Small Molecule Inhibitors

In the nucleus, the formation of the Tcf-P-catenin complex is a prerequisite for target gene activation. Targeted interference with this complex formation in colon cancer cells has been shown to effectively abrogate activation of target genes and to inhibit their growth in vitro (Tetsu and McCormick 1999; van de Wetering et al. 2002).

Accordingly, small compounds that can specifically disrupt the Tcf-P-catenin complex in vivo hold immense potential for the treatment of Wnt-driven tumors.

Earlier crystallographic studies of Tcf-P-catenin complexes demonstrate that the N-terminal acidic domain of Tcf proteins binds, with a high affinity (Kd = ~10 nM), to an extended region encompassing R3-10 of P-catenin (Graham et al. 2000, 2001). However, this basic groove of the P-catenin Arm repeat domain also interacts with other molecules. For instance, comparison of crystal structures of P-catenin bound to E-cadherin, APC or Axin reveals that these ligands bind along the basic groove of P-catenin in a very similar manner to Tcf proteins (Ha et al. 2004; Huber and Weis 2001; Xing et al. 2003, 2004). These observations raise concern that any small molecule capable of disrupting the Tcf-P-catenin complex would potentially interfere with P-catenin-cadherins, P-catenin-APC and/or P-catenin-Axin interactions. Interaction with E-cadherin is essential for cell-cell adhesion, while complex formation with APC or Axin controls P-catenin levels in normal tissues. Disruption of such interactions is likely to have undesirable consequences. Hence, small-molecule inhibitors must selectively interfere with the Tcf-P-catenin complex without disrupting other essential interactions of P-catenin.

The first small-molecule disruptors of the Tcf-P-catenin complex were successfully identified using a high-throughput immunoenzymatic detection of the proteinprotein interaction (Lepourcelet et al. 2004). Among 7,000 purified natural compounds, eight displayed reproducible, dose-dependent inhibition of the Tcf-P-catenin interaction with IC50 < 10 |M. Two most potent compounds (PKF115-584 and CGP049090; Fig. 3) consistently scored as inhibitors of the Tcf-P-catenin interaction in nearly every secondary assay, including disruption of Tcf-P-catenin complexes in vitro, and inhibition of colon cancer cell proliferation, P-catenin-dependent reporter activity and P-catenin-mediated axis duplication in Xenopus embryos. Significantly, these compounds share a common core chemical structure, implying that they exert their effects in a similar mechanism. However, the precise molecular mechanisms by which these active compounds block the interaction between Tcf and P-catenin are unclear. Unfortunately, these small molecules also interfere with APC-P-catenin interactions, limiting their therapeutic potential as anticancer drugs (Lepourcelet et al. 2004). Nevertheless, this pioneer work underscores the

Fig. 3 Chemical structures of small-molecule inhibitors of P-catenin signaling. RKF115-584 and CGP049090 abrogate the Tcf/p-catenin interaction, while ICG-001 interferes with P-catenin binding to CBP, leading to inhibition of P-catenin signaling

Fig. 3 Chemical structures of small-molecule inhibitors of P-catenin signaling. RKF115-584 and CGP049090 abrogate the Tcf/p-catenin interaction, while ICG-001 interferes with P-catenin binding to CBP, leading to inhibition of P-catenin signaling

PKF 115-584

CGP049090

ICG-001

PKF 115-584

CGP049090

ICG-001

feasibility of targeting protein-protein interactions among the essential components of oncogenic Wnt/P-catenin signaling in cancer drug discovery.

Given the high-affinity and dynamic binding of Tcf proteins to P-catenin, and the fact that substantially overlapping regions in the basic groove of P-catenin interact with multiple molecules, screening or designing inhibitors that are highly specific for the Tcf-P-catenin interaction may face a major challenge. Additionally, one might envision that disrupting binding of P-catenin to essential transcriptional co-activators represents potential opportunities for small-molecule-mediated intervention of P-catenin activity in tumor cells.

Another class of drugs with inhibitory effects on P-catenin activity has been isolated by a high-throughput cell-based assay and found to disrupt the interaction of P-catenin with CBP (Eguchi et al. 2005; Emami et al. 2004). SW480 colon cancer cells express truncated APC and hence show elevated basal activities of the P-catenin reporter TOPFLASH, which contains three optimal Tcf/Lef-binding sites driving luciferase expression (Korinek et al. 1997). Among 5,000 structurally divergent synthetic compounds, three closely related small molecules were found to display a dose-dependent inhibition of TOPFLASH activity in SW480 cells, with an IC50 of 3 |M, without significantly affecting the mutant reporter FOPFLASH. The lead compound ICG-001 (Fig. 3), with a molecular weight of 548, selectively binds to the N-terminal portion of CBP and interferes with its interaction with P-catenin. Curiously, this compound also binds to the corresponding region of the CBP homolog p300, but does not seem to disrupt its association with P-catenin. Through a microarray analysis, ICG-001 treatment of SW480 cells was found to inhibit expression of a subset of P-catenin target genes including cyclin D1 and the anti-apoptotic gene survivin (Ma et al. 2005). ICG-001 also exhibited selective growth inhibitory effects and cytotoxicity in colon carcinoma cells with activated P-catenin, but not in normal colonic epithelial cells in vitro. More importantly, treatment with this compound dramatically decreases tumor growth in vivo in both APCMin and SW620 xenograft mouse models of cancer (Emami et al. 2004).

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