Inhibitors of the Cdc34 acidic loop: A computational investigation integrating molecular dynamics, virtual screening and docking approaches

Abstract

Among the different classes of enzymes involved in the ubiquitin pathway, E2 ubiquitin-conjugating enzymes occupy a central role in the ubiquitination cascade. Cdc34-like E2 enzymes are characterized by a 12-14 residue insertion in the proximity of the catalytic site, known as the acidic loop. Cdc34 ubiquitin-charging activity is regulated by CK2-dependent phosphorylation and the regulatory mechanism involves the acidic loop. Indeed, the phosphorylation stabilizes the loop in an open conformation that is competent for ubiquitin charging. Cdc34 is associated with a variety of diseases, such as hepatocellular carcinomas and prostatic adenocarcinomas. In light of its role, the discovery of potential inhibitory compounds would provide the mean to effectively modulate its activity. Here, we carried out a computational study based on molecular dynamics, virtual screening and docking to identify potential inhibitory compounds of Cdc34, modulating the acidic loop conformation. The molecules identified in this study have been designed to act as molecular hinges that can bind the acidic loop in its closed conformation, thus inhibiting the Cdc34-mediated ubiquitination cascade at the ubiquitin-charging step. In particular, we proposed a pharmacophore model featuring two amino groups in the central part of the model and two lateral aromatic chains, which respectively establish electrostatic interactions with the acidic loop (Asp 108 and Glu 109) and a hydrogen bond with Ser 139, which is one of the key residues for Cdc34 activity.

Keywords: Cdc34; Docking; E2 conjugating enzyme; MD, molecular dynamics; Sc, Saccharomyces cerevisiae; UBC, ubiquitin-binding domain; Ub, ubiquitin; Ubiquitin; Ubiquitination; Virtual screening.

Graphical abstract

Fig. 1

Cdc34 three-dimensional structure. The acidic loop is represented in yellow, while the catalytic cysteine in red. The three residues (P110, I137 and N138) selected to define the binding site for virtual screening are depicted in cyan (right panel). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Autodock results upon clustering with Autodock internal routines. The first 20 molecules from the Autodock energy-ranking list are depicted with different colors. The residues of the acidic loop are shown as yellow sticks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Histogram distribution of the distances calculated between the docked molecules and the residues selected for the Autodock grid construction (P110, I137 and N138).

Fig. 4

Molecules identified by the post-clustering ranking according to both structural and energetic criteria. In the left panel, the first 20 high-ranking molecules after clustering and a portion of Cdc34 are depicted with surface representation of molecule C08743791. In the right panel, the high-ranking clustered molecules are shown superimposed to underline the presence of two recurrent amino groups. The selection step has been carried out post-processing Autodock output with Phyton Pymol API tools.

Fig. 5

Examples of binding poses for the molecules. The left panel and right panels show the orientation of molecule C08743791 (4,6-dimethyl-N-[3-(5-phenyl-1H-pyrazol-3-yl)-1H-1,2,4-triazol-5-yl]pyrimidin-2-amine), and molecule C20209924 (5-methyl-N-{2-[3-(3-pyridinyl)-1H-1,2,4-triazol-5-yl]ethyl}-2-indolinecarboxamide), respectively. The first molecule is an example of molecule with an orientation suited for the interaction with both the acidic loop and the surrounding structural components (V143 and V147), while the latter is only interacting with the acidic loop and belongs to the subset of compounds that has been discarded upon the final selection step with API tools.

Fig. 6

Interactions between the selected molecules and D108 and E109 residues of the acidic loop. Molecule C08743791 (4,6-dimethyl-N-[3-(5-phenyl-1H-pyrazol-3-yl)-1H-1,2,4-triazol-5-yl]pyrimidin-2-amine) is reported as an example and the hydrogen bonds with D108 and E109 of the acidic loop are highlighted.

Fig. 7

Interactions between the selected molecules and S139. (Left panel) Aromatic rings are highlighted as a recurrent feature of the high-ranking molecules. (Right panel) The aromatic ring of compound C29375629 (2-(2-bromo-4,6-difluoro-anilino)-2-oxo-ethyl]) is shown to interact with S139.

Fig. 8

Interactions between the selected molecules and valine residues in Cdc34 catalytic cleft. The aromatic ring of molecule C08743791 (4,6-dimethyl-N-[3-(5-phenyl-1H-pyrazol-3-yl)-1H-1,2,4-triazol-5-yl]pyrimidin-2-amine) is reported as an example of interaction with V143 and V147 located on helix α3 of Cdc34.

Fig. 9

Pharmacophore model for Cdc34 inhibitors. The three main areas required for the inhibition of the acidic loop of Cdc34 are highlighted.

Fig. 10

Binding free energy of the top four compounds of the post-clustering ranking. The first bar (light blue) refers to the total binding free energy (full list reported in Table 1), the second bar (light yellow) is the internal energy obtained as the sum of the H-bonds (and VdW contributions) (olive green) and the electrostatic component (gray). The last column (red) refers to the torsional energy. Binding free energy = internal energy + torsional energy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)