Antivirals for allosteric inhibition of Zika virus using a homology model and experimentally determined structure of envelope protein
© The Author(s) 2017
Received: 24 May 2017
Accepted: 22 July 2017
Published: 28 July 2017
An approach to inhibiting enveloped flaviviruses is to deter the ability of the envelope protein(s) binding onto glycoproteins. In previous work, using a small ~100-amino acid homology model of Zika virus envelope protein (ZVEP), we proved the susceptibility of Zika virus to inhibition. In this work, we verify the efficacy of the homology model based antiviral search method using a larger protein (>400 amino acids) and comparing the results with the experimentally determined one (PDB ID:5IRE).
By examining how glycan molecules, small-molecule probes and screened ligands that have a high affinity to ZVEP, we report the mechanics of ZVEP to inhibition via allosteric blockage of the glycan-binding domain while proposing even more possibly potent inhibitors. The small molecular probes based study using the homology model and subsequently verified using actual experimental structure, 5IRE, revealed that ZVEP is druggable. A pharmacophore analysis followed by screening showed at least four ligands that allosterically binds to the glycan binding domain constituted by residues VAL 153 and ASN 154 in 5IRE. Based on further selection criteria ZINC40621658 was identified to have high potential to be a strong antiviral candidate for Zika virus inhibition.
The World Health Organization estimates that Zika disease, an infection caused by Zika virus, to reach epidemiological levels [1, 2]. An infection with the virus causes Zika Fever [3, 4], and yet, no vaccines or drugs are available to prevent or treat an infection . The connections of the virus to microcephaly [1, 6] and neurological conditions in infected adults, including cases of the Guillain–Barré syndrome [7, 8], make Zika potentially destructive.
Among several approaches to developing antiviral drugs targeting the Zika flavivirus, inhibition of the envelop protein is considered to be promising. Computational and experimental work have proven that blocking envelope protein is a viable approach to inhibiting flaviviruses [9–11], which include Zika, Dengue, Encephalitis, and West Nile.
To perform in silico inhibitor screening, experimentally determined structures of the protein(s) responsible for virus’ virulence are essential; and in many instances, these structures are not available . So, there is a need for a technique to reliably screen for drugs in the absence of three-dimensional structural data. Nevertheless, advances in biotechnology have enabled rapid availability of nucleotide sequence data that could be used to develop homology models that in turn could be utilized for inhibitor screening. This work looks at finding antivirals targeting ZVEP using a homology model and then comparing the efficacy of the potential inhibitors with the experimentally determined NMR structure of ZVEP 5IRE . The methods can be applied to druggable proteins of any emerging pathogen even in the absence of complete structural information.
Based on the target-template alignment, homology models were built via Promod-II . Conserved coordinates between the target and the template were copied from the template to the model. Insertions and deletions were remodeled using a fragment library. Then, side-chains were rebuilt. Lastly, the geometry of the model was regularized by using force fields. The overall and per-residue model quality was assessed using the QMEAN4  and Global Model Quality Estimation (GMQE) scoring functions as shown in Fig. 1c, d. Each residue is assigned a QMEAN reliability score between zero and one, describing the expected similarity to the native structure and higher numbers indicate a greater reliability of the residues. GMQE combines properties from the target-template alignment, and the score is expressed as above reflecting the expected accuracy of a model built with that alignment and template.
Dengue virus envelope protein (WNEP, PDB ID: 3j27) was identified to be the best template to be used for the development of the final ZVEP homology model and a three-dimensional model for the target protein was generated (Fig. 1b). Model quality assessment tools were used to assess the reliability of the resulting models are given in the inset (Fig. 1c, d). The predicted ZVEP homology structure (yellow—Fig. 1b), when overlaid with that of 5IRE for comparison (blue—Fig. 1b), shows >50% structural identity preserved indicating the model’s utility as a proxy for studying ZVEP behavior.
Active site analysis
Druggability assessment of homology model and experimental structure of ZVEP
The hotspot distribution when the druggability analysis was applied to ZVEP 5IRE is depicted in Fig. 3c. The druggability analysis revealed 203 small-molecule binding hotspots ranging from a minimum ∆G of −2.34 kcal/mol and maximum of −1.00 kcal/mol. Throughout the protein surface, 105 binding hotspots of isopropanol was detected with the lowest binding free energy of −2.34 kcal/mol. Again, isobutene (51 hotspots, −1.85 kcal/mol), isopropylamine (15 hotspots; −1.94 kcal/mol), acetamide (6 hotspots, −1.90 kcal/mol), and acetate (26 hotspots, −1.99 kcal/mol) enrichment were more isolated. The analysis predicted the presence of two druggable domains (Fig. 3c). The probe occupancy grid distribution across the ZVEP surface of the homology model at the vicinity of the receptor—suggesting active site compositions of a potential drug candidate—are depicted in Fig. 3d.
The analysis suggests that the homology model predicts the potential druggability of ZVEP which was further confirmed by 5IRE. The primary active probe for ZVEP is isopropanol while the secondary being isobutene.
Chemical characteristics of select compounds screened via ELIXIR-A
Affinity (kcal/mol) on homology model
Affinity (kcal/mol) on 5IRE
All the four ZINC candidates depicted in Table 1 had higher binding affinities for the site than the glycan NAG—indicating that the ligands would bind tighter to the location as opposed to the glycan. It should be noted that the efficacy of ZINC33683341, the more conservative candidate of the three that has the highest affinities on 5IRE has been already analyzed using in vitro assay as described previously and verified to inhibit Zika virus  successfully. A close inspection of the binding confirmations of ZINC33683341 on 5IRE suggests the inhibition action is allosteric, i.e., although the ligand bound transverse to the primary glycan binding site (Fig. 4b), the binding action was able to trigger inhibition. Excitingly, based on binding affinities with the homology model and in silico verification of interactions on 5IRE, ZINC40621658 seems to hold even more promise (Fig. 4c–e). The distribution of ZINC40621658 (Fig. 4c) on 5IRE (Fig. 4e) is consistent with that predicted by the homology model (Fig. 4d). A close examination of the interaction diagram reveals that ZINC40621658 would bind at proximity to the glycan binding site and the inhibition would be allosteric (since all conformations of NAG make hydrogen bonding and close interactions with residues 153–154 on 5IRE as depicted in Fig. 2f, g whereas ZINC40621658 form interactions with several residues between residues 145–148 and 364–373. Due to more attractive affinities, spatial distribution, and desirable pharmacophore properties, ZINC40621658 has the potential to be a strong antiviral candidate for Zika virus inhibition and has to be experimentally verified for its efficacy.
How ligands and small molecule drug-like probes interact with ZVEP was analyzed using a homology model developed using West Nile envelope protein as the template. The small molecular probes based study using the homology model and subsequently verified using actual experimental structure, 5IRE, revealed that ZVEP is druggable. A pharmacophore analysis followed by screening showed at least four ligands that allosterically binds to the glycan binding domain constituted by residues VAL 153 and ASN 154 in 5IRE. Based on further selection criteria ZINC40621658 was identified to have high potential to be a strong antiviral candidate for Zika virus inhibition and has to be experimentally verified for its efficacy. We believe that identification these compounds that have a high affinity to the glycan receptor is a decent starting point for drug discovery targeting ZVEP.
Of the five potential inhibitors that were screened, only one was experimentally verified. To ascertain the efficacy, all five should be subjected to experimental verification. Although this work targeted inhibition of the envelope, primarily due to advantages of the drug not being required to penetrate the virus, inhibition via this route requires relatively high ligand concentrations (~100 µM).
TF was responsible in performing a portion of the simulations and ligand screening. SF analyzed data and wrote the manuscript. Both authors read and approved the final manuscript.
Some of this work was conducted with the help of high-performance research computing resources provided by Texas A&M University (http://hprc.tamu.edu).
Authors confirm that they have read BioMed Central’s guidance on competing interests and declare that none of the authors have any competing interests.
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