Molecular docking analysis of netropsin and novobiocin with the viral protein targets HABD, MTD and RCD

Dengue, West Nile and Zika virus belongs to the family flaviviridae and genus flavivirus. It is of interest to design and develop inhibitors with improved activity against these diseases. We used the helicases target to screen for potential inhibitors against these viruses using molecular docking analysis. NS3 helicases of flavivirus family of viruses such as Dengue, West Nile and Zika are prime targets for drug development. The computer aided molecular docking analysis of netropsin and novobiocin with the viral protein targets HABD, MTD and RCD is reported for further consideration.

during the last two decades [7,8]. Without doubt, Zika diseases seeks attention after West Nile infection, dengue, which appeared in 1999 and chikungunya which appeared in 2013 [8]. The Zika contamination has a spot with the assortment flavivirus and is generally vectored by Aedes mosquitoes [9] found over the continents [10- -12].
Helicases are omnipresent engine proteins that catalyze the unwinding of double-stranded DNA or RNA by ATP hydrolysis. They move along a nucleic acid phospho-di-ester spine and separate the complementary strands by utilizing energy got from ATP hydrolysis. [13] RNA helicases are important particles for RNA metabolic procedures, for example, ribosome biogenesis, joining and interpretation. It has been demonstrated that the helicases are connected to different segments of the macromolecular machines and they have critical role in numerous ©Biomedical Informatics (2019) 234 processes [14]. Hence, the consideration of helicases as potential viral drug targets is realized. The viral proteome is an extended polypeptide chain in the ER. It is further known that NS2A, NS2B, NS4A, and NS4B are structural layer proteins essential for viral replication through possible protein-protein and protein-lipid associations. The functions played by NS1, NS2A and NS4A in viral replication are complex to understand [15]. NS1 has different oligomerization states dependent on its glycosylation status [16][17][18]. The methyltransferase is in charge of topping the incipient genomic RNA by successively utilizing S-adenosyl methionine as the methyl donor. This is done by consecutive methylation on the N7 molecule with the top guanine and the 2'O particle of the ribose in the main adenine [19][20]. Defect in capping decrease viral multiplication to cause infection that are gradually subtle to the natural resistant reaction as they stimulate higher interferon (IFN) flagging and immunizer reactions [21]. Moreover, the positive sense of viral RNA is discharged in the cytoplasm during infection. NS5 protein initially deciphers it as a negative sense strand before utilizing the negative strand (with regards to a dsRNA middle of the road) to organize excess of positive sense RNA. The viral mRNA is then used to express the poly protein by host cell ribosomes. It is also known that a hetero-duplex framed by a DNA format and an RNA primer is formed [22]. This component is available in the DENV NS5 full-length protein whose preference for dsDNA is like ssRNA [23]. In the context of these available data it is of interest to screen HABD (helicase ATP binding domain), MTD (methyl transferase domain) and RCD (RNA catalytic domain) for potential inhibitors as drug candidates.

Multiple sequence alignment (MSA) of the conserved domain:
The conserved domain sequences of dengue virus, zika virus and Japanese encephalitis virus were retrieved from the genome database in NCBI and BLASTp analysis was completed. The FASTA formats of the retrieved sequences were used for further analysis. Multiple sequence alignment (MSA) of Helicase ATP binding domain (201 amino acid residues), Methyl transferase domain (262 amino acid residues) and RNA Catalytic domain (149 amino acid residues) was completed using ClustalW3 and Clustal Omega. Various domains were manually assigned and confirmed by using Pfam, Prosite, SMART, PANTHER and InterProScan.

Three-dimensional structure prediction by I-TASSER:
The protein sequence (201) of Helicase ATP binding domain (HABD), the protein sequence (262) of Methyl transferase domain (MTD) and the protein sequence (149) of RNA Catalytic domain (RCD) were downloaded from the Swiss Pro database. The threedimensional model was produced utilizing the I-TASSER server which creates a 3D model of inquiry arrangement by different threading arrangements and iterative necessary gathering reenactment [24]. We used this server because its accessibility, composite methodology of displaying an execution in CASP rivalry. I-TASSER technique incorporates general strides of ©Biomedical Informatics (2019) threading, and auxiliary get together, display determination, refinement, and structure-based comments [25]. An optional structure was developed by PSIPRED [26] utilizing the structure library LOMETS [27]. Z-score checked the nature of the formal arrangement followed by threading arrangements [24] using Monte Carlo simulation [28]. The reenactment incorporates Cα/side chain connection, H-securities, hydrophobicity, spatial controls from threading template [27] and arrangement based on contact expectations from SVMSEQ [29]. The adaptations produced amid the refinement reenactment process were bunched by SPICKER [30]. Furthermore, the normal of three-dimensional directions of all the grouped structure was determined to acquire bunch centroids. In the refinement process, the chosen bunch centroids were again used to perform further reenactment, which evacuates steric conflicts to refine the topology of the group centroids. The known structures in PDB were recognized by TM-adjust [31]. The final structural models were generated by REMO [32] in which group centroids of second-round reenactment were utilized. The useful analogs were ranked based on TM-score, RMSD, arrangement character, and the inclusion of the structural arrangement. The nature of the model was dictated by C-score (certainty score), which is -5 to 2. It depends on threading arrangement and the combination of auxiliary for refinement reenactments.

Preparation of ligands and protein targets:
The 3D structures of HABD, MTD and RCD were built using I-TASSER. The hydrogen atoms having polar nature were then included. The buildup structures less in numbers were removed and the fragmented side chains were later replaced by Auto Dock Tools (ADT) version 1.5.6 downloaded from the Scripps Research Institute. Further, particles having Gasteiger charges were included and the non-polar hydrogen iotas were added to the protein structure. The built structures were then stored in PDBQT format in ADT [33]. 3D structures of netropsin and novobiocin were drawn using ChemBioDraw Office 12.0. The 3D co-ordinates of the ligands was then created and stored in PDBQT format using ADT [33].

Receptor grid formation:
Networks predetermine matrix maps of restricting energies in various particle types (for example, hydrogen holding oxygen, carbons, and aliphatic carbons in a macromolecule (for example a RNA/DNA, protein)) before docking [34]. These network maps are then utilized in AutoDock 4.2 docking computations to characterize the absolute restricting vitality for a ligand with a macromolecule [33]. Network mapping computes the vital parameters over the protein nuclear information and determines the directions of the HABD, MTD, and RCD for docking. Likewise, lattice mapping manages an appropriate surface topology for the iotas of mixes for association with the HABD, MTD and RCD dynamic sites. Network mapping is a necessity to guide netropsin, and novobiocin mixes to search for their locale for binding with the HABD, MTD and RCD dynamic sites. The network measurements for the HABD protein was 58 × 48 × 52 matrix focused with separating 1.00 Å between the framework focused on the ligand for protein (59.686, 70.660 and 46.318 directions). The framework measurements for MTD protein was 48 × 52 × 58 matrix focused with dispersing 1.00 Å between the network focuses; however fixated on the ligand for protein (61.363, 69.710 and 54.870 co-ordinates). The lattice measurements for malate synthase protein was 52 × 52 × 55 framework focused with dividing 1.00 Å between the framework focuses yet fixated on the ligand for protein (60.820, 65.487 and 70.722 directions). The network was made for selecting promising cooperation for docking between ligands and targets [35].
©Biomedical Informatics (2019)  (Figures 2 A-F). The comparative study provided useful insights on conserved domains, which are present in all three viruses. This is useful to study targets for antiviral drug design.

Three-dimensional structure models:
The molecular model of HABD, MTD, and RCD proteins was developed using I-TASSER. The models obtained from server incorporate supporting structure with certainty score (0 to 9), anticipated dissolvable openness. We obtained five models with C-score, top ten formats from PDB used in the arrangement; top ten PDB basic analogs, useful analogs of protein, and restricting site deposits. The HABD model (Figure 3A) was chosen with Cscore 0.92, TM-score 0.60 ± 0.14 and RMSD 7.3 ± 4.2 Å. MTD (Figure 3B) was selected as the best-expected model with C-score 1.17, TM-score 0.87±0.07, and RMSD 3.6±2.5å. MTD (Figure 3 C) was chosen as the best model with C-score 0.99, TM-score 0.85±0.08, and RMSD 2.8±2.1 Å. C-score with higher esteem mirror a model for better quality [18]. Standardized Zscore commonly evaluates threading. A normalized Z-score >1 esteem mirror a specific arrangement. TM-adjust distinguished 5xdrA, 2px5A and 4v0qA2 in PDB library as the best scoring models from I-TASSER with a TM-score of 0.792, 0.979 and 0.979, respectively.

Conclusion:
We report the molecular docking analysis of netropsin and novobiocin against the helicases targets of Dengue, West Nile and Zika viruses. The analysis shows that netropsin and novobiocin bind to viral targets HABD, MTD and RCD with high binding ability for further in vitro and in vivo studies.