Identification of a potent antagonist of smoothened in hedgehog signaling

Junwan Fan,Chen Liu,Yongjun Wang,Steven Y Cheng,Haowen Li,Zichen Zhao,Wenyan He,Lun Kuang,Wei Chen

doi:10.1186/s13578-021-00558-9

Junwan Fan, Chen Liu + Show 7 more

Open Access

https://doi.org/10.1186/s13578-021-00558-9

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Abstract

BackgroundHedgehog signaling is essential to the regulation of embryonic development, tissue homeostasis, and stem cell self-renewal, making it a prime target for developing cancer therapeutics. Given the close link between aberrant Hedgehog signaling and cancers, many small molecular compounds have been developed to inhibit Smoothened, a key signal transducer of this pathway, for treating cancer and several such compounds have been approved by the United States Food and Drug Administration (GDC-0449 and LDE-225). However, acquired drug resistance has emerged as an important obstacle to the effective use of these first generation Hedgehog pathway blockers. Thus, new Smoothened inhibitors that can overcome such resistance is an urgent need going forward.ResultsWe established the Smoothened/βarrestin2-GFP high-throughput screening platform based on the mechanistic discovery of Hedgehog signaling pathway, and discovered several active small molecules targeting Smoothened including 0025A. Here we show that 0025A can block the translocation of βarrestin2-GFP to Smoothened, displace Bodipy-cyclopamine binding to wild-type Smoothened or mutant Smoothened-D473H, reduce the accumulation of Smo on primary cilia and the expression of Gli upon Hedgehog stimulation. In addition, we show that 0025A can effectively suppress hair follicle morphogenesis and hair growth in mice.ConclusionsOur results demonstrate that 0025A is a potent antagonist targeting Smoothened wild-type and mutant receptors in the Hedgehog signaling pathway and may provide a new therapy for refractory cancers.

Highlights

Hedgehog signaling is essential to the regulation of embryonic development, tissue homeostasis, and stem cell self-renewal, making it a prime target for developing cancer therapeutics
We show that several active hits including 0025A are discovered. It can block the translocation of βarr2-GFP to Smo, displace Bodipy-cyclopamine binding to wild-type Smo or mutant Smo-due to Asp473 to His mutation (D473H), effectively reduce the accumulation of Smo on primary cilia and the expression of Gli upon Hh stimulation as well as suppress hair follicle morphogenesis and hair growth in mice
Our results demonstrate that 0025A is a potent antagonist targeting Smo receptors in the Hh signaling pathway and may provide a therapy for refractory cancers

Summary

Results

Identification of compound 0025A As we showed previously, βarr2-GFP was distributed evenly in the cytoplasm when it was expressed alone in U2OS cells, which are commonly used for high-throughput cancer drug screens [31] (Fig. 1a). To examine the inhibitory role of 0025A, we induced the serum-starved NIH3T3 cells by 20% Shh-CM with vehicle, 0025A (0.01, 0.1, 1, 10 μM) or GDC-0449 (1 μM) for 24 h, and observed that 0025A dose-dependently reduced the mRNA and protein level of Gli. We detect the expression of Ptch, which is another target gene of Hh signaling pathway [39], and found that 0025A could dose-dependently inhibit the mRNA level of Ptch induced by 20% Shh-CM (Additional file 1: Fig. S1). 5 μm of Gli in hair follicles in the dorsal skin sections by the immunostaining of Gli (Additional file 2: Fig. S2) These results demonstrated that 0025A could suppress Shh signaling mediated hair growth in vivo

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Materials and methods