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A Putative Frizzled 7-Targeting Compound Acts as a Firefly Luciferase Inhibitor
Julia Kinsolving1, Lukas Grätz1, Jan Hendrik Voss1
1Section of Receptor Biology & Signaling, Dept. Physiology & Pharmacology, Karolinska Institutet, Stockholm S-171 77, Sweden.
Abstract:
The Frizzled family (FZD1-10) 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.
Insights
Compound 28, a potential cancer therapeutic targeting Frizzled 7 (FZD7) and WNT signaling, was found to be a luciferase inhibitor, not a WNT pathway inhibitor. This highlights the necessity of counter screens in drug discovery.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Frizzled family (FZD1-10) receptors regulate WNT signaling, crucial for cell proliferation.
- Aberrant FZD7 and WNT/β-catenin signaling are implicated in intestinal cancers, making FZD7 a therapeutic target.
- Targeting FZD7 offers a promising strategy for developing novel cancer treatments.
Purpose of the Study:
- To evaluate compound 28, identified via virtual screening, as a potential FZD7 inhibitor for cancer therapy.
- To validate the inhibitory effects of compound 28 on WNT/β-catenin signaling.
- To investigate the mechanism of action of compound 28 and its specificity.
Main Methods:
- Structure-based virtual screening to identify potential FZD7 inhibitors.
- TOPFlash reporter gene assay to assess WNT/β-catenin signaling inhibition.
- Pharmacological validation using Firefly luciferase (Fluc) inhibition assays.
- Fluc-independent assays including bioluminescence resonance energy transfer (BRET) biosensors and quantitative PCR (qPCR).
Main Results:
- Compound 28 was identified as a potent inhibitor of Firefly luciferase (Fluc) with an IC50 of 30 nM.
- Compound 28 did not inhibit WNT/β-catenin signaling as measured by Fluc-independent assays.
- Fluc-independent assays demonstrated that compound 28 failed to inhibit WNT-3A-induced FZD7 conformational changes and Axin2 gene transcription.
Conclusions:
- Compound 28 acts as a luciferase inhibitor, not a specific FZD7 or WNT pathway inhibitor.
- The study underscores the critical importance of implementing counter screens to validate findings from initial compound screening.
- False positives due to assay interference necessitate rigorous validation to ensure therapeutic candidate efficacy and specificity.
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