Novel guanosine derivatives against Zika virus polymerase in silico.

The Zika virus (ZIKV) outbreak, which started in the year 2015, is considered the fastest and most widely spread outbreak reported for this flavivirus. The polymerase domain of the NS5 protein has been targeted in other viral infections and is recognized as a suitable target in ZIKV infection. Different novel modified compounds against ZIKV NS5 have been tested in silico. A few structures have been solved for ZIKV polymerase and deposited in the protein data bank website. Two of these solved structures (with a resolution of less than 1.9  A) are used in this study to test the binding of 74 novel compounds in silico. Molecular docking is used to quantify the binding affinities of ZIKV polymerase and compare it to the hepatitis C virus NS5B. A total of 19 novel compounds revealed results that are either similar to or better than the physiological molecule, guanosine triphosphate. Water molecules are found to facilitate the binding of the compounds to ZIKV RNA-dependent RNA polymerase (RdRp) structures. The presented 19 novel compounds represent good binders to ZIKV RdRp and could be suitable candidates for developing a new and effective anti-ZIKV polymerase nucleotide inhibitor.

INTRODUCTION

n class="Chemical">Sepan>venty years ago, in Upan> class="Chemical">ganda, the Zika virus (ZIKV) wn class="Chemical">as reported for the first time.1, 2 About six decades later, newer incidents of emergence recorded in Nigeria, Senegal, and Gabon.3, 4, 5 Despite its spread, the reported ZIKV infections were not like the latest emerging outbreak in the year 2015 in Latin America.6 ZIKV is transmitted through body fluids and sexually. A direct link between newborn microcephaly and pregnant women infection was confirmed in the year 2016.7, 8, 9, 10 Mosquito bites are the main route of spread of ZIKV infections along with sexual intercourse.11, 12 The mild symptomatic ZIKV infection can be easily detected in body fluids like blood urine and saliva.10, 13 Severe neurological diseases and sterility are reported in some patients as the virus targets all cells of the nervous system.14

The n class="Species">ZIKVpan> n class="Chemical">genome is a single‐stranded RNA that encodes a 3400 amino acid polyprotein, which is processed by viral and host proteases to ten functional proteins.15 The nonstructural 5 (NS5) protein is the most widely conserved protein and has been targeted in previous viral infections like hepatitis C virus (HCV).16, 17, 18 The ZIKV RNA‐dependent RNA polymerase (RdRp) domain of the NS5 protein has been targeted by anti‐HCV drugs (repurposing).19, 20, 21 This protein domain is vital in the virus life cycle as it builds the new RNA strand from the complementary strand (the externalized RNA to the host cell) by utilizing the free nucleotides in the cytoplasm.15, 22 Targeting such a protein with nucleotide inhibitors stops the virus life cycle and eradicates the infection. The active site of NS5 polymerase lies in the palm subdomain (motif C), where two consecutive aspartates (D665 and D666) protrude from a beta‐turn structure and are surface accessible.19, 23 Nucleotide inhibitors mimic the native nucleotides in their ability to selectively target the active site of NS5 to be added into the primer RNA strand. Once bound to the protein, nucleotide inhibitors block the polymerization process and cause protein inhibition.24, 25, 26 Guanosine derivatives were studied against HCV polymerase and a candidate, IDX‐184, was under the clinical trials phase IIb before this was halted due to a side effect in the year 2013.18 This gave better results compared with the uridine derivative (sofosbuvir), adenine derivative (MK‐0608), and the wide‐range antiviral ribavirin against HCV and human coronaviruses in silico.16, 18

Molecular modeling reprepapan class="Chemical">n class="Chemical">senpan>ts a successful method un class="Chemical">sed to predict the drug/tarn class="Chemical">get binding potency and mode of interaction. Based on a suitable model, it can differentiate between active and inactive inhibitors and suggest new compounds (in silico screening).27, 28, 29, 30, 31

A few solved structures have n class="Chemical">bepan>en deposipan> class="Chemical">ted in the protein data bank recenn class="Chemical">tly for the ZIKV NS5 protein.32, 33, 34, 35, 36, 37 In this study, the author used two structures with PDB codes 5U04 and 5WZ3 as a drug target due to their appropriate resolution (1.9 and 1.8 Å, respectively) compared with other solved structures of ZIKV NS5. AutoDock Vina was used as the docking calculation method after cross‐docking with HCV NS5B RdRp. Seventy‐four new compounds were tested in this study against ZIKV NS5 RdRp structures. These compounds were derivatives of the guanine nucleotide.

MATERIALS AND METHODS

Ligands preparation for the docking study

Structures of the lin class="Chemical">gapan>nds are prepared using SCIGRESS 3.4 tools.31 The modifications were based on previous work, where three groups of modifications were introduced in the 2′ position in the ribose ring of the guanosine derivative.38 Structural geometry optimization wn class="Chemical">as performed using the following scheme; classical mechanical geometry optimization (MM3 force field) followed by the semi‐empirical parameterization method 6 (PM6) and finally, quantum mechanical density functional theory using the B3LYP functional. All these calculations were performed on the 3.4 GHz intel core‐i7 processor PC (12 GB RAM) using SCIGRESS 3.4 software.39, 40, 41, 42

Target retrieval, preparation, and docking

The solved structures of n class="Species">ZIKVpan> polymern class="Chemical">ase (n class="Chemical">NS5 RdRp domain) deposited in the protein data bank37 were examined, and two were selected, namely (PDB ID: 5U04 and 5WZ3). The structures were solved by x‐ray crystallography over the last 2 years with a resolution of 1.9 and 1.8 A, respectively. The HCV NS5B RdRp solved structure (PDB ID: 2XI3) was retrieved for comparison with ZIKV structures.43 Ions and ligands were removed using SCIGRESS docking preparation tools. The grid box was chosen to be of a 10 Å side length cube for all the structures. The box centers were selected to be at the active site (D665 & D666 and D318 & D319 in ZIKV and HCV NS5 RdRp, respectively). The grid boxes centers for 5U04, 5WZ3, and 2XI3 structures were (21.3 × 70.1 × 96.5), (52.1 × 2.0 × 80.2), and (9.8 × 5.6 × 10.0) Å, respectively. Missing hydrogen atoms were added using the SCIGRESS docking preparation tools, as the targets used in this study were solved by x‐ray crystallography. AutoDock Vina implemented on SCIGRESS 3.4 software was used in this study with the flexible target's active site and the flexible ligand approach.30, 44 The binding affinities were represented for the best complexes with the aid of PyMOL, Maestro, and Microsoft Excel software.45, 46, 47

RESULTS

Ligand preparation

n class="Chemical">Sepan>venty‐four modified pan> class="Chemical">guanosine derivatives (see Table S1) were sketched and optimized using the same procedure of that of Elfiky.38 The modified compounds were bn class="Chemical">ased on modifying the 2′‐position of the ribose ring of the guanosine triphosphate (GTP). The addition of a bulky group at this position gave good results against HCV NS5B RdRp in previous studies.38, 48

Binding affinity calculation

AutoDock Vina implemepan class="Chemical">nted on SCIGRESS 3.4 software wnpan> class="Chemical">as used in this study to calculate the binding energies between the ligands and the target polymerase for both ZIKV and HCV. Figure 1A‐C shows the calculated binding energies (docking scores) for the three groups of modifications (groups I, II, and III, respectively) against ZIKV NS5 RdRp (orange line) and HCV NS5B RdRp ((PDB ID: 2XI3) blue line). Average values, with the standard deviations as error bars, are represented in Figure 1 for ZIKV solved structures (PDB ID: 5U04 and 5WZ3).

Figure 1

The docking scores calculated by AutoDock Vina software for three groups of modifications to GTP, group I (A), group II (B), and group III (C). The docking study was performed using two ZIKV RdRp solved structures (PDB ID: 5WZ3 and 5U04), and one HCV RdRp solved structure (PDB ID: 2XI3) for comparison. The average values of the docking scores for ZIKV RdRp are represented by the orange line while HCV RdRp docking values in blue lines. GTP, guanosine triphosphate; HCV, hepatitis C virus; RdRp, RNA‐dependent RNA polymerase; ZIKV, Zika virus

The docking scores calculapapan class="Chemical">n class="Chemical">ted by AutoDock Vinclass="Chemical">pan>a software for three groups of modifications to pan> class="Chemical">GTP, group I (A), group II (B), and group III (C). The docking study was performed using two n class="Species">ZIKV RdRp solved structures (PDB ID: 5WZ3 and 5U04), and one HCV RdRp solved structure (PDB ID: 2XI3) for comparison. The average values of the docking scores for ZIKV RdRp are represented by the orange line while HCV RdRp docking values in blue lines. GTP, guanosine triphosphate; HCV, hepatitis C virus; RdRp, RNA‐dependent RNA polymerase; ZIKV, Zika virus

Table 1 summarizes the inpapan class="Chemical">n class="Chemical">teractionpan>s n class="Chemical">between the top‐ranked lin class="Chemical">gands and ZIKV NS5 RdRp structures. The selection is based on the docking scores. Values equal to or less than (better) the binding energy of the parent nucleotide (GTP) are listed in Table 1. Group I of the modifications have six compounds that are equal to or better binders compared with GTP. These molecules include the modified GTP at the 2′ position with fluoromethyl, difluoromethyl, trifluoromethyl, 2,2‐difluoroethyl, 2,2,2‐trifluoroethyl, and selenanylmethyl. On the other hand, 10 compounds from group II have better (compared with GTP) docking scores. These molecules include the modified GTP at the 2′ position with ethyloxidanyl, phosphanyl, phenyloxidanyl, 3,5‐dihydroxyphenyl, (2,6‐dihydroxyphenyl)oxidanyl, (2‐hydroxyphenyl)oxidanyl, (3‐hydroxyphenyl)oxidanyl, (2,6‐difluorophenyl)oxidanyl, (3‐fluorophenyl)oxidanyl, and (4‐fluorophenyl)oxidanyl. Finally, group III has only three compounds that have better binding energies than ZIKV RdRp structures compared with GTP. These compounds have two modifications to the 2′ position, which are two methyl groups, ethyl and fluorenyl, and two fluorenyl groups. The interacting amino acids with the ligands are also listed in Table 1 with the number of water molecules that take part in the interactions. Some water molecules mediate the interaction by binding to both the ligand and protein binding pocket.

Table 1

GTP selected modifications, with the average docking scores less than (better) or equal to GTP, calculated using AutoDock Vina for ZIKV structures (PDB ID: 5U04 and 5WZ3)

R1	Substitution name	Average docking scores (kcal/mol) ± SD	H2O and amino acids involved in H‐bond formation
			5U04	5WZ3
GTP	The parent compound	−6.30 ± 0.60	H 2 O (5), N612, D665, S798	H 2 O (13), D540, D665 (2), D666
Group I (R1)
2	Fluoromethyl	−6.35  ± 0.25	H 2 O (9), T608, D666, I799,	H 2 O (7), N612 (3), S712
3	Difluoromethyl	−6.45  ±  0.05	H 2 O (7), D665, D666, S712	H 2 O (7), N612 (2), D665, S712
4	Trifluoromethyl	−6.35  ±  0.85	H 2 O (7), T608, D666, S712	H 2 O (8), N612 (2), S663(2), D666
6	2,2‐Difluoroethyl	−6.65  ±  0.35	H 2 O (7), T608, N612, D666, S712	H 2 O (10), N612 (2), S663 (3), D665, S712 (4)
7	2,2,2‐Trifluoroethyl	−6.35  ±  0.55	H 2 O (7), D666, S712, I799	H 2 O (9), D540, S663, D665, 2 S712
15	Selenanylmethyl	−6.45  ±  0.15	H 2 O (6), T608, N612, S663, D665, D666	H 2 O (9), D540 (2), D665 (3), D666, S712, I799
Group II (R2)
2	Ethyloxidanyl	−6.45  ±  0.25	H 2 O (7), S663, D665, D666, S712, W797, S798	H 2 O (8), W539, D540 (4), N612 (2), S663, D665
5	Phosphanyl	−6.35  ±  0.25	H 2 O (5), N612, D665, S712, I799	H 2 O (10), W539, D540, N612, S663, 2 D665, S798
9	Phenyloxidanyl	−6.60  ±  0.30	H 2 O (9), D540, N612, D665 (3), D666, I779	H 2 O (9), D540, N612, D665 (3), D666, I779
13	3,5‐Dihydroxyphenyl	−6.50  ±  0.30	H 2 O (7), T608 (2), S663 (2), D665, D666 (2)	H 2 O (12), D540, S603, S663 (2), D665, D666
16	(2,6‐Dihydroxyphenyl)oxidanyl	−6.65  ±  0.15	H 2 O (8), T608 (2), Y609, D666 (2), S712	H 2 O (9), D535, N612 (2), D665, S712
17	(2‐Hydroxyphenyl)oxidanyl	−6.50  ±  0.50	H 2 O (8), N612, D665, S712 (2)	H 2 O (9), D535 (2), N612 (2), S663, D665, K691
19	(3‐Hydroxyphenyl)oxidanyl	−6.30  ±  0.60	H 2 O (6), T608, D665, D666 (2)	H 2 O (9), D535, D540, N612, S663 (2), D665
36	(2,6‐Difluorophenyl)oxidanyl	−6.30  ±  0.05	H 2 O (7), T608, D665 (2), D666 (3), S712	H 2 O (9), D540 (2), D665 (3), S712 (2)
39	(3‐Fluorophenyl)oxidanyl	−6.40  ±  0.10	H 2 O (6), T608 (2), D665, D666, S712 (2)	H 2 O (14), D540, S712
40	(4‐Fluorophenyl)oxidanyl	−6.40  ±  0.40	H 2 O (6), D665, D666, S712, W797, I799	H 2 O (8), W539, D540 (3), N612, S663, D665 (3)
Group III (R3 and R4)
1	Methyl + methyl	−6.40  ±  0.60	H 2 O (7), D666 (2), S712, S798	H 2 O (11), N612 (3), D665
3	Ethyl + fluoranyl	−6.45  ±  0.15	H 2 O (5), T608, N612, D666, S712 (2), I799	H 2 O (7), W539, D540 (3), S663 (2)
4	Fluoranyl + fluoranyl	−6.30  ±  0.30	H 2 O (5), T608, D665, D666 (3)	H 2 O (8), D540, N612, S663 (2), D665 (2)

Note: The H bonds formed between the ligands and the proteins are listed with their number. The amino acids involved in the interaction are listed with the number in brackets for the number of H bonds if greater than 1. It is based on a baseline value below it the values are marked as bold or underlined.

Abbreviations: GTP, guanosine triphosphate; ZIKV, Zika virus.

n class="Chemical">GTPpan> n class="Chemical">selected modifications, with the average docking scores less than (better) or equal to GTP, calculated using AutoDock Vina for ZIKV structures (PDB ID: 5U04 and 5WZ3)

n class="Chemical">Npan>on class="Chemical">te: The H bonds formed n class="Chemical">between the ligands and the proteins are listed with their number. The amino acids involved in the interaction are listed with the number in brackets for the number of H bonds if greater than 1. It is based on a baseline value below it the values are marked as bold or underlined.

Abbreviations: n class="Chemical">GTP, class="Chemical">pan> class="Chemical">guanosine triphosphate; ZIKV, Zika virus.

Figure 2A and 2B shows the docked structures of papan class="Chemical">n class="Chemical">GTP to npan> class="Species">ZIKV NS5 RdRp solved structures (PDB ID: 5WZ3 and 5U04, respectively). GTP is represented by atom type in stick color: carbon in green, hydrogen in white, nitrogen in blue, oxygen in red, and sulfur in orange. Water molecules are represented by red spheres. The ZIKV RdRp protein is represented by a rainbow‐colored cartoon. The R groups of the amino acids that take part in the binding to GTP are represented by lines of the same color as the cartoon. Polar interactions are represented by dashed yellow lines.

Figure 2

The docking complexes of GTP to ZIKV NS5 RdRp (PDB ID: 5WZ3 (A) and 5U04 (B)). ZIKV RdRp is represented by the colored cartoon while the amino acids involved in the interactions are represented by lines and labeled by their three‐letter code. GTP is represented according to the atom type in stick color ; C, green; H, white; O, red; N, blue; P, orange. Water is represented bye red spheres while yellow dashed lines represente the polar contact. GTP, guanosine triphosphate; NS5, non‐structural 5; RdRp, RNA‐dependent RNA polymerase; ZIKV, Zika virus

The docking complexes of papan class="Chemical">n class="Chemical">GTP to npan> class="Species">ZIKV NS5 RdRp (PDB ID: 5WZ3 (A) and 5U04 (B)). ZIKV RdRp is represented by the colored cartoon while the amino acids involved in the interactions are represented by lines and labeled by their three‐letter code. GTP is represented according to the atom type in stick color ; C, green; H, white; O, red; N, blue; P, orange. Water is represented bye red spheres while yellow dashed lines represente the polar contact. GTP, guanosine triphosphate; NS5, non‐structural 5; RdRp, RNA‐dependent RNA polymerase; ZIKV, Zika virus

DISCUSSION

Sugn class="Chemical">gepan>sting a popan> class="Chemical">tent inhibitor against n class="Species">ZIKV RdRp is the primary goal of this study, Figure 1 shows that almost all compounds have good binding energies to ZIKV polymerase that are comparable to that for HCV RdRp. For the groups I and III of the modifications, the docking scores of ZIKV are almost in the same range as HCV except for compound 7 in group III that has a bit higher docking score value (−5.2 kcal/mol). On the other hand, group II of the modifications has some compounds that bind more like HCV, and other compounds that have less binding potency compared to HCV. Overall, the docking scores for the modifications lie between −6.65 and −5.1 for ZIKV polymerase while the values for HCV RdRp are between −7.7 and −5.2 (see Table S1). This implies the effectiveness of the modified compounds in competing for the active site of ZIKV polymerase.

Table 1 shows the n class="Chemical">bepan>st compounds based on their binding energies (docking scores) to ZIKV RdRp structures. A total of 19 compounds show n class="Chemical">better values for binding ZIKV NS5 RdRp compared with the parent compound GTP. These compounds have a better chance to bind to the active site of the polymerase upon its presence in solution with GTP, the parent, and physiological molecule.

The amino acids ipan class="Chemical">nvolved in the binding of the top‐ranked ligands to pan> class="Species">ZIKV polymerases are listed in Table 1. We can notice that the active site aspartates (D665 and D666) mediate the interaction in almost all binding trials. This supports the conservation of these residues in viral and even human polymerases.

The n class="Species">ZIKVpan> solved structure hn class="Chemical">as n class="Chemical">water molecules surrounding the binding pocket. The author decided to perform the docking experiment without removing the water molecules as water may affect the binding of the hydrophilic ligands to the hydrophilic active site aspartates. Surprisingly, the number of water molecules that interact with the ligands varies from five (GTP, compound 5 in group II and compounds 3 and 4 of group III) up to 14 (compound 39 in group II). It has been reported that water mediates the dynamics of biomolecules.49 Figure 2 shows how the water molecule interacts in a network facilitating and supporting the binding of GTP into ZIKV NS5 RdRp. Panel A of Figure 2 represents the GTP docked into the ZIKV RdRp structure (PDB ID: 5WZ3) while panel B represents the structure (PDB ID: 5U04). The water molecules network through H‐bonds. Besides this, water connects with both the ligand (GTP) and the protein (ZIKV NS5 RdRp) polar residues through the formation of polar contacts. These polar interactions stabilize the protein‐ligand complexes facilitating the polymerase function. The water network that is formed upon ligand‐protein docking is key for the effectiveness of the ligand as an inhibitor.

CONCLUSION

RdRp is sugn class="Chemical">gepan>sted to n class="Chemical">be a suitable target for HCV and other viruses due to its vital role in the replication of the virus. Due to the conservation of the RdRp's active site, it is also targeted in ZIKV and other viral infections. Previous studies show that anti‐HCV NS5B drugs are able to bind to ZIKV RdRp (repurposing trials) but with relatively lower affinity compared with HCV. In this study, the author presents 19 modified guanosine derivatives that show in silico effectiveness against ZIKV NS5 RdRp. Further experimental work is suggested to characterize these modifications further and apply it to the virus in vitro and in vivo assays.

CONFLICT OF INTERESTS

The authors declare that there are no conflict of inn class="Chemical">terests.

Supplementary information

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