Anticancer Activity of (S)-5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)- N-(1-oxo-1-((pyridin-4-ylmethyl)amino)propan-2-yl)-1 H-indole-2-carboxamide (RS4690), a New Dishevelled 1 Inhibitor

Abstract

Wingless/integrase-11 (WNT)/β-catenin pathway is a crucial upstream regulator of a huge array of cellular functions. Its dysregulation is correlated to neoplastic cellular transition and cancer proliferation. Members of the Dishevelled (DVL) family of proteins play an important role in the transduction of WNT signaling by contacting its cognate receptor, Frizzled, via a shared PDZ domain. Thus, negative modulators of DVL1 are able to impair the binding to Frizzled receptors, turning off the aberrant activation of the WNT pathway and leading to anti-cancer activity. Through structure-based virtual screening studies, we identified racemic compound RS4690 (1), which showed a promising selective DVL1 binding inhibition with an EC₅₀ of 0.74 ± 0.08 μM. Molecular dynamic simulations suggested a different binding mode for the enantiomers. In the in vitro assays, enantiomer (S)-1 showed better inhibition of DVL1 with an EC₅₀ of 0.49 ± 0.11 μM compared to the (R)-enantiomer. Compound (S)-1 inhibited the growth of HCT116 cells expressing wild-type APC with an EC₅₀ of 7.1 ± 0.6 μM and caused a high level of ROS production. These results highlight (S)-1 as a lead compound for the development of new therapeutic agents against WNT-dependent colon cancer.

Keywords: DVL1; PDZ domain; WNT pathway; cancer; virtual screening.

Figure 1

Compound RS4690 (1) achieved from virtual screening studies.

Figure 2

In silico docking results of (S)-1 (cyan) and (R)-1 (orange) in complex with the DVL1 PDZ binding site. Residues involved in interactions are reported as white sticks. The PDZ is depicted as light blue cartoon. H-bonds are reported as yellow dotted lines.

Figure 3

Equilibrium binding experiment between PDZ domain of DVL1 and the C-terminal portion of TMEM88 in the absence and presence of (S)-1 or (R)-1 at 1 µM (right panel) and 5 µM (left panel). Lines are the best fit for a hyperbolic function.

Figure 4

The dose response curves are representative for DVL recruitment inhibition experiment (left panel) and WNT pathway activity measurement (right panel).

Figure 5

Confocal immunofluorescence showing the recruitment of DVL1-GFP (green) by HA-FZD4 (red) at the PM of HEK293 cells in the presence of 7 μM of compound 1 (racemic mixture), (S)-1, (R)-1 or the corresponding volume of vehicle (DMSO). (Magnification bar = to 14 μm).

Figure 6

ROS production in HCT116 cells upon treatment with different concentrations of (R)-2 or (S)-2 for 48 h. The asterisks show the differences between the effects induced by the two drugs.

Figure 7

Key contacts of compound (S)-2 in the DVL1 PDZ binding pocket. H-bonds are not shown for the sake of clarity.