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.

中文翻译：

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一种推定的卷曲 7 靶向化合物充当萤火虫荧光素酶抑制剂


G 蛋白偶联受体的 Frizzled 家族 （FZD_1-10） 调节介导增殖输入的 WNT 信号传导。在肠癌中经常观察到 FZD₇ 失调和 WNT/β-catenin 信号传导过度。因此，开发靶向 FZD₇ 的癌症治疗药物具有吸引力。基于结构的虚拟筛选已经确定了化合物 28，它基于基于荧光素酶的报告基因 TOPFlash 测定抑制 WNT/β-catenin 信号传导。然而，经药理学验证，化合物 28 反而是一种有效的萤火虫荧光素酶 （Fluc） 抑制剂 （IC₅₀ = 30 nM），与 TOPFlash 测定中报道的化合物 28 介导的抑制的 IC₅₀ 相匹配。此外，我们采用了不依赖 FUC 的分析、FZD₇ 聚焦生物发光共振能量转移生物传感器和定量 PCR，以强调化合物 28 无法抑制 WNT-3A 诱导的 FZD₇ 构象动力学和 WNT 靶基因 Axin2 的转录。因此，我们强调了抗体筛选的重要性，以验证干扰用于化合物筛选的检测技术的化合物。