specific symptoms of SARS-CoV-2 infection, but they can also probably inhibit viral enzymes. We recommend further experimental evaluations in vitro and in vivo on these FDA-approved drugs to assess their potential antiviral effect on SARS-CoV-2.

The world is currently experiencing an emerging pandemic called COVID-19 (caused by SARS-CoV-2), to which no effective antiviral drugs or vaccines have been approved to date [1] . A recent hypothesis has proposed that COVID-19 may have three phases. Some of the drugs are probably more effective in each phase separately. These three phases are called the viral early infection phase, the pulmonary phase, and the hyper-inflammation phase [2] . In the early infection phase, antiviral drugs are probably the best option. In the second phase, due to the involvement of the immune system, the lungs become involved. Some symptoms, such as cough, shortness of breath, and hypoxia, are observed in this phase. Blood clots are also reported mostly in the second phase. In the hyper-inflammation phase, the cytokine storm is triggered by the activation of the immune system. The cytokine storm leads to more severe damage to the lungs, kidneys, heart, and other organs. In this phase, the anti-inflammatory category of drug candidates is probably better to be more investigated. Given that these phases overlap, no single drug is expected to be sufficient for all three phases, and a combination of drugs would probably be more efficient [2] .

The rapid global spread of this virus has underscored the need to develop anti-Coronavirus therapies. Several approaches and strategies are typically used to detect a potential antiviral treatment against various infections, such as the new Coronavirus. One possible common approach is applying the existing broad-spectrum antiviral drugs using standard assays.

Screening the previously approved chemical compounds by bioinformatics tools is another fast J o u r n a l P r e -p r o o f method in antiviral drug discovery. In this method, medications are evaluated for their potency to inhibit some essential elements of the new viruses [1, 3] .

The 3CLpro is the prime enzyme responsible for proteolysis. It cleaves the viral polyprotein into distinct functional components [4] . The essential value of 3CLpro in the virus life cycle makes it a suitable target for developing effective antiviral drugs against different Coronaviruses [5, 6] .

3Clpro offers unconventional Cys catalytic residues with a unique diversification. Differently from other chymotrypsin-like enzymes and many SER (or Cys) hydrolases, including catalytic Cys-His Dyad instead of a canonical Ser (Cys)-His-Asp (Glu) triad8. The Cys145 and His41 catalytic residues in 3Clpro are entombed on the protein surface in an active site cavity. This cavity can contain four substrates in P1' to P4 positions and is flanked by both Domains I and II residues [7] . Another essential non-structural protein of the Coronavirus is the RNA-dependent RNA polymerase (RdRp, also known as nsp12) [8] . RdRp catalyzes the viral RNA synthesis and thus plays a pivotal role in the SARS-CoV-2 replication and transcription process, probably along with nsp7 and nsp8 as co-factors [9, 10] . Among coronaviruses, particularly in SARS-CoV-2, essential sites such as template entry and binding, polymerase activity reaction site followed by the exit through the tunnel (thumb) are highly conserved. Tyr618, Cys622, Asn691, Asn695, Met755, Ile756, Leu757, Leu758, Ser759, Asp760, Asp761, Ala762, Val763, Glu811, Phe812, Cys813 and Ser814 are the critical residues of interaction in the RDRP active site. The residue of active sites are adjoining aspartates, i.e. Asp761 and Asp762, participate in specific RdRp enzyme reactions [11] .

Different anti-RNA polymerase drugs currently on the market have been previously approved for use against various viruses, including Ribavirin [12] , Remdesivir [13] , Galidesivir [14] , and Tenofovir [15] . They are presently being examined against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). For the 3CLpro target, several studies and current clinical trials have proposed the Lopinavir [16] , Ritonavir [17] , Darunavir [18] , Ganovo [19] , ASC09F [20] , and Cobicistat [21] . Ritonavir/Lopinavir (LPV) is one of the most commonly reported clinical trials for COVID-19. Even though some data indicate somewhat efficacy for LPV, its severe side effects are considerable. [22, 23] These confirm that RdRp and 3CLpro can be recommended as valuable targets for drug design against SARS-CoV-2, and inhibition of their activity seems a promising strategy to cure SARS-CoV-2 infection. In this study, we used a target-based virtual screening approach to identify novel inhibitors of SARS-CoV-2 RdRp and 3CLpro.

J o u r n a l P r e -p r o o f Methods

Drug repurposing using virtual screening (VS) techniques is one of the rapid and most promising strategies to candidate drugs against the Coronavirus [24] .

In this study, 3D structures of 1760 FDA-approved drugs were retrieved from the NCBI PubChem database [25] . In fact, there were three-dimensional structures for approximately 2,500 approved small molecule drugs (not proteins, etc); Therefore, We first removed some of the structures from our selection set including, the two-component structures, tiny compounds weighing less than 100 kDa, and the large-complex compounds with a high number of rotatable bonds. The remaining small molecules were filtered and selected for further docking analysis, including the 1760 small molecule drugs. The conjugate gradient geometry optimization was performed using Open Babel [26] and MMFF94 force fields for each drug geometry. [27] 

RdRp and 3c-like proteinase (3CLpro) (from reference sequence of Accession number NC_045512) protein sequences were retrieved from the NCBI database. Then, homology modeling predictions were carried out using the Swiss model server (https://swissmodel.expasy.org/). The structures were energetically minimized using SPDB viewer software [28] and visualized by the CHIMERA UCSF v1.14 software [29] . The binding sites (active sites) in target proteins were identified by evaluating protein grooves in CHIMERA UCSF software 22 [30] and considering the previous studies [19, 31] . Since recently crystallography structures of the proteins were reported in PDB databank, we performed superimposing to check our homology modelling similarity with the crystallography results.

Superimposing of the modelled structure with deposited crystallography structures available in PDB (Protein Databank) revealed the root mean square deviation (RMSD) value of <2 angstroms (among 0.3 to 1.5 angstrom), which meant a perfect fit. Therefore, modelling has insignificant impacts on our overall results compared to using crystallography structures.

J o u r n a l P r e -p r o o f

All nonpolar hydrogens were merged. Partial atomic charges were then assigned using the Gasteiger-Marsili approach for accurate ionization and tautomeric states of residues. Besides, charges were added to models, and Kollman United Atom charges and atomic salvation parameters were performed.

Molecular docking was carried out to evaluate possible energy of interactions, hydrogen bonds, 

An100 ns MD simulation for RdRp and 3clpro was used to confirm the docking results for identified candidate antiviral drugs. Molecular dynamics (MD) is a mathematical tool for analyzing the system dynamic structural behaviour; in this process, atoms and molecules interact as a time-based function. The simulations of MD take the versatility of goals into account. The structural parameter RMSD and the number of intermolecular H-bonds have been used for determining the stability, dynamics and compactness of protein-drug complexes [36] .

Six drugs were chosen for MD analysis based on binding energy and the number of hydrogen bonds in docking analysis. Six simulations were performed using the GROMACS 5.1.4

J o u r n a l P r e -p r o o f simulation suite for FDA-approved drugs containing Nilotinib, Imatinib, and dihydroergotamine for 3clpro and Lapatinib, Dexasone, and Relategravir for RdRp. The gromos54a7 force field was utilized for the complexes [37] . The ATB server was used for the preparation of the coordinates and topology of ligands [38] . The complexes were then solvated with TIP3P water molecules in a truncated octahedron periodic box with an 8 Å radius buffer zone of water molecules around the complexes using Gmx Editconf&Solvate softwares. Then counter ions have been added with the tool of Gromacs to neutralize the overall system charge. The surface charge of the structure was neutralized by adding several sodium ions. Reduction of energy on the structures was performed with 50,000 steps using the steepest descent method for eliminating van der Waals interactions and formation of hydrogen bonds between water molecules and the complex. In the next step, the system temperature was gradually increased from 0 to 310 ° K for 500 ps at constant volume, and then at constant pressure for 500 ps the system was equilibrated. Molecular dynamics simulations were performed at a temperature of 310 K and a duration of 100 nanoseconds. Nonbonded interactions with 10 Å intervals were calculated by the PME method. The SHAKE algorithm was used to limit the hydrogen atom bonds to increase computational speed. Finally, the simulation information was saved at 0.2 ps intervals for analysis.

Molecular docking was performed on FDA-approved drugs to determine the potential drug candidates for inhibiting the SARS-CoV-2. The docking was based on the recognition of the binding pocket of Homology Modeled RdRp and 3CLpro enzymes. The SWISS online server modeled the viral proteins. The number of ligands selected from the PubChem database of NCBI for screening was 1760. All these drugs were docked against the two target enzymes of SARS-CoV-2 and ranked based on their binding affinity. The compounds with a binding affinity of −8.5 or less were considered better compounds, possibly inhibiting the RdRp enzyme. The binding affinity of −8.1 or less was considered the selection criterion against the 3CLpro protein.

We used AutoDock Vina to dock the drugs to achieve more accurate medicines related to the two viral essential components. We first selected the top 100 medications for each viral target based on the order of their affinity energies. Depending on the rate of changes in the affinity energies among the drugs ordered, we selected 58 candidate drugs against the active site of the RdRp enzyme with an affinity of -8.5 or less and 69 candidate drugs against the active site of the J o u r n a l P r e -p r o o f 3CLpro enzyme with affinity binding. -8.1 or less. We observed that 20 drugs had binding affinity energy less than -9 against the RdRp target. However, only seven drugs had binding affinity energy less than -9 against the 3CLpro. They are likely to provide promising drugs against SARS-CoV-2. All the candidate drugs were then classified into several categories (Tables No.1 and No.2). We sought further studies on COVID-19 drugs to validate our identified drugs. We found that some of these candidate drugs have already been introduced or validated by various other studies, including in-silico, preclinical, and clinical trials. These verifying studies are available in Table No .4. We also compared the two identified drug lists using the online Venn diagram tool. Supplementary Figure S1 depicts the Venn diagram comparing the two drug lists against RdRp and 3CLpro. We found that 32 drugs were shared between the two drug lists. They seem to be promising since they would probably inhibit both of the essential viral components. Supplementary Table S1 lists these 32 shared drugs (vs. RdRp and 3CLpro). Raltegravir, an anti-HIV drug, was discovered to be the best compound against RdRp based on binding energy (-9.5 kcal/mole). Doxazosin (−9.3 kcal/mol) was also a BPH drug that appeared Tables No.1 and No.2. Here we discuss some of our candidate drugs previously introduced or validated by other types of studies, including in-silico, preclinical, and clinical trials.

According to our results, some antiviral drugs were detected against 3CLpro including, Bictegravir, Dolutegravir, Raltegravir, and Indinavir; among them, Raltegravir was identified to have interaction with RdRp too. Indinavir was previously suggested as a repurposing candidate against nCoV-2019 [9, 10]. Dolutegravir is an Anti-HIV drug that has already been registered in clinical trials for COVID-19 treatment [39, 40] . Raltegravir was also reported as a possible drug against multi-targets, including 3CLpro targets in in-silico studies [41, 42] . However, Bictegravir, an anti-HIV drug, has not been studied in-silico or registered as any clinical trial.

We identified several antibacterials as potential candidates against RdRp and 3CLpro. These antibacterials included Eravacycline, Sultamicillin, Cefpiramide, Ceftobiprole, Cefoperazone, Novobiocin, Alatrofloxacin, Ceftolozane, and Ceftriaxone. Besides, Eravacycline is an antibiotic previously proposed by a virtual docking screening study [43] . The use of antibiotics is beneficial for patients with COVID-19 in two ways. Since bacterial diseases are the main challenges for patients admitted to the intensive care units (ICU), they can probably play dual roles as antiviral and antibacterial [44] . Due to the adverse side effects of both types of drugs on J o u r n a l P r e -p r o o f the immune system and the body, only one drug with two functions is likely to lead to fewer side effects.

New evidence suggests that Cytokine storm Syndrome (CSS), a systemic inflammatory response, threatens a subset of patients with COVID-19 [45] . Acute Respiratory Distress Syndrome (ARDS) is also rooted in the pathogenesis of inflammatory mediators. It appears to be necessary to prevent increased inflammation for limiting the possible progression of ARDS [46] . Some of the drugs that have gained acceptable affinity scores in docking are classified as antiinflammatory drugs in the DrugBank database [47] . Among these in-silico detected drugs,

Hesperidin has been previously introduced in other in-silico studies to have antiviral potency by inhibiting SARS-CoV-2 main protease, PLpro (papain-like protease), and helicase (Nsp13) [19, [48] [49] [50] [51] [52] . It has also been previously computationally predicted to have a binding affinity to the ACE II receptor, so it might probably help treat COVID-19 in this way [53] . It has already treated cells against the influenza type-A virus in vitro by upregulating P38, JNK, and enhancing cell-autonomous immunity [54] . The two other inflammatory drugs, available on our result list, have also been suggested by other in-silico docking studies, including Diosmin [48] and Rutin [48, 50] . Rutin is predicted to inhibit the viral helicase (Nsp13) [52] .

Asthma and airway allergies have similar pathogenetic mechanisms to some respiratory tract infections [55] , and the main manifestations are related to respiration in both COVID-19 and allergy/asthma. Montelukast, an antiasthmatic drug identified in our result list, is registered for clinical trial against COVID-19 (NCT04389411). Antiasthmatic drugs stabilize mast cells to reduce the release of cytokines. They alleviate the inflammatory cell infiltration into the lungs [56] . Montelukast, a cysteinyl leukotriene receptor antagonist (cysLT), has anti-inflammatory effects. It reduces cytokine production. Montelukast may reduce the inflammatory response in severe cases of COVID-19. It might limit the progression of the disease [57] .

A previous study has shown that an anti-allergy drug interfered with SARS-CoV replication. The SARS-CoV is a positive-strand RNA virus. The drug was called cyclosporin and was inhaled orally [58] . Some anti-allergy drugs have also appeared in our results table with appropriate docking scores (binding energies), such as Chromoglycic acid and Zafirlukast. They inhibit the release of chemical mediators from our sensitized mast cells and are used to prevent asthma. [59] Chromoglycic acid played a therapeutic role in Balb/c mice infected with influenza A (H5N1) J o u r n a l P r e -p r o o f compared to the PBS treated group. However, it did not affect the viral load [60] . It also has been predicted to have a binding affinity against the SARS-CoV-2 Nsp16 by another in-silico Study [61] . We recommend them to be further examined for their possible antiviral effect on the SARS-CoV-2 virus. Since the most severe symptoms of COVID-19 are respiratory distress, the use of certain anti-allergy medications may reduce the severity of respiratory manifestations of COVID-19 infection and may help breathe in patients with COVID-19.

Acute myocardial injury has been reported in some severe cases of COVID-19 [62] . Patients with chronic cardiovascular disease are among the most susceptible groups in severe COVID-19

and have the highest morbidity rate among COVID-19 severe cases [63] . Interestingly, ten cardiovascular drugs have appeared with proper docking scores (binding energies) in our results (Tables No.1 and No.2). Some previous studies have reported the effect of some of these cardiovascular drugs on various viral infections, including SARS-CoV, HCMV, and SARS-CoV-2. For example, Avatrombopag is predicted to bind to ACEII and ACEI (in-silico).

Avatrombopag likely blocks SARS-CoV-2 interaction with host receptors [64] . Conivaptan, known as hyponatremia treatment, is also previously predicted to bind to 3CLpro in-silico, and it has also scored adequately on our result list [52] .

An ARB is reported to prevent the aggravation of acute lung injury in mice infected with SARS-CoV, which is closely related to SARS-CoV-2 [65] . Eltrombopag is a Thrombopoietin Receptor

Agonist and improves the low number of platelet counts in ITP and treats Thrombocytopenia.

Interestingly, platelets have been shown to play a role in defense against respiratory viruses.

Activated platelets engulf HIN1 virions and secrete antiviral molecules to destroy virions. The H1N1 virus is close to SARS-CoV-2. We can probably assume that it may show beneficial effects in SARS-CoV-2 Treatment. The Eltrombopag is also used in the Treatment of HCV and HIV-1. It is also an iron chelator and can prevent virus replication in human cytomegalovirus (HCMV). Interestingly an in-vitro study has confirmed its effect against SARS-CoV-2 in Vero cells [66] [67] [68] [69] .

It has been reported that SARS-CoV-2 can also induce infection-associated Coagulopathies. receptor) is an anticoagulant drug. The usage of these drugs is recommended in one letter for COVID-19 [72] . Since COVID-19 pneumonia and myocardial infarction (MI) are concomitant, Ticagrelor seems to contribute to patient survival for various reasons. One reason is that the PLATO study has shown that sepsis and pulmonary infections were less common in individuals using Ticagrelor. It prevents DIC development by reducing pro-inflammatory factors and platelet reactivation [73] ; besides, it reduces lung injury in pneumonia by reducing thromboinflammatory factors [74] . Surprisingly, recently it has also been reported as an antibacterial that acts against some antibiotic-resistant gram-positive bacteria [75] .

Edoxaban, a direct oral anticoagulant (DOAC), has also appeared on our results. OACs are indicated for preventing thrombosis in susceptible patients and treating venous thromboembolism (VTE) [76] . The use of some antiviral drugs potentially enhances the OACs level in plasma. In one study, patients on OAC with COVID-19 started antiviral drugs, and their OAC plasma levels were measured and compared with those documented before treatment.

Patients treated with both antiviral and OAC drugs showed an alarming increase in OAC plasma Levels. Physicians probably had better replace OACs with other anticoagulant medicines to prevent bleeding complications while using them concomitant with antivirals in COVID-19 [76] .

It is crucial to adjust the serum levels of some anticoagulant drugs in the proper range, as both high and low levels might cause coagulation problems. The fact that taking some antiviral drugs can alter the serum stability of them makes it even more challenging to monitor the amount of them in patients' blood. As a result, if we can propose a drug with both antiviral and anticoagulant effects for coagulation problems, it will probably be more comfortable to monitor the treatment [76, 77] . If Edoxaban shows sufficient antiviral effect in further in-vitro and invivo studies, it will likely be a suitable resort to overcome the dilemma.

Some drugs related to benign prostatic hyperplasia (BPH) or male erectile dysfunctional impotence have been identified to inhibit SARS-CoV-2 in our analysis, such as Dutasteride, Doxazosin, and Tadalafil. Dutasteride has been previously predicted to inhibit the main viral protease and E channel in-silico [78, 79] . Doxazosin is also predicted to inhibit the viral Mpro.

The inhibiting effect of Doxazosin was validated by the MD trajectory clustering approach insilico [80] . Tadalafil is predicted to have potential against nsp1 by the DeepDTA method and has J o u r n a l P r e -p r o o f also shown affinity as a 2'-O-methyltransferase inhibitor [81] . Besides, this category of drugs can be suitable choices against COVID-19 since they are androgen related. Androgen decrease has been associated with reduced ACE2 activity [82] . Besides, in type II pneumocytes, TMPRSS2 prims the viral spike surface, enabling the cell viral entry. Androgen receptor regulates the TMPRSS2 gene. The TMPRSS2 expression is also associated with an increase in androgen receptor (AR) [83] . It also blocks 5-AR isoform 3, which is expressed in the respiratory epithelium and fibroblasts. Based on these reasons, an androgen antagonist like Dutasteride could be a therapeutically beneficial drug for COVID-19. However, some cautions should be considered, and more preclinical studies seem to be required since inhibition of 5-AR impair the regeneration capacity of the respiratory epithelium [84] . We observed drugs previously prescribed for migraine pains among the shared drugs, such as Ergotamine (a calcitonin gene-related peptide antagonist). Ergotamine was detected as a possible inhibitor for SARS-CoV-2 RNA-dependent RNA polymerase in our analysis. It has also been reported to have affinity binding for three viral proteins in COVID-19 by other in-silico studies [89] . It has been the drug of choice in some migraine sufferers who have long duration or infrequent headaches over 50 years. One study found that the over-release of neuropeptides, such as the calcitonin gene-related peptide (CGRP), may lead to an abnormal vascular response seen in acute lung injury. Therefore, the CGRP blockade may be helpful in some lung injuries. [90, 91] Headaches are also symptoms that emerge in a subset of patients with COVID-19, although they do not occur isolated [92] .

In addition to previous drug categories, some drugs related to other drug categories appeared on our result list. One of these categories was anti-diabetic drugs, including Linagliptin (a DPP4 inhibitor). Considering that diabetes is a risk factor for critical manifestations of COVID-19 and increases the risk of severe symptoms in people with COVID-19, a clinical trial has been registered to assess its efficacy and safety in diabetic patients with COVID-19 (NTC04371978).

Chronic hyperglycemia and inflammation can also lead to abnormalities in the immune system [93] . Based on our analysis results, we predict that taking Linagliptin can probably benefit these patients as an antiviral agent. Besides, recently a challenge was posed by a hypothesis against may contribute to the viral entry in SARS-CoV-2. However, they do not provide experimental data in this regard. There is also evidence that DPP4 inhibitors may modulate inflammation and have anti-fibrotic activity [96] . Therefore using Linagliptin, in patients even without type 2 diabetes can probably prevent the sustained cytokine storm indirectly [97] . However, some have presented nuanced debates that we must not rush to use DPP4 inhibitors since they may suppress the immune system or cause other life-threatening conditions [98, 99] .

Diamorphine (heroin) is another drug that has properly scored against viral proteins in our analysis. Consuming high doses of some opioids and specifically, heroin affects the brain stem negatively and reduces the respiratory rate and has complications to the lungs and respiration system [100, 101] . However, it can still be used therapeutically in patients with a terminal J o u r n a l P r e -p r o o f disease (perhaps in severe pain to prevent neurotic shock). It can probably be investigated for patients in advanced and painful stages of cancers with moderate COVID-19 [102] .

Drospirenone is another drug that has appeared on our list. Drospirenone is used to control acne and PMDD. It is a synthetic progestin contraceptive that contains estrogen and progesterone.

Although the safety of its use is still controversial and it may increase venous thromboembolism, it is confirmed by another in-silico study to bind the three viral target proteins, including RdRp, Mpro, and PLpro [78] .

This study has identified 69 small molecule drugs with higher binding affinity and interaction with the RdRp and 3clpro proteins active pocket residues. The top 10 small molecule drugs with docking binding energies lower than 9.2 kcal/mol for RdRp and lower than 8.9 kcal/mol are shown in Table 3 . Moreover, the six drugs were selected for MD simulation, including Nilotinib, Imatinib, and dihydroergotamine for 3clpro and Lapatinib, Dexasone, Relategravir for RDRP. 

Several studies support that patients infected by SARS-CoV-2 are at risk of cytokine storm, inflammatory alterations, and disseminated intravascular coagulation. The lungs are the main target organ for the virus; patients develop acute lung damages, which can end to respiratory failure, although the defects in other organs, heart, nervous system, and skin are also reported. In this study, two crucial viral enzymes, RdRp and 3CLpro, were selected to dock against FDAapproved drugs. We identified and repurposed several medicines. We then categorized them and Relategravir may be effective drugs to treat COVID-19 with need more confirming experimental studies. We hope that they limit the morbidity and mortality associated with the recent severe acute respiratory syndrome pandemic. The H bonds are represented with green dashed lines. J o u r n a l P r e -p r o o f 

Drug treatment options for the 2019-new coronavirus (2019-nCoV)

SARS-CoV-2 and COVID-19: between pathophysiology complexity and therapeutic uncertainty

Reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor

Common and unique features of viral RNA-dependent polymerases

Coronavirus main proteinase: target for antiviral drug therapy

Ul-Haq, Identification of chymotrypsin-like protease inhibitors of SARS-Cov-2 via integrated computational approach

Structural plasticity of SARS-CoV-2 3CL M pro active site cavity revealed by room temperature X-ray crystallography

Structure of the RNA-dependent RNA polymerase from COVID-19 virus

Potential therapeutic agents for COVID-19 based on the analysis of protease and RNA polymerase docking

Discovering drugs to treat coronavirus disease 2019 (COVID-19)

SARS-CoV-2 RNA Dependent RNA polymerase (RdRp)-A drug repurposing study

Lopinavir/ Ritonavir, Ribavirin and IFN-beta Combination for nCoV Treatment

Remdesivir in adults with severe COVID-19: a randomised, double-blind

A Study to Evaluate the Safety, Pharmacokinetics and Antiviral Effects of Galidesivir in Yellow Fever or COVID-19

Randomized Clinical Trial for the Prevention of SARS-CoV-2 Infection (COVID-19

scRNA-seq Profiling of Human Testes Reveals the Presence of ACE2

Receptor, a Target for SARS-CoV-2 Infection

Clinical efficacy of lopinavir/ritonavir in the treatment of Coronavirus disease 2019

Lack of Antiviral Activity of Darunavir against SARS-CoV-2, medRxiv

Prediction of the SARS-CoV-2 (2019-nCoV) 3C-like protease (3CL pro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates

The COVID-19 epidemic

Focusing on the unfolded protein response and autophagy related pathways to reposition common approved drugs against COVID-19

Lopinavir/ritonavir f or the treatment of COVID-19: A living systematic review protocol, medRxiv

Therapeutic Management of COVID-19 Patients: A systematic review

Drug repurposing using computational methods to identify therapeutic options for COVID-19

PubChem as a public resource for drug discovery

Open babel: an open chemical toolbox

A sobering assessment of small-molecule force field methods for low energy conformer predictions

Swiss-PDB viewer (deep view)

UCSF Chimera-a visualization system for exploratory research and analysis

MODELLER, and IMP: an integrated modeling system

In Silico Identification of a Potent Arsenic Based Approved Drug Darinaparsin against SARS-CoV-2: Inhibitor of RNA dependent RNA polymerase (RdRp) and Necessary Proteases

Software for molecular docking: a review

Small-molecule library screening by docking with PyRx

Optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface leads the pathways of drugdesigning

Discovery studio modeling environment, release 3.5, Accelrys Discovery Studio

Moncayo-Palacio, Targeting the 3CLpro and RdRp of SARS-CoV-2 with phytochemicals from medicinal plants of the Andean Region: molecular docking and molecular dynamics simulations

Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone ϕ, ψ and side-chain χ1 and χ2 dihedral angles

An automated force field topology builder (ATB) and repository: version 1.0

Predicting commercially available antiviral drugs that may act on the novel coronavirus (SARS-CoV-2) through a drug-target interaction deep learning model

Targeting SARS-Cov-2: A systematic drug repurposing approach to identify promising inhibitors against 3C-like Proteinase and 2'-O-RiboseMethyltransferase

In silico identification and docking-based drug repurposing against the main protease of SARS-CoV-2, causative agent of COVID-19

Computational Drug Discovery and Repurposing for the Treatment of COVID-19: A Systematic Review

Fast Identification of Possible Drug Treatment of Coronavirus Disease-19

COVID-19) Through Computational Drug Repurposing Study

What are the risk factors and agents responsible for bacterial infections in ICUs?

Clinical features of patients infected with 2019 novel coronavirus in

Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome

0: a major update to the DrugBank database for

main protease (Mpro) inhibitors from natural polyphenols: An in silico strategy unveils a hope against CORONA

Identification of potential molecules against COVID-19 main protease through structure-guided virtual screening approach

An investigation into the identification of potential inhibitors of SARS-CoV-2 main protease using molecular docking study

Revealing the potency of citrus and galangal constituents to halt SARS-CoV-2 infection

Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods

Citrus fruits are rich in flavonoids for immunoregulation and potential targeting ACE2

A dual character of flavonoids in influenza A virus replication and spread through modulating cell-autonomous immunity by MAPK signaling pathways

The Use of Antiallergic and Antiasthmatic Drugs in Viral Infections of the Upper Respiratory Tract

Recent advances in the development of anti-allergic drugs

As a potential treatment of COVID-19: Montelukast

The inhaled corticosteroid ciclesonide blocks coronavirus RNA replication by targeting viral NSP15, bioRxiv

Pharmacokinetic profile of zafirlukast

The therapeutic effects of sodium cromoglycate against influenza A virus H5N1 in mice

Potential drugs targeting Nsp16 protein may corroborates a promising approach to combat SARS-CoV-2 virus

Association of Coronavirus Disease 2019 (COVID-19) With Myocardial Injury and Mortality

Cardiovascular Implications of Fatal Outcomes of Patients With Coronavirus Disease

Repurposing of approved drugs with potential to block SARS-CoV-2 surface glycoprotein interaction with host receptor

Interactions of coronaviruses with ACE2, angiotensin II, and RAS inhibitors-lessons from available evidence and insights into COVID-19

The Thrombopoietin Receptor Agonist Eltrombopag Inhibits Human Cytomegalovirus Replication Via Iron Chelation

Promoting platelets is a therapeutic option to combat severe viral infection of the lung

Identification of antiviral drug candidates against SARS-CoV-2 from FDA-approved drugs

Prioritisation of potential anti-SARS-CoV-2 drug repurposing opportunities based on ability to achieve adequate target site concentrations derived from their established human pharmacokinetics, medRxiv

Clinical characteristics of coronavirus disease 2019 in China

Disseminated intravascular coagulation in patients with 2019-nCoV pneumonia

Ticagrelor Can Be an Important Agent in the Treatment of Severe COVID-19 Patients with Myocardial Infarction

Cerebral astroblastoma: analysis of six cases and critical review of treatment options

Astroblastoma: report of two cases with unexpected clinical behavior and review of the literature

Patterns of care and treatment outcomes of patients with astroblastoma: a National Cancer Database analysis

Direct oral anticoagulant plasma levels striking increase in severe COVID-19 respiratory syndrome patients treated with antiviral agents. The Cremona experience

Monitoring the distribution of warfarin in blood plasma

Computational Molecular Docking and Virtual Screening Revealed Promising SARS-CoV-2 Drugs

Pharmaceutical Targeting the Envelope Protein of SARS-CoV-2: the Screening for Inhibitors in Approved Drugs

Profiling Molecular Simulations of SARS-CoV-2 Main Protease (Mpro) Binding to Repurposed Drugs Using Neural Network Force Fields

Computational Guided Drug Repurposing for Targeting 2'-O-Ribose Methyltransferase of SARS-CoV-2

Predicting the angiotensin converting enzyme 2 (ACE2) utilizing capability as the receptor of SARS-CoV-2

Androgen receptor and androgen-dependent gene expression in lung

Effects of 5-alpha reductase inhibitors on lung function: A reason for discontinuation during COVID-19 pandemic?

Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection

FDA approved drugs with broad anti-coronaviral activity inhibit SARS-CoV-2 in vitro

Silico Identification of Widely Used and Well Tolerated Drugs That May Inhibit SARSCov-2 3C-like Protease and Viral RNA

Dependent RNA Polymerase Activities, and May Have Potential to Be Directly Used in Clinical Trials

Targeting the SARS-CoV-2 Main Protease to Repurpose Drugs for COVID-19

Drug repurposing of approved drugs Elbasvir, Ledipasvir, Paritaprevir, Velpatasvir, Antrafenine and Ergotamine for combating COVID19

Role of calcitonin gene-related peptide (CGRP) in ovine burn and smoke inhalation injury

Ergotamine in the acute treatment of migraine: a review and European consensus

Central nervous system manifestations of COVID-19: A systematic review

COVID-19 and diabetes: can DPP4 inhibition play a role?

Fatal toxicity of chloroquine or hydroxychloroquine with metformin in mice

The dipeptidyl peptidase-4 inhibitor linagliptin attenuates inflammation and accelerates epithelialization in wounds of diabetic ob/ob mice

DPP4 inhibition: preventing SARS-CoV-2 infection and/or progression of COVID-19?

Dipeptidyl peptidase-4 (DPP4) inhibition in COVID-19

Is DPP4 inhibition a comrade or adversary in COVID-19 infection

Letter to the Editor in response to the article "COVID-19 and diabetes: Can DPP4 inhibition play a role?

The impact of heroin illicit market in the framework of COVID 19 pandemic

Understanding heroin overdose: a study of the acute respiratory depressant effects of injected pharmaceutical heroin

The therapeutic use of heroin: a review of the pharmacological literature

Rutin; a new drug for the treatment of increased capillary fragility

Telmisartan as tentative angiotensin receptor blocker therapeutic for COVID-19

Angiotensin receptor blockers as tentative SARS-CoV-2 therapeutics, Drug development research

Combination therapy with ombitasvir/paritaprevir/ritonavir for dialysis patients infected with hepatitis C virus: a prospective multi-institutional study

Eltrombopag is a potential target for drug intervention in SARS-CoV-2 spike protein, Infection

Molecular docking and dynamics simulation of FDA approved drugs with the main protease from 2019 novel coronavirus

Silico Identification of Widely Used and Well Tolerated Drugs That May Inhibit SARSCov-2 3C-like Protease and Viral RNA

Dependent RNA Polymerase Activities, and May Have Potential to Be Directly Used in Clinical Trials

The thrombopoietin receptor agonist eltrombopag inhibits human cytomegalovirus replication via iron chelation

Acute and 14-day hepatic venous pressure gradient response to carvedilol and nebivolol in patients with liver cirrhosis

Direct oral anticoagulant plasma levels' striking increase in severe COVID-19 respiratory syndrome patients treated with antiviral agents: The Cremona experience

Factor Xa Inhibition Reduces Coagulation Activity but Not Inflammation Among People With HIV: A Randomized Clinical Trial, Open forum infectious diseases

Combined Deep Learning and Molecular Docking Simulations

Approach Identifies Potentially Effective FDA Approved Drugs for Repurposing Against SARS-CoV

FDA approved drugs with broad anti-coronaviral activity inhibit SARS-CoV-2 in vitro

Repurposing of clinically developed drugs for treatment of Middle East respiratory syndrome coronavirus infection

Targeting the SARS-CoV-2 Main Protease to Repurpose Drugs for COVID-19

Drug repurposing of approved drugs Elbasvir, Ledipasvir, Paritaprevir, Velpatasvir, Antrafenine and Ergotamine for combating COVID19

Prevalence of respiratory conditions among people who use illicit opioids: a systematic review

Graphical abstract-docking drugs against the viral RdRp and 3CLpro enzymes. The candidate drugs were classified into several categories

The authors thank the Proteomics Research Center of Shahid Beheshti University of Medical Sciences for their support in conducting this work (NO.22784).

The authors declare no conflict of interest.