Hit-to-Lead Short Peptides against Dengue Type 2 Envelope Protein: Computational and Experimental Investigations

Abstract

Data from the World Health Organisation show that the global incidence of dengue infection has risen drastically, with an estimated 400 million cases of dengue infection occurring annually. Despite this worrying trend, there is still no therapeutic treatment available. Herein, we investigated short peptide fragments with a varying total number of amino acid residues (peptide fragments) from previously reported dengue virus type 2 (DENV2) peptide-based inhibitors, DN58wt (GDSYIIIGVEPGQLKENWFKKGSSIGQMF), DN58opt (TWWCFYFCRRHHPFWFFYRHN), DS36wt (LITVNPIVTEKDSPVNIEAE), and DS36opt (RHWEQFYFRRRERKFWLFFW), aided by in silico approaches: peptide-protein molecular docking and 100 ns of molecular dynamics (MD) simulation via molecular mechanics using Poisson-Boltzmann surface area (MMPBSA) and molecular mechanics generalised Born surface area (MMGBSA) methods. A library of 11,699 peptide fragments was generated, subjected to in silico calculation, and the candidates with the excellent binding affinity and shown to be stable in the DI-DIII binding pocket of DENV2 envelope (E) protein were determined. Selected peptides were synthesised using conventional Fmoc solid-phase peptide chemistry, purified by RP-HPLC, and characterised using LCMS. In vitro studies followed, to test for the peptides' toxicity and efficacy in inhibiting the DENV2 growth cycle. Our studies identified the electrostatic interaction (from free energy calculation) to be the driving stabilising force for the E protein-peptide interactions. Five key E protein residues were also identified that had the most interactions with the peptides: (polar) LYS36, ASN37, and ARG350, and (nonpolar) LEU351 and VAL354; these residues might play crucial roles in the effective binding interactions. One of the peptide fragments, DN58opt_8-13 (PFWFFYRH), showed the best inhibitory activity, at about 63% DENV2 plague reduction, compared with no treatment. This correlates well with the in silico studies in which the peptide possessed the lowest binding energy (-9.0 kcal/mol) and was maintained steadily within the binding pocket of DENV2 E protein during the MD simulations. This study demonstrates the use of computational studies to expand research on lead optimisation of antiviral peptides, thus explaining the inhibitory potential of the designed peptides.

Keywords: dengue; envelope protein; molecular docking; molecular dynamics; peptide; plaque assay.

Figure 1

Binding pocket of DENV2 E protein. DET4 (in magenta) binds at the DI-DIII hinge (DI in green and DIII in blue) is illustrated here. Coloured figure is available online.

Figure 2

Plot of 100 ns MD simulation for the DN580pt_8-13 peptide fragment. Slight overlapping between the complex data (blue) and the E protein data (orange). Coloured figure is available online.

Figure 3

Plot of 100 ns MD simulation for the DN580pt_8-11 peptide fragment. The peptide was stable for the first 10 ns and then quickly destabilized and moved out of the pocket by 35 ns simulation. Coloured figure is available online.

Figure 4

Plot of 100 ns MD simulation for the DN580pt_9-9 peptide fragment. The complex data (blue) overlap with the E protein data (orange) throughout the simulation. Coloured figure is available online.

Figure 5

Plot of 100 ns MD simulation for the DN580pt_10-6 peptide fragment. The complex data (blue) overlap with the E protein data (orange). Coloured figure is available online.

Figure 6

Combined free energy decomposition per residue of DN58opt_8-13, DN58opt_10-6, and DN58opt_9-9. The positive and negative values indicate the unfavourable and favourable contributions for the binding, respectively.

Figure 7

Schematic representation of the binding interactions between the DENV2 E protein (circled residues—in green/orange/pink/red/purple) and the peptide fragments (line representation): (a) DN58opt_10-6; (b) DN58opt_8-13; (c) DN58opt_9-9, peptide fragments. Coloured image is available online.

Figure 8

Vero cells viability (in percentage viability, Viability %) following 48 h exposure to DN58opt_8-13, DN58opt_9-9, and DET4, at various concentrations (0–400 µM).

Figure 9

Antiviral activity of synthesised short peptide fragments against DENV2 using plaque formation assay. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 10

Illustration of six-residue peptide fragmentation of DN58opt as an example. The same procedure was applied to 7-, 8-, 9- and 10-amino-acid peptides: (a) sliding windows of 6 amino acids, with 1 amino acid step size; (b) a different illustration of the sliding windows. Greyed residues are not a 6-amino-acid fragment; thus, it was rejected. The bold red line is where the peptide was cut.