A Putative Frizzled 7-Targeting Compound Acts as a Firefly Luciferase Inhibitor

Abstract

The Frizzled family (FZD_1-10) of G protein-coupled receptors regulates WNT signaling mediating proliferative input. Dysregulation of FZD₇ and exaggerated WNT/β-catenin signaling is frequently observed in intestinal cancers. Therefore, it is attractive to develop therapeutics targeting FZD₇ for cancer treatment. Structure-based virtual screening has identified compound 28, which inhibited WNT/β-catenin signaling based on the luciferase-based reporter gene TOPFlash assay. However, upon pharmacological validation, compound 28 rather acts as a potent Firefly luciferase (Fluc) inhibitor (IC₅₀ = 30 nM), matching the reported IC₅₀ for compound 28-mediated inhibition in the TOPFlash assay. Moreover, we employed Fluc-independent assays, a FZD₇-focused bioluminescence resonance energy transfer biosensor and quantitative PCR, to emphasize the inability of compound 28 to inhibit the WNT-3A-induced conformational dynamics in FZD₇ and transcription of Axin2, a WNT target gene. Thus, we underline the importance of counter screens to validate compounds that interfere with the detection technology used for compound screening.

Figure 1

Compound 3 abrogates the basal and WNT-3A-induced TOPFlash signal. (A) Chemical structure of compound 3 (corresponding to compound 28). The 2-aminothiazole group is circled in orange. (B) Two plasmids, Renilla luciferase (Rluc) and M50 Super 8x TOPFlash (Fluc), are used in combination to transfect ΔFZD_1–10 HEK293T cells. (C) Schematic of the TOPFlash assay used to quantify WNT/β-catenin pathway activation induced by WNT-3A-induced activation of HiBiT-FZD₇. (D) Reported TOPFlash ratio response in ΔFZD_1–10 HEK293T cells transfected with HiBiT-FZD₇. 10 μM compound 3 was added and subsequently WNT-3A (300 ng/mL) or vehicle and incubated for 24 h. Data are presented as means ± SEM of three biological replicates (**p < 0.01, n.s. not significant) (one-way ANOVA with Dunnett’s multiple comparisons test). Parts of this figure were created with BioRender.com released under a Creative Commons Attribution-NonCommerical-NoDrivs 4.0 International license.

Figure 2

Compound 3 is a potent Firefly luciferase inhibitor. (A) Schematic of the counter assay performed in ΔFZD_1–10 HEK293T cells where Fluc was transiently transfected in the absence of WNTs or FZDs driven solely by a CMV promoter. (B) A concentration–response curve highlights the effect of increasing concentrations of compound 3 on luminescence emerging from soluble Fluc. Data are shown as raw luminescence counts of three biological replicates with means ± SEM. IC₅₀ = 25 nM (pIC₅₀ 7.625 ± 0.02) (C) Schematic of the counter assay performed in ΔFZD_1–10 HEK293T cells where Rluc was transiently transfected in the absence of WNTs or FZDs. (D) Concentration–response curve shows the lack of an effect of increasing concentrations of compound 3 on luminescence emerging from soluble Rluc. Data are are shown as raw luminescence counts of three biological replicates with means ± SEM from three biological replicates. (E) Schematic of the counter assay in ΔFZD_1–10 HEK293T cells where Fluc and Rluc were transfected in the presence of 10 μM CHIR99021. (F) Concentration–response curve demonstrating the inhibitory action of compound 3 on TOPFlash activity in the presence of 10 μM CHIR99021. Data are presented as TOPFlash ratios (Fluc/Rluc) with means ± SEM of three biological replicates. IC₅₀ is 33 nM (pIC₅₀ of 7.47 ± 0.08). All conditions contain 10 nM C59. Parts of this figure were created with BioRender.com released under a Creative Commons Attribution-NonCommerical-NoDrivs 4.0 International license.

Figure 3

Orthogonal assays depict the lack of effect of compound 3 on the WNT-induced conformational rearrangements or bona fide WNT target gene, Axin2. (A) The kinetic profile of the unimolecular FZD₇-DEP sensor upon WNT-3A stimulation and DMSO (purple) vs WNT-3A stimulation with compound 3 (green) are shown. Experiments were conducted in ΔFZD_1–10 HEK293T cells transiently transfected with FZD₇-DEP-Clamp. Data are presented as ΔBRET ratios from three independent experiments with means ± SEM. (B) A concentration response curve depicts the effect of increasing concentrations of compound 3 on cytosolic Nluc (TK-Nluc). Data are presented as normalized Nluc counts to conditions in the absence of compound 3 (=1) with means ± SEM of three biological replicates. (C) Mouse embryonic fibroblasts (MEFs) were kept unstimulated or stimulated with either 200 ng/mL WNT-3A, or 10 μM compound 3 in the presence of 10 nM C59 for 24 h. Afterward, cells were analyzed for expression Axin2 mRNA by quantitative PCR (qPCR). Data is reported as fold-increase relative to baseline conditions (serum-free medium with C59) and log2 transformed. Error bars indicate mean ± SEM of six biological replicates (*p < 0.05, n.s. not statistically significant; one-way ANOVA with Dunnett’s multiple comparisons test).