Identification of a potent antagonist of smoothened in hedgehog signaling

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. 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.

We 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.

Our 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.