Targeting the ubiquitin-conjugating enzyme E2D4 for cancer drug discovery-a structure-based approach

Abstract

Cancer progression is a global burden. The incidence and mortality now reach 30 million deaths per year. Several pathways of cancer are under investigation for the discovery of effective therapeutics. The present study highlights the structural details of the ubiquitin protein 'Ubiquitin-conjugating enzyme E2D4' (UBE2D4) for the novel lead structure identification in cancer drug discovery process. The evaluation of 3D structure of UBE2D4 was carried out using homology modelling techniques. The optimized structure was validated by standard computational protocols. The active site region of the UBE2D4 was identified using computational tools like CASTp, Q-site Finder and SiteMap. The hydrophobic pocket which is responsible for binding with its natural receptor ubiquitin ligase CHIP (C-terminal of Hsp 70 interacting protein) was identified through protein-protein docking study. Corroborating the results obtained from active site prediction tools and protein-protein docking study, the domain of UBE2D4 which is responsible for cancer cell progression is sorted out for further docking study. Virtual screening with large structural database like CB_Div Set and Asinex BioDesign small molecular structural database was carried out. The obtained new ligand molecules that have shown affinity towards UBE2D4 were considered for ADME prediction studies. The identified new ligand molecules with acceptable parameters of docking, ADME are considered as potent UBE2D4 enzyme inhibitors for cancer therapy.

Keywords: ADME; Active site; Cancer; Homology modelling; Protein-protein docking; Virtual screening.

Fig. 1

Bio-chemical pathway of UBE2D4 and its role in cancer progression. Ubiquitin proteasomal degradation process involves binding of ubiquitin with ubiquitin-activating enzyme (E1) in the presence of ATP to form a thio-ester bond. Activated ubiquitin is transferred to UBE2D4 enzyme maintaining thio-ester bond with the elimination of E1 enzyme. This reaction is followed by covalent attachment of UBE2D4 with an ubiquitin CHIP- ligase (E3) which results in polyubiquitination reaction and leads to cell cycle progression

Fig. 2

Putative conserved domains and conserved motifs of the UBE2D4 protein. PLN00172 ubiquitin conjugating enzyme; provisional, UBCc ubiquitin-conjugating enzyme E2, catalytic (UBCc) domain. Basic local alignment search tool illustrates that the UBE2D4 contains domains like, ubiquitin (Ub) thio-ester intermediate interaction domains, E3 interaction domains present in the UBc domain

Fig. 3

Alignment of UBE2D4 protein with template sequences. Pair wise sequence alignment carried out with CLUSTALW server. The conserved residues are represented as asterisk and highly similar residues as colon, similar residues as period. Alignment of UBE2D4 with the template protein gives 95% sequence similarity

Fig. 4

Ramachandran plot of UBE2D4 protein. The red-coloured field in the plot indicates energetically the most favoured region. The yellow field represents the additionally allowed region, the light yellow represents the generously allowed and the white field indicates the disallowed region

Fig. 5

ProSA z-score plot of the UBE2D4 protein. ProSA plot—the energy profile of UBE2D4 obtained using ProSA web server analysis showing z-score of −5.49. The ProSA–web z-scores of all protein chains in PDB determined by X-ray crystallography are shown in light blue and by NMR spectroscopy in dark blue

Fig. 6

ProSA energy profile of UBE2D4 protein. The ProSA analysis of the model shows maximum residues in the negative energy region. The negative ProSA energies of UBE2D4 protein indicate a reliable arrangement of amino acids in the 3D structure

Fig. 7

The VERIFY_3D profile of UBE2D4 protein. VERIFY_3D analyzes the compatibility of an atomic UBE2D4 model (3D) with its own amino acid sequence (1D). The scores of a sliding 21-residue window (from −10 to +10) are added and plotted for individual 147 residues. 85% of the residues have an average 3D–1D score >0.2

Fig. 8

The 3D model of the UBE2D4 protein. The 3D structure of the UBE2D4 protein, which shows four alpha helices and four beta sheets

Fig. 9

The binding site region in UBE2D4 protein predicted using SiteMap. The UBE2D4 protein is represented in solid ribbon form. The active site shown in mesh fashion represents hydrogen bond acceptors in red, hydrogen bond donors in blue and hydrophobic region in yellow

Fig. 10

Protein-protein docking of UBE2D4 and its natural substrate ubiquitin ligase CHIP. The protein-protein docking (molecular surface figure) of the UBE2D4 with its natural substrate ubiquitin ligase CHIP was carried out using PATCH DOCK server. The UBE2D4 protein active site residues LYS 66, VAL 67, SER 80, SER 83, ILE 84 and ARG 90 bind with the ubiquitin ligase CHIP

Fig. 11

Binding interactions of UBE2D4 protein with the ligand molecules. The protein is represented in green solid ribbon form and the ligand in blue stick form. The hydrogen bond interactions are represented in black and Pi–cation interactions in orange. UBE2D4 protein amino acid residues are represented in green yellow sticks. The interactions in the protein-ligand complex are explained by the 2D diagram (D1 to D10). The non-ionic interactions of the ligand molecules with protein amino acid residues are shown in pink lines

Fig. 11

Binding interactions of UBE2D4 protein with the ligand molecules. The protein is represented in green solid ribbon form and the ligand in blue stick form. The hydrogen bond interactions are represented in black and Pi–cation interactions in orange. UBE2D4 protein amino acid residues are represented in green yellow sticks. The interactions in the protein-ligand complex are explained by the 2D diagram (D1 to D10). The non-ionic interactions of the ligand molecules with protein amino acid residues are shown in pink lines

Fig. 11

Binding interactions of UBE2D4 protein with the ligand molecules. The protein is represented in green solid ribbon form and the ligand in blue stick form. The hydrogen bond interactions are represented in black and Pi–cation interactions in orange. UBE2D4 protein amino acid residues are represented in green yellow sticks. The interactions in the protein-ligand complex are explained by the 2D diagram (D1 to D10). The non-ionic interactions of the ligand molecules with protein amino acid residues are shown in pink lines

Fig. 11

Binding interactions of UBE2D4 protein with the ligand molecules. The protein is represented in green solid ribbon form and the ligand in blue stick form. The hydrogen bond interactions are represented in black and Pi–cation interactions in orange. UBE2D4 protein amino acid residues are represented in green yellow sticks. The interactions in the protein-ligand complex are explained by the 2D diagram (D1 to D10). The non-ionic interactions of the ligand molecules with protein amino acid residues are shown in pink lines

Fig. 12

SASA graph of the UBE2D4 protein before and after docking. The decreased SASA values of the UBE2D4 protein active site is shown in the graph. The residues PRO 64, VAL 67, ASP 84, ARG 90 and ASP 117 show less surface accessibility after docking. The maroon line after docking and the blue line represents before docking