Expanding the inhibitor space of the WWP1 and WWP2 HECT E3 ligases

Abstract

The HECT E3 ubiquitin ligases 1 (WWP1) and 2 (WWP2) are responsible for the ubiquitin-mediated degradation of key tumour suppressor proteins and are dysregulated in various cancers and diseases. Here we expand their limited inhibitor space by identification of NSC-217913 displaying a WWP1 IC₅₀ of 158.3 µM (95% CI = 128.7, 195.1 µM). A structure-activity relationship by synthesis approach aided by molecular docking led to compound 11 which displayed increased potency with an IC₅₀ of 32.7 µM (95% CI = 24.6, 44.3 µM) for WWP1 and 269.2 µM (95% CI = 209.4, 347.9 µM) for WWP2. Molecular docking yielded active site-bound poses suggesting that the heterocyclic imidazo[4,5-b]pyrazine scaffold undertakes a π-stacking interaction with the phenolic group of tyrosine, and the ethyl ester enables strong ion-dipole interactions. Given the therapeutic potential of WWP1 and WWP2, we propose that compound 11 may provide a basis for future lead compound development.

Keywords: SAR; WWP1; WWP2; drug discovery; ubiquitin ligase inhibitors.

Graphical abstract

Figure 1.

High throughput DSF screen of the NCI Diversity Set VI compound library against WWP1 and WWP2. Assays were carried out using 10 µM compound, containing 0.1% DMSO and normalised by ΔT_m calculated from controls. Positive hits (green) displayed had an average ΔT_m and associated errors above a threshold of three times the standard deviation of their respective controls (A) Single-shot DSF screen of WWP2-LH (top) and WWP1-L34H (bottom) against NCI Diversity Set VI compounds. (B) Triplicate DSF screen of 24 confirmed hit compounds for WWP1-L34H and WWP2-LH.

Figure 2.

ELISA autoubiquitination assay screen of PAINS-filtered DSF hit compounds against WWP1 (A) and WWP2 (B). Enzyme inhibition was measured at a compound concentration of 500 µM (1% DMSO), with a hit threshold (hits shown in green) of less than 50% relative activity, normalised to their respective 0% and 100% WWP1-L34H and WWP2-FL controls.

Figure 3.

Synthesised NSC-217913 against WWP1 and WWP2. (A) Chemical structure of NSC-217913 and associated WWP1 IC₅₀ (B) Dose-dependent ELISA autoubiquitination assay of synthesised NSC-217913. Compound inhibition was measured on a log scale from 1000 – 10 nM (1% DMSO), normalised to 0% and 100% WWP1-L34H (blue triangle) and WWP2-FL (red square) controls. IC₅₀ values were calculated from non-linear regression curves fitted in GraphPad.

Scheme 1.

Synthetic route to access 6, 7, 9, 11, 13 and 15. i: Ethylene glycol (2.04 M), 175 °C, 18 h, 45%. ii: PCl₅ (7.0 eq), POCl₃ (0.44 M), 90 °C – 200 °C, 18 h, 32%. iii: NH₄OH (25% aq., 0.27 M), 120 °C, 2 d., 31%. iv: Thiocarbonyldiimidazole (2.0 eq), dioxane (0.2 M), 120 °C, 36 h, 34%. v: Br-R (1.05 eq), NaOH (1.5 eq), EtOH (0.067 M), RT, 18 h, Ar or N₂, 24 – 76%. vi: NaOH (0.15 M, methanol), CH₂Cl₂: MeOH (14:11 v/v, 0.063 M), RT, 18 h, 69%.

Figure 4.

2D and 3D ligand poses of compound 11 docked to (A) WWP1-2L34H and (B) WWP2-LH. Key interacting residues are represented as sticks and coloured associated with their hydrophilic (blue), hydrophobic (green) or positively (purple) and negative (orange) charged characteristics, with the binding pocket surface (grey) also shown. Interactions including π-π stacking (green), hydrogen bonding (pink), halogen bonding (orange) and electrostatic (red) are given in angstroms (Å). 2D and 3D images were created using the Maestro Schrodinger Suite and PYMOL, respectively.