Results and Discussion Identification of WIKI as a Small Molecule Inhibitor of Wnt/?catenin Signaling
To make an assay for Wnt/?catenin signaling suitable for high throughput screening, we generated A375 melanoma cells stably infected with a ?catenin-activated luciferase reporter (BAR) [23,36] and selected populations in which luciferase activity is increased at least 4,000-fold by WNT3A. We tested the robustness of our assay by calculating the Z-factor (Z9) values [40] using probes that are known to enhance (U0126 [41], Riluzole [42], and GSK3B inhibitor IX [43]) or inhibit (XAV-939 [33]) Wnt/�catenin signaling (Figure S1A). For all control probes, we found the Z9 values to be greater than .45 (Figure S1A), a value considered robust in high throughput screening assays [40]. Following validation of our assay, we then screened A375 melanoma cells at two concentrations of a small molecule library in the presence of a twenty percent effective concentration (EC20) dose of WNT3A. We focused on small molecules that reduced expression of the luciferase reporter at a low dose (330 nM) and that did not kill cells at a high dose (10 mM) relative to controls treated with dimethyl sulfoxide (DMSO), with the expectation that these criteria would filter out compounds that inhibited BAR due to cellular toxicity. Five compounds met our criteria for further study by significantly decreasing Wnt/?catenin signaling without causing toxicity at either dose (Fig. 1A).

We next asked whether any of the five compounds preferentially modulated Wnt/?catenin signaling by comparing the repression of BAR in A375 cells relative to luciferase reporters for the Nuclear Factor Kappa B (NF-kB), Transforming Growth Factor Beta (TGF?, and Retinoic Acid (RA) signaling pathways (Fig. 1B). Of the five candidate Wnt/?catenin inhibitors that we tested, WIKI4 (left panel, Fig. 1C) was the only inhibitor of BAR that did not also inhibit the reporters for NF-kB, TGF? and RA (Fig. 1B). Furthermore, WIKI4 has demonstrated activity in one of nine published assays ( summary.cgi?cid = 2984337), supporting our contention that WIKI4 is not a general inhibitor of activity in high throughput screening assays. We then demonstrated that WIKI4 inhibits Wnt/?catenin signaling in several other cell lines, including DLD1 colorectal cancer cells (Fig. 1D), NALM6 B cells (Figure S1B), U2OS osteosarcoma cells (Figure S1B) and hESCs (Figure S1C). In all cell types tested, we observed that WIKI4 potently inhibited Wnt/?catenin signaling and that its half-maximal response dose was ,75 nM. We next investigated whether WIKI4 is sufficient to inhibit expression of Wnt/?catenin target genes in DLD1 colorectal carcinoma cells, which express a truncated form of the Wnt/atenin inhibitor APC [44]. We found that incubation of DLD1 cells overnight with either WIKI4 or the structurally distinct TNKS inhibitor, XAV-939 (right panel, Fig. 1C) [33], resulted in decreased steady-state abundance of AXIN2, and TNFRSF19 (Fig. 1E), which is consistent with WIKI4 acting as an inhibitor of Wnt/?catenin signaling. Furthermore, we observed that WIKI4 is sufficient to inhibit WNT3A-dependent increases in the expression of AXIN2 and TNFRSF19 in hESCs (Figure S1D, S1E). Thus we have identified WIKI4 as a new inhibitor of Wnt/ ?catenin signaling that regulates the pathway in several cell types. To determine which chemical groups in WIKI4 are required for its ability to inhibit Wnt/?catenin signaling, we next performed a structure activity relationship analysis (Figure S2). WIKI4 has a molecular weight of 522 and a calculated partition coefficient of 4.8, putting it near the limits of “druglikeness” by Lipinski’s Rule of Five [45]. WIKI4’s mass and complexity is greater than XAV939 (Fig. 1C), and identification of small active WIKI4 analogs could provide more opportunities for modification while maintaining its druglike properties. To identify less complex WIKI4 analogs and to determine which portions of WIKI4 are required for activity, we searched for commercially available analogs. We queried the ZINC [46] and eMolecule ( databases and identified 62 WIKI4 analogs for further testing (Table S3). We assayed the Wnt/?catenin inhibitory activity of a subset of these compounds (Figure S2). Our results indicate that the traizole’s 4-pyridyl and 4-methoxyphenyl groups tolerate some modification, but the latter group could not be removed (Figure S2A). Additionally, substitution of the 1,8-naphthalimide group with a phthalimade group eliminated activity as did replacement of the 1,8-naphthalimide group with a methyl or phenyl group (Figure S2B).WIKI4 Inhibits the Cellular Responses to Wnt/?catenin Signaling
We next asked whether cells treated with an effective dose of WIKI4 would show a reduction in Wnt/?catenin-mediated responses at the cellular level. As DLD1 colorectal cancer cells require ?catenin signaling for growth in limiting culture experiments [47], these cells provide an excellent functional model of the pathway in which to test small molecules. We found that WIKI4 inhibits growth of DLD1 cells relative to DMSO controls in media containing low serum (Fig. 2A).Figure 2. WIKI4 inhibits the functional outcomes of Wnt/?catenin signaling. (A) WIKI4 inhibits colony formation of DLD1 colorectal cancer cells. DLD1 cells were plated individually in 0.5% serum containing medium, and treated with the indicated concentrations of WIKI4 and XAV-939. This experiment is representative of three independent experiments and the error bars represent standard deviation of three technical replicates. (BF) WIKI4 prevents Wnt3A-dependent differentiation of H1 human embryonic stem cells (hESCs). (B) Culturing hESCs for six days with Wnt3A causes marked morphological changes that are rescued by treatment with WIKI4. Scalebar = 500 mm. expression of markers of undifferentiated hESCs following Wnt3A stimulus. hESCs were stimulated with the indicated treatments and expression of GCTM2 and CD9 was assessed by flow cytometry following six days of treatment. (D-F) The effect of WIKI4 treatment on the expression of genes that are altered during Wnt3A-dependent differentiation of hESCs was assessed by qPCR. hESCs were treated for the indicated conditions for six days, and then analyzed by qPCR for markers of undifferentiated stem cells (NANOG, POU5F1) (D), endoderm (SOX17, GATA6) (E), and mesoderm (T, KDR) (F). The data was normalized to 100,000 copies of GAPDH and plotted as a ratio to the untreated hESCs (cultured in KSR media). The data in the experiments presented in B-F are representative of three independent experiments and the error represents standard deviation of technical replicates. In B-F, LCM = control L cell CM, WNT3A = Wnt3a CM; both 50% (vol/vol) in KSR medium. that WIKI4 inhibits a known cellular response to Wnt/?catenin signaling. Given that cellular responses to Wnt/?catenin signaling are diverse and context-dependent, we next examined the effects of WIKI4 on hESCs. Activation of Wnt/?catenin signaling in hESCs alters their cell fate and causes them to differentiate into early mesoderm and endoderm lineage cells [16,17].

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