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Short CommunicationEffective Anti-SARS-CoV-2 RNA Dependent RNA Polymerase Drugs Based on DockingMethods: The Case of Milbemycin, Ivermectin, and Baloxavir MarboxilAli Hassan Daghir JanabiDepartment of Veterinary Microbiology, College of Veterinary Medicine, University of Al-Qadisiyah, DiwaniyahCity, IraqAbstractBackground: Severe Acute Respiratory Syndrome-coronavirus 2 (SARS-CoV-2) is a* Corresponding author:Ali Hassan Daghir Janabi, Ph.D.,Department of VeterinaryMicrobiology, College ofVeterinary Medicine, Universityof Al-Qadisiyah, Diwaniyah City,IraqTel: 96 47810069889E-mail:[email protected]: 6 Jun 2020Accepted: 15 Jul 2020new virus with a global pandemic. Yet, no vaccine or efficient treatments are foundagainst the disease. The viral RNA dependent RNA Polymerase (RdRP) is a suitabletarget for developing antiviral agents. SARS-CoV-2 RdRP was employed to test itsbinding activity with some drugs.Methods: Using some docking methods, RdRP was targeted by Milbemycins (MMs),Ivermectin (IMT), Baloxavir Marboxil (BM), and Tadalafil (TF), a phosphodiesterasetype 5 inhibitor.Results: MM-A3 5-oxime (MMA35O), MM-A3 (MMA3), MM-A4 5-oxime (MMA45O),IMT, BM, and TF showed the highest binding affinity to RdRp.Conclusion: The drugs used in the present computational investigation are effectiveagainst the SARS-CoV-2 RdRP with high affinity values especially, milbemycins, ivermectin, and Baloxavir marboxil, which could further be studied in laboratory andclinical trials for saving millions of lives around the world.Avicenna J Med Biotech 2020; 12(4): 246-250Keywords: Baloxavir, COVID-19, Ivermectin, TadalafilIntroductionA few drugs have been used for treating Coronavirus Disease 2019 (COVID-19) patients such as Remdesivir (RDS) and Favipiravir (FPV) which are considered as nucleotide and nucleoside, respectively, inhibitors that interfere with the assembly of the viral RNA.RDS is used for the treatment of Ebola 1 and Marburgviruses 2 and SARS and MERS infections 3. In a compassionate-clinical use of RDS in hospitalized COVID19 patients breathing ambient air or on oxygen support,clinical improvement was seen in 36 of 53 patients(68%) 4 which is still a humble recovery especially inthe case of more severe and critical patients. Like RDS,FPV (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is a nucleoside analogue used as an antiviral drugthat halts the replication of the viral RNA due to itsincorporation in the newly-made RNA strand of widerange viruses 5. FPV was recorded to induce inhibitionto seasonal and avian influenza viruses even thosestrains that are resistant to neuraminidase inhibitors asapproved using cell culture and experimental animals 6.Due to the availability of the RdRP 3D structure 7,docking methods in the present work were employed toenhance production of drugs that bind with high affinities to the binding sites of the enzyme. This might246246block the activity of the enzyme, halt the viral RNAbuilding, and eventually stop the viral replication.Materials and MethodsIn the current study, drugs (Table 1) were employedto target active binding sites of the SARS-CoV-2 RdRP(PDB ID: 6m71; https://www.rcsb.org/), after removing cofactors from the structure using PyMOL 8. MMs,members of the macrocyclic lactone family, were selected according to a report that IMT, a member of thesame family, was effective in minimizing the replication of the COVID-19 in cell culture 9. For BM, theselection was according to its inhibition effects on thepolymerase-based initiation of mRNA of the influenzavirus 10. In the case of TF selection, it was previouslyTable 1. Drugs utilized for the docking and their PubChem IDsPubChem CID91617476982834391617829ChemSpider ID7988461124081896110635Copyright 2020, Avicenna Journal of Medical Biotechnology. All rights reserved.DrugMilbemycin A3 5-oximeMilbemycin A3Milbemycin A4 5-oximeIvermectinBaloxavir marboxilTadalafilVol. 12, No. 4, October-December 2020

Janabi AHDreported to prevent infection of a cell line by the Human Immunodeficiency Virus (HIV) 11. PyRx-PythonPrescription (0.8) and Autodock vina plugin 12 wereused for the prediction of the affinity values. In details,Standard Database Format (SDF) files of the ligandswere downloaded from https://pubchem.ncbi.nlm.nih.gov/ and were converted into PDB file type via the useof BABEL plugin in the PyRX software. IMT wasdownloaded from the http://www.chemspider.com/ dueto unavailability of the 3-dimensional structure in thehttps://pubchem.ncbi.nlm.nih.gov/. Then, both proteinand ligand files were prepared for the docking processusing PyRx software for removing waters, in which thePyRx software gets rid of water molecules automatically before generating pdbqt files. Grid boxes were optimized for free searching by the ligands on the wholeenzyme molecule (Active and non-active sites). Thedissociation constant (Kd) is predicted via auto dockbuilt in the PyRx software that converts the affinityenergy values (kcal/mol) into Kd according to the following equation:DeltaG R*T*lnKiDeltaG: Affinity energy (kcal/mol)R: Gas constantT: Room temperature (298.15 K 25 C)Ki: Dissociation constantPyMOL 8 was used to create the docking images. Theactive binding sites are located on motifs A to G; however, A and C motifs of the RdRP are considered important for the RNA template involvement. These sitesare clamped by critical motifs F and G that ease theentry of this template via a groove to the active sites inthe motifs A and C 7.Interactions (2 and 3D models) between the RdRPand the ligands were visualized using Discovery StudioVisualizer v20.1.0.19295 (BIOVIA). The images weregenerated using Discovery Studio Visualizer v20.1.0.19295 (BIOVIA), Microsoft PowerPoint, and GIMPv2.10.18.ResultsThe outcomes of the present investigation shown intable 2 unveiled the highest affinity values for MMA35O, MMA3, MMA45O, and IMT when binding toRdRp followed by BM and TF. The chemical structures of these ligands are displayed in figure 1.The results showed that the palm subdomain in motif B of the RdRP is engaged by the MMA35O (Figure2A). The bonds of the attachment between the ligandand the protein are shown in figures 3-5A. MMA3 revealed its binding to the bindings sites in the RNAtemplate tunnel groove of the F motif of the RdRP enzyme (Figure 2B). The linkages between the ligand andthe enzyme are displayed in figures 3-5B. Moreover,MMA45O displayed attachment to the binding siteslocated in the B and F motifs in the way of RNA template entry (Figure 2C). The ligand-protein basedbonds are shown in figures 3-5C. IMT revealed binding to the binding sites present in the C and F motifs inthe inlets of RNA template and NTPs and the outlet ofthe product hybrid (Figure 2D). For the linkages, theligand-enzyme connection is shown in figures 3-5D.For the BM, the binding site location was in the B andF motifs of the inlets of RNA template and NTPs andTable 2. Autodock Vina binding affinity values of drugs to theCOVID-19 RdRpDrugsMilbemycin A3 5-oximeMilbemycin A3Milbemycin A4 5-oximeIvermectinBaloxavir marboxilTadalafilBinding affinity values (kcal/mol)-10.1-10.1-9.2-9.2-9.2-8.9Figure 1. Chemical structures of the study ligands.Avicenna Journal of Medical Biotechnology, Vol. 12, No. 4, October-December 2020247

Computational Anti-COVID-19 RNA Polymerase LigandsFigure 2. RNA dependent RNA polymerase (grey, surface-hydrophobicity mode) of the SARS-CoV-2 (COVID-19) in complex withdifferent ligand drugs (red); A) MMA35O binds to the B motif; B)MMA3 binds to the F motif; C) MMA45O binds to the B and F motifs; D) IMT binds to the C and F motifs; E) BM binds to the B and Fmotifs; F) TF binds to the B and F motifs.Figure 3. Zoom-in images of the RNA dependent RNA polymerase(grey) of the SARS-CoV-2 (COVID-19) with different ligand drugs(cyan); A) MMA35O binds to the B motif; B) MMA3 binds to the Fmotif; C) MMA45O binds to the B and F motifs; D) IMT binds to theC and F motifs; E) BM binds to the B and F motifs; F) TF binds tothe B and F motifs.the outlet of the product hybrid (Figure 2E). The adherence of the ligand to the protein is displayed in figures 3-5E. Finally, TF provided binding properties atthe same location of that from BM (Figure 2F). Theligand-enzyme bonds are shown in figures 3-5F.DiscussionThe active sites of the RdRP, also known as nsp12,are located in the motifs A to G 7. This activity is probably due to different directions taken by the majorcomponents; RNA template, NTPs, and product hybrid, involved in the building of the new viral RNAstrands. The finding of the present study revealed thatthe palm subdomain in the motif B of the RdRP isbound by the MMA35O. The palm subdomain (Residues T582-P620 and T680-Q815) is an important partof the core region of the RdRP that participates in thestability of the enzyme structure along with fingers248248Figure 4. Interactions (3D-model) of ligands with the RNA dependentRNA polymerase of the SARS-CoV-2 (COVID-19) based on hydrogen bond style visualized by Discovery Studio Visualizer v20.1.0.19295; A) MMA35O; B) MMA3; C) MMA45O; D) IMT; E) BM; F)TF.subdomain (Residues L366-A581 and K621-G679) anda thumb subdomain (Residues H816-E920) 7. Hypothetically, the presence of the MMA35O in the boundregion may increase the chances of halting the enzymeactivity especially when this region is considered adjacent to the passage of the RNA building materials.MMA3 showed its binding to the F motif of the RdRPenzyme. The F motif is a critical part through whichthe RNA template is expected to enter to the activesites in the motifs A and C to engage in the process ofthe RNA replication. This attachment region is locatedin the active groove that is used as an RNA templateentry passage. Typically, this means that the occurrence of the ligand in this pocket might enhance thechance of disrupting the replication of the viral RNAstrand.Another molecule that expressed binding to RdRP isMMA45O. The drug attached to the binding sites in themotifs B and F. The bound ligand was seen sitting inthe groove, RNA template passage entry to the motif Aand C, clamped by the F motif. Such occasion mightinterfere with the activity of the RdRP, thereby stopping its activity and eventually finishing the cycle ofviral replication. Motif A (Residues 611-TPHLMGWDYPKCDRAM-626) has a conserved divalent-cationbinding residue (D618) which is similar to that in otherviral RdRP such as in HCV (D220) and poliovirus(D233). IMT revealed binding to motif C and F. MotifC consists of residues of 753-FSMMILSDDAVVCFN767 including the catalytic residues of 759-SDD-761 7.In the present study, Baloxavir marboxil displayedsuccessful binding to the active sites in the B and Fmotifs of the RdRP. Baloxavir marboxil suppresses theactivity of the viral replication of the influenza virus byinhibiting the polymerase PA subunit 13. Since the target of this drug is the viral polymerase, this might be agreat drug of choice for treating COVID-19 patients.The TF showed strong binding activity to the polymer-Avicenna Journal of Medical Biotechnology, Vol. 12, No. 4, October-December 2020

Janabi AHDFigure 5. Interactions (2D-model) of ligands with the RNA dependent RNA polymerase of the SARS-CoV-2 (COVID-19) based on hydrogen bondstyle visualized by Discovery Studio Visualizer v20.1.0.19295; A) MMA35O; B) MMA3; C) MMA45O; D) IMT; E) BM; F) TF.ase of the virus. Tadalafil is an inhibitor to the phosphodiesterase type 5 component utilized to manageerectile dysfunction 14. This could be applied as a greatdrug of choice for treating COVID-19 cases especiallywhen this drug is used to treat pulmonary arterial hypertension 15.ConclusionThe current work provided a group of drugs thatcould be further studied in vitro, in vivo, and in clinicaltrials to find whether they are effective against theSARS-CoV-2 (COVID-19) virus and thereby, quicklyhelping to manage the pandemic around the world.2018;9(2):e00221-18.4. Grein J, Ohmagari N, Shin D, Diaz G, Asperges E, Castagna A, et al. Compassionate use of remdesivir forpatients with severe Covid-19. N Engl J Med 2020;382(24):2327-2336.5. Furuta Y, Komeno T, Nakamura T. Favipiravir (T-705),a broad spectrum inhibitor of viral RNA polymerase.Proc Jpn Acad Ser B Phys Biol Sci 2017;93(7):449-463.6. Sleeman K, Mishin VP, Deyde VM, Furuta Y, KlimovAI, Gubareva L V. In vitro antiviral activity of favipiravir (T-705) against drug-resistant influenza and 2009A(H1N1) viruses. Antimicrob Agents Chemother 2010;54(6):2517-2524.AcknowledgementNo specific fund was allocated to this work.7. Gao Y, Yan L, Huang Y, Liu F, Zhao Y, Cao L, et al.Structure of the RNA-dependent RNA polymerase fromCOVID-19 virus. Science 2020;368(6492):779-782.Conflict of InterestNone is declared.8. The PyMOL Molecular Graphics System. No Title [Internet]. Schrödinger, LLC.; 2020. Available from: https://pymol.org.References1. Tchesnokov EP, Feng JY, Porter DP, Götte M. Mechanism of inhibition of ebola virus RNA-dependent RNApolymerase by remdesivir. Viruses 2019;11(4):326.2. Warren TK, Jordan R, Lo MK, Ray AS, Mackman RL,Soloveva V, et al. Therapeutic efficacy of the smallmolecule GS-5734 against Ebola virus in rhesus monkeys. Nature 2016;531(7594):381-385.3. Agostini ML, Andres EL, Sims AC, Graham RL, Sheahan TP, Lu X, et al. Coronavirus susceptibility to theantiviral remdesivir (GS-5734) is mediated by the viralpolymerase and the proofreading exoribonuclease. MBio9. Caly L, Druce JD, Catton MG, Jans DA, Wagstaff KM.The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antiviral Res 2020;178:104787.10. Heo YA. Baloxavir: First global approval. Drugs 2018;78(6):693-697.11. Guo J, Liang H, Toro R, Wu Y. Effects of tadalafil andsildenafil on HIV infection in vitro. J Hum Virol Retrovirology 2015;2(1):00030.12. Dallakyan S, Olson AJ. Small-molecule library screeningby docking with PyRx. Methods Mol Biol 2015;1263:243-250.Avicenna Journal of Medical Biotechnology, Vol. 12, No. 4, October-December 2020249

Computational Anti-COVID-19 RNA Polymerase Ligands13. O’Hanlon R, Shaw ML. Baloxavir marboxil: the newinfluenza drug on the market. Curr Opin Virol 2019;35:14-18.14. Coward RM, Carson CC. Tadalafil in the treatment oferectile dysfunction. Ther Clin Risk Manag 2008;4(6):2502501315-1330.15. Henrie AM, Nawarskas JJ, Anderson JR. Clinical utilityof tadalafil in the treatment of pulmonary arterial hypertension: An evidence-based review. Core Evid 2015;10:99-108.Avicenna Journal of Medical Biotechnology, Vol. 12, No. 4, October-December 2020

portant for the RNA template involvement. These sites are clamped by critical motifs F and G that ease the entry of this template via a groove to the active sites in the motifs A and C 7. Interactions (2 and 3D models) between the RdRP and the ligands were visualized using Discovery Studio Visualizer v20.1.0.19295 (BIOVIA). The images were