key: cord-0866546-e8oyso24
authors: Elfiky, Abdo A.; Elshemey, Wael M.
title: Molecular dynamics simulation revealed binding of nucleotide inhibitors to ZIKV polymerase over 444 nanoseconds
date: 2017-09-18
journal: J Med Virol
DOI: 10.1002/jmv.24934
sha: 98b77e8790850cf2580be9ee65e23b5b96b0a04a
doc_id: 866546
cord_uid: e8oyso24

In the year 2015, new Zika virus (ZIKV) broke out in Brazil and spread away in more than 80 countries. Scientists directed their efforts toward viral polymerase in attempt to find inhibitors that might interfere with its function. In this study, molecular dynamics simulation (MDS) was performed over 444 ns for a ZIKV polymerase model. Molecular docking (MD) was then performed every 10 ns during the MDS course to ensure the binding of small molecules to the polymerase over the entire time of the simulation. MD revealed the binding ability of four suggested guanosine inhibitors (GIs); (Guanosine substituted with OH and SH (phenyl) oxidanyl in the 2′ carbon of the ribose ring). The GIs were compared to guanosine triphosphate (GTP) and five anti‐hepatitis C virus drugs (either approved or under clinical trials). The mode of binding and the binding performance of GIs to ZIKV polymerase were found to be the same as GTP. Hence, these compounds were capable of competing GTP for the active site. Moreover, GIs bound to ZIKV active site more tightly compared to ribavirin, the wide‐range antiviral drug.

in Brazil and Caribbean countries. 3, 4 By the year 2016, more than 80 countries reported ZIKV infection worldwide. 5 The World Health Organization (WHO) declared it as a public health emergency of international concern in February 2016. 5 ZIKV is transmitted through the bites of Ades mosquitos (Aedes africanus, Aedes aegypti, Aedes luteocephalus, and Aedes albopictus). [6] [7] [8] It can be detected in body fluids like saliva, urine, and blood. 9,10 Slight fever, rash, arthralgia, and conjunctivitis are common symptoms of ZIKV infection. 5, 11 Strong links between ZIKV infection and microcephaly and neuronal disorders in newborn infants of ZIKV-infected women were confirmed in March 2016. 12 Non-structural 5 (NS5) viral protein have two domains; the helicase domain and the RNA-dependent RNA polymerase (RdRp) domain which is vital for viral replication. 13, 14 The polymerase domain has a highly conserved active site in different viruses including hepatitis C virus (HCV), West Nile virus, and Middle East Respiratory Syndrome

Coronavirus. [15] [16] [17] [18] [19] The antiviral activity of different nucleotide inhibitors were studied against the RdRp of HCV. Sofosbuvir is an example of a successful nucleotide inhibitor that was approved by Food and Drug Administration (FDA) in December 2013. [20] [21] [22] [23] The same compound was also studied as a possible inhibitor against ZIKV. [24] [25] [26] Molecular modeling is usually used to mimic the behavior of macromolecules. 27, 28 Molecular docking (MD) is important in testing the binding of small molecules to protein active site and in virtual screening. 18 These in silico techniques were used to study viral proteins. 18, 27, [29] [30] [31] [32] [33] Molecular dynamics simulation (MDS) offers a means to study protein dynamics in silico. It is able to mimic and predict the dynamical properties of protein in solution 28 in addition to suggesting possible interaction potency. [34] [35] [36] [37] Nucleotide inhibitors (in its active triphosphate form) were sketched and optimized using SCIGRESS 3.0 software. 29, 40 Optimization was done using Molecular Mechanics force field MM3 41 followed by quantum mechanics (B3LYP functional). 42 Supplementary Figure S1 shows the structures of the inhibitors in 2D representation. 43 

NAMD software 34 was used to perform MDS on the Cy-Tera super computer facility of the Cyprus Institute of Science (Project no. pro15b114s1). ZIKV model was solvated (explicitly) using TIP3P water model. 34 Two chlorine ions were added to neutralize the protein charge at pH 7. The protein in the solvent system was equilibrated at room temperature (310 K) then the solvent was minimized for 100 ps. Normal pressure and temperature (NPT) ensemble was used to relax the system for a total period of 5 ns, after that normal volume and temperature (NVT) ensemble was used in the production run for 444 ns. In the production run, the box size was set to (90.32 × 90.32 × 90.32) Å 3 based on the fluctuations during the 5 ns of NPT ensemble. Every 10 ns of the production run, the structure of the protein was retrieved for docking study. Therefore, the docking study was performed for 45 times during different dynamic states (different conformations). The retrieved structures were used to check the solvent accessible surface area (SASA) and radius of gyration (R of G). These two parameters were used to check conformational changes during the course of MDS. AutoDock Vina software 44 was used to perform the docking study using default parameters. The protein is treated as rigid as we need to test the contribution of dynamics of the protein into inhibitor binding. The calculated binding affinity (in kcal/mol) were used for comparison between different nucleotide inhibitors.

As reported before anti-HCV drugs were capable of binding to ZIKV polymerase active site. 24, 25 In the current study, MDS was performed on the ZIKV RdRp model for a period of 444 ns. Despite five solved structures of ZIKV, NS5 RdRp were deposited in the Protein Data Bank in the last few months, the comparatively built model used in this study had very strong sequence and hence structural conservation ( Figure 1A ). Sequence comparison between the five solved structures (PDB IDs: 5WZ3, 5TFR, 5TMH, 5TIT, and 5U04) and the model used in this study is shown in Supplementary Figure S2 . Figure 1B shows the root mean square deviation (RMSD) over the carbon alpha atoms of the polymerase versus time. Saturation was achieved after 200 ns with about 12 Å RMSD. During the course of MDS, two distances were recorded. These were the distance between carbon alpha of the active site D124 and the two alpha carbons of D6 and G46 (green and red curves in Figure 2A , respectively). D6 lied in the center of alpha helix (α1 in Figure 2B ) near the active site. On the other hand, G46 lied at the end of the arm consisting of β1, β2, and its connecting loop (fingers tip).

It was reported that this arm takes part in the interaction with the RNA Figure 2F ), the distance reduces to 24 Å (see Supplementary Video S1) in the next 150 ns of MDS (step ii in Figure 2F ) and finally it is increases again to about 28 Å for the rest of the simulation time (step iii in Figure 2F ). This implies a conformational change in the distal end of the arm that contains G46 and suggests its functional characteristics. Based on structural alignment made between ZIKV and HCV polymerases, this arm is a part of the fingers domain, where the fingers' tips interact with the viral RNA during the polymerization process. 45 Figure 2B illustrates the secondary structural elements of the ZIKV RdRp model before performing the MDS. It has nine alpha helices and six beta sheets. The active site aspartates are protruding from the beta turn between β3 and β4. The distance between the active site D124 and each of D6 and G46 is labeled at selected times during the MDS ( Figure 2C-E) . Figures 3A and 3B shows the calculated values of surface accessible surface area (SASA) in Å 2 and the radius of gyration (R of G) in Å. These values were calculated each 10 ns of the MDS using visualizing molecular dynamics (VMD) software. 46 SASA was calculated as relative exposure (0 to 1), that is, the exposure of each amino acid in the protein relative to per residue SASA values.

The latter is calculated for each amino acid alone in the maximum speed molecular surface (MSMS) algorithm implemented in VMD software. 47 The solvent radius was chosen to be 1.4 Å (radius of water molecule). On the other hand, radius of gyration was calculated using the following formula: Figure S3) . Hence, we can conclude that the arm movement has not contributed against binding of the small molecules to ZIKV polymerase.

As reported by the authors in earlier studies, IDX-184 shows promising results compared to other drugs. 22, 25, 27, 48 The new suggested compounds have also promising average binding affinities to ZIKV NS5 RdRp compared to other drugs ( Figure 4 ). Upon binding, the suggested ligands will stop the polymerization process and impair the virus life cycle.

In the previous studies, authors suggested the ability of anti-HCV drugs to bind and subsequently inhibit ZIKV polymerase. 25, 26 This was based on structural similarity between HCV and ZIKV polymerases. In this study, the authors simulated the dynamics of ZIKV polymerase in water over 444 ns. The results supported our previous suggestion. In addition, four new compounds were introduced as promising binders to ZIKV polymerase active site based on the calculated binding affinities. On the other hand, the arm movement that occurred after 200 ns did not affect the binding of all of the small molecules. Future work is suggested to test the arm movement effect on the viral RNA upon polymerase binding. 

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