Cyclopamine, a steroidal alkaloid, inhibits Hedgehog (Hh) signaling by directly binding to Smoothened (Smo), a key receptor in the Hh pathway. This study shows that cyclopamine specifically blocks Hh signaling by binding to the heptahelical bundle of Smo, which is crucial for its function. The binding of cyclopamine also helps reverse the retention of partially misfolded Smo in the endoplasmic reticulum (ER), likely through effects on protein conformation. These findings reveal the mechanism of cyclopamine's teratogenic and antitumor activities and suggest that small molecules can regulate Smo function. Cyclopamine is derived from plants in the genus Veratrum and has been linked to developmental malformations in sheep. It is known to inhibit Hh signaling by interfering with the initial events of Hh signal reception, which involves the transmembrane proteins Patched (Ptch) and Smo. Ptch inhibits Smo function, and cyclopamine appears to act in a Ptch-independent manner. The study demonstrates that cyclopamine binds directly to Smo, affecting its conformation and function. This binding is sensitive to Ptch function, providing biochemical evidence for Ptch's effect on Smo structure. The study used photoaffinity and fluorescent derivatives of cyclopamine to show that it specifically cross-links Smo. These results indicate that cyclopamine interacts with Smo in a manner that is structurally and functionally relevant to its inhibitory effects. Additionally, cyclopamine binding to Smo is localized to the heptahelical bundle, and the binding is sensitive to Ptch function. These findings suggest that the regulation of Smo activity by Ptch may involve endogenous small molecules. The study also shows that cyclopamine can alter the conformation of SmoA1, a constitutively active form of Smo, by promoting its exit from the ER. This suggests that cyclopamine may modulate Smo activity by altering its structure and localization. The results indicate that cyclopamine's mechanism of action involves direct binding to Smo and that this binding is influenced by Ptch activity. The study provides evidence that cyclopamine binds to Smo and that this binding is essential for its inhibitory effects on Hh signaling. These findings have implications for the treatment of Hh-related diseases, including cancers such as medulloblastoma and basal cell carcinoma. Cyclopamine's ability to specifically inhibit Hh signaling makes it a potential therapeutic agent for these conditions. The study also highlights the importance of understanding the molecular mechanisms underlying Smo regulation and the potential of small molecules in modulating these processes.Cyclopamine, a steroidal alkaloid, inhibits Hedgehog (Hh) signaling by directly binding to Smoothened (Smo), a key receptor in the Hh pathway. This study shows that cyclopamine specifically blocks Hh signaling by binding to the heptahelical bundle of Smo, which is crucial for its function. The binding of cyclopamine also helps reverse the retention of partially misfolded Smo in the endoplasmic reticulum (ER), likely through effects on protein conformation. These findings reveal the mechanism of cyclopamine's teratogenic and antitumor activities and suggest that small molecules can regulate Smo function. Cyclopamine is derived from plants in the genus Veratrum and has been linked to developmental malformations in sheep. It is known to inhibit Hh signaling by interfering with the initial events of Hh signal reception, which involves the transmembrane proteins Patched (Ptch) and Smo. Ptch inhibits Smo function, and cyclopamine appears to act in a Ptch-independent manner. The study demonstrates that cyclopamine binds directly to Smo, affecting its conformation and function. This binding is sensitive to Ptch function, providing biochemical evidence for Ptch's effect on Smo structure. The study used photoaffinity and fluorescent derivatives of cyclopamine to show that it specifically cross-links Smo. These results indicate that cyclopamine interacts with Smo in a manner that is structurally and functionally relevant to its inhibitory effects. Additionally, cyclopamine binding to Smo is localized to the heptahelical bundle, and the binding is sensitive to Ptch function. These findings suggest that the regulation of Smo activity by Ptch may involve endogenous small molecules. The study also shows that cyclopamine can alter the conformation of SmoA1, a constitutively active form of Smo, by promoting its exit from the ER. This suggests that cyclopamine may modulate Smo activity by altering its structure and localization. The results indicate that cyclopamine's mechanism of action involves direct binding to Smo and that this binding is influenced by Ptch activity. The study provides evidence that cyclopamine binds to Smo and that this binding is essential for its inhibitory effects on Hh signaling. These findings have implications for the treatment of Hh-related diseases, including cancers such as medulloblastoma and basal cell carcinoma. Cyclopamine's ability to specifically inhibit Hh signaling makes it a potential therapeutic agent for these conditions. The study also highlights the importance of understanding the molecular mechanisms underlying Smo regulation and the potential of small molecules in modulating these processes.