Abstract
Introduction
Dengue remains an overwhelming burden globally, where the disease is now endemic in 100 countries worldwide. It was reported that the host's glycolytic pathway was proven to be essential for the replication of the causative agent of the disease, the dengue virus (DENV). We are particularly interested in human hexokinase isoform 2 (HK2) as an anti-DENV drug target since the expression of this enzyme was proven to be upregulated in DENV-infected cells, thus activating the pathway to support their life cycles.
Methods
In this study, a ligand-based virtual screening programme, Ultra-Fast Shape Recognition with Atom Types (USRCAT) was used to obtain compounds similar to glucose (GLC), the substrate for HK2. The analogues of GLC were then docked into the binding pockets of the structure of HK2 (PDB ID: 2NZT), by using AutoDock Vina programme. Meanwhile, the recombinant human HK2 was expressed in bacterial system to be utilised for the subsequent enzyme inhibition study. A full-length cDNA encoding HK2 was cloned into pETite N-His SUMO Kan vector and later transformed into E.coli BL21(DE3) and BL21(DE3) pLysS cells separately, for protein expression studies with varying parameters, which includes time of incubation, growth media, cell strains and concentrations of inducer.
Results
The ligand-based virtual screening resulted in 20 similar molecules to GLC with similarity scores ranging from 0.85-0.90. The hits, which were docked into the HK2 binding sites exhibited binding energies ranged from -6.2 to -5.8 kcal/mol and -6.5 to -5.7 kcal/mol on binding pockets 1 and 2 (Figure 1), respectively. The recombinant HK2 in pETite N-His SUMO Kan vector has successfully been constructed, which was approximately 5 kb in size. The bacterially-expressed HK2 has also successfully been expressed in both E.coli BL21(DE3) and BL21(DE3)pLysS cells, which showed the presence of the protein that was approximately 120 kDa in size.
Conclusions
Taken together, the identification of potential inhibitors of recombinant human HK2, as well as the successful expression of the enzyme have paved the way in discovering future anti-DENV therapeutics. The effects of these potential inhibitors on the activity of recombinant human HK2 is being pursued by structural and biochemical studies.
