In silico Docking Study of Some Coumarin Derivatives as Potential Inhibitors on Different Dengue Viral Proteins

GABRIELA TATARINGA1*, BALASUBRAMANIAN SATHYAMURTHY2, ION SANDU3,4, ANA MARIA ZBANCIOC1* 1Grigore T. Popa University of Medicine and Pharmacy Iasi, Organic Chemistry Department, 16 Universitatii Str., 700115 Iasi, Romania; 2Ramaiah College of Arts, Science and Commerce, Department of Biochemistry, 7th Main Road, 560054, Bangaluru, Karnataka, India 3Alexandru Ioan Cuza University of Iasi, Arheoinvest Interdisciplinary Platform, Scientific Investigation Laboratory, 11 Carol I Blvd., 700506 Iasi, Romania 4 Romanian Inventors Forum, 3 Sf. Petru Movila Str., Bloc L11, III/3, 700089 Iasi, Romania

In this study, the binding efficiency of 10 coumarin derivatives with some selected proteins from Dengue virus through in silico method was done. By virtual screening and docking results, we have found that the hybrid derivative between coumarin and isatin has the most convenient binding activity for the seven selected proteins.
Keywords: coumarin derivatives, molecular docking, dengue virus Coumarin (benzopyran-2-ona) is an oxygen heterocyclic compound that has a bicyclic structure with lactone carbonyl group, firstly isolated in 1820 by Vogel.
Coumarins represent a class of compounds with a broad spectrum of biological activities: anticancer, anti-HIV, antimicrobial, antithrombotic, antiviral, antioxidant and antiinflammatory [2,3]. Coumarin ring can be considered as a privileged scaffold and an ideal framework for the design of compounds that can interact with different targets [4][5][6].
On the other side, isatin is a versatile chemical skeleton able to participate to a broad range of synthetic reactions, being an important molecule in synthetic medicinal chemistry. Recently, isatin and its derivatives have gained great attention due to its biological potential like antibacterial, antifungal, antimalarial, antiviral, anti-HIV, anti-mycobacterial, anticancer [7][8][9].
Dengue virus (DENV) is a member of the Flaviviridae family. Dengue virus is an enveloped RNA virus containing a positive-sense and single strand genome. Upon DENV entry into the target cells, the RNA genome is translated to a single polyprotein associated with the host endoplasmic reticulum. The polyprotein is cleaved by host and viral proteases into three structural proteins (capsid, premembrane and envelope) and seven non-structure proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) [10][11][12][13][14].
Currently, there is no vaccine or antiviral treatment for dengue fever. The patients are only recommended to drink plenty of fluids or take medicines like acetaminophen to avoid dehydration from vomiting and high fever.
Thus, it is very important to develop new antiviral substances against dengue viruses.
Chemical synthesis is a very important tool to insert new chemical groups in order to obtain new derivatives with improved profile from biological effect point of view. * email: gtataringa22@yahoo.com; sweetyanamaria@yahoo.com Impressed by the important biologically active profile of coumarin and isatin derivatives and as a part of our interest in the synthesis and screening of potentially bioactive compounds, we performed the evaluation of anti Dengue potential of some coumarin derivatives, previously synthesized, via molecular docking approach.

Synthesis of coumarin derivatives
The synthesis of the target compounds was reported previously [15].

Preparation of Dengue viral proteins
The protein data bank (PDB) was used to obtain the three-dimensional structure of the macromolecule [16][17][18][19]. The structures are downloaded and saved either in mm CIF or PDB format. Proteins of dengue virus were used for this study. The 3D structure of all the fourteen proteins were downloaded from PDB and saved in PDB format. The downloaded proteins were viewed in Py-Mol viewer.

Preparation of ligands
Ten synthesized ligands with coumarin ring, were selected for this study (Table I). Ligands were constructed using ChemSketch. The constructed ligands were optimized to add the hydrogen bonds and the obtained structures were saved in mol for docking analysis and named as A, B and C respectively.

Docking study
Docking studies were conducting using iGEMDOCK software. The proteins and the ligands were loaded and the out path was set. Standard docking parameters were used for docking (population size=200, generations =70 and Number of solutions =2). The docking process was initiated. After the docking process, the best docking pose for the individual ligands can be obtained for all the seven dengue viral proteins.

Results and discussions
Two acetohydrazide and eight Schiff bases with coumarin skeleton were selected (Table 1).
From docking study, we listed binding affinity of 10 compounds based on ligand binding energy (Tables 2 and  3). The lower energy scores represent better target proteinligand binding affinity compared to higher energy score.
Visualization of hydrogen bond interaction between the compounds I-V and various target proteins were shown in Table 4.
The interactions of the designed compounds with the amino acids in the active site of proteins are listed in Table  5.
A close-up view of the profile for Dengue virus selected protein with the investigated ligands is shown in the figures 1-7.
The 3D structure coordinates of seven proteins of Dengue virus are optimized and 10 coumarin derivatives were used for these proteins via molecular docking approach. Their total binding energy was calculated using iGEMDOCK. Evaluations of binding conformation of 10 compounds with Dengue viral proteins are performed using     iGEMDOCK. From docking study, we listed binding affinity of 10 compounds based on ligand binding energy ( Table  2). The binding pose for each ligand molecule into the capsid dengue viral protein is analyzed and the one having lowest ligand binding energy with these proteins among the different poses are generated. The lower energy scores represent better protein-ligand target binding affinity compared to higher energy score.
Dengue virus envelope protein mediates the binding of the virus to the host cell surface receptors. Thus, interference of this interaction would be a potential avenue for antiviral therapy. A small hydrophobic channel has been identified in the DENV envelope protein and this site has been identified as a target for small-molecule inhibitors. The compound Vb was found to have the lowest ligand binding energy (-113.61 kcal/mol) compared to other analogs for envelope protein.
NS3 helicase is an essential enzyme for viral replication and has been identified as a potential drug target. The enzyme shows both ATP-hydrolysis activity as well as RNA duplex unwinding activity. 2-((4-propyl-2-oxo-2H-chromen-7 -y l ) o x y ) -N ' -( 5 -n i t r o -2 -o x o i n d o l i n -3 -y l i d e n e ) acetohydrazide (Vb) showed notably strong docking with DENV NS3 helicase (total binding energy is -144.65 kcal/ mol).
NS2B-NS3 is a trypsin-like serine protease that cleaves the dengue protein into individual peptides necessary for viral replication. DENV protease has been identified as a primary target for the development of dengue antiviral drugs. The compound IVb has the lowest total binding energy (-106.81 kcal/mol); the binding site of DENV NS2B-NS3 protease involves the binding site of ARG(54).
The compound Vb was found to have lower ligand binding energy than other analogs for envelope protein, NS1 protein, transmembrane domain of NS2A protein, NS3 helicase protein and NS5 protein.
Regarding to transmembrane domain of NS2A protein interaction, evaluations of the results showed that, also the compound Vb forms two hydrogen bonds at positions GLY(3), ASP(1), with bond energies, -3.5 and -11.8 kcal/ mol respectively.
We further analyzed the docked pose for finding the binding mode of compound Vb in to transmembrane domain of NS2A protein, NS3 helicase protein, NS5 protein to validate the reasonable binding conformations.

Conclusions
Our molecular docking studies explored the possible binding modes of 10 compounds with coumarin ring with seven Dengue viruse proteins. It revealed that all the investigated compounds show medium affinity for all the proteins. Especially the compound Vb (2-((4-propyl-2-oxo-2H chromen-7-yl)oxy)-N-(5-chloro-2-oxoindolin-3ylidene)acetohydrazide) shows best results compared to other compounds. Comparing the binding energy and the binding site residues, we found that all compounds differ either in their binding modes or with the binding site residues for hydrogen bond formation. The conclusion drawn from our virtual screening and docking result was that the compound Vb has highest binding affinity with most of the proteins and it can be used as an effective target compound for Dengue virus. Though, there are many reports on the in vitro analysis of these compounds and its antioxidant properties, but there are no in silico studies that predict the binding and active regions especially with these proteins. Our study is probably the first such attempt to predict the binding site. However, validation of our results through in vivo and in vitro experiments along with animal models will enlighten hope for the future development of more potent drugs for the treating Dengue.