The
smoothened receptor (Smo) plays a key role in Hedgehog (Hh) signaling pathway and it has been regarded as an efficacious therapeutic target for
basal cell carcinoma (BCC) and
medulloblastoma (MB). Nevertheless, the resistance mutation and active mutants of Smo have put forward the requirement of finding more effective inhibitors. Herein, we performed metadynamics simulations on Smo bound with
vismodegib (Smo-Vismod) and with
cholesterol (Smo-CLR), respectively, to explore the inhibition mechanism of
vismodegib. The simulation results indicated that
vismodegib-induced shifts of TM5, TM6, and TM7, which permitted the extracellular extension of TM6 and extracellular loop3 (ECL3) to enter the extracellular
cysteine-rich domain (CRD) groove. Therefore, an open CRD groove that has not been noticed previously was observed in Smo-Vismod complex. As a consequence, the occupied CRD groove prevents the binding of
cholesterol. In addition, the HD and ECLs play crucial roles in the interaction of CRD and TMD. These results reveal that TM5, TM6, and TM7 play important roles in allosteric inhibition the activation of Smo and disrupting
cholesterol binding by
vismodegib binding. Our results are expected to contribute to understanding the allosteric inhibition mechanism of Smo by
vismodegib. Moreover, the detailed conformational changes contribute to the development of novel Smo inhibitors against resistance mutation and active mutants of Smo.