PF-5274857 is a Smo antagonist with an IC50 of 2.7 nM. The Ki of PF-5274857 is 4.6 nM. PF-5274857 completely inhibits Shh-induced Hh pathway activity with an IC50 of 2.7 nM measured by the transcriptional activity of Smo downstream gene Gli1 in MEF cells. PF-5274857 is shown to bind to Smo with an IC50 of 5.8 nM. The μ-opioid receptor is weakly inhibited by PF-5274857 with a dissociation constant of 36 μM subsequently determined in a functional assay. In tumor tissue, PF-5274857 downregulates Gli1, Gli2, Ptch1, and Ptch2 gene expression levels to various degrees with maximal effects being achieved between 6 and 12 hours postdose, whereas PF-5274857 has little effect on Smo levels. In skin tissue, downregulation of Gli1 and Gli2 is also observed with a similar time course by PF-5274857. The model-derived drug concentration for half maximal inhibition of the tumor Gli1 mRNA production rate (IC50) by PF-5274857 is determined to be 8.9 nM in the Ptch+/−p53+/− medulloblastoma allograft mice, which mathematically corresponds to tumor regression of 119% TGI after 6 days of plasma exposure at this concentration. In the Ptch+/−p53−/− medulloblastoma allograft mice, the IC50 value is estimated to be 3.5 nM, consistent with the Ptch+/−p53+/− results. PF-5274857 is a potentially attractive clinical candidate for the treatment of tumor types including brain tumors and brain metastasis driven by an activated Hh pathway. [1]