PF-5274857

2 years -80 in solvent

Oral administration

Gli-Luc/MEF cells

ca.30 mg/kg

5.8 nM, 5.8 nM, 5.8 nM, 5.8 nM, 5.8 nM, 5.8 nM

PF-5274857 shows significant dose-dependent tumor growth inhibition (TGI) and induces tumor regression at high doses(>10 mg/kg)., PF-5274857 downregulates Gli1, Gli2, Ptch1, and Ptch2 gene expression levels to various degrees with maximal effects being achieved between 6 and 12 hours post-dose (Gli1 is the most sensitive gene), 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 also able to cross the blood–brain barrier in rats with 4 hours post-dose. [1]

Biochemical assays, HEK293 cells overexpressing human Smo (amino acids 181-787) are grown in Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% FBS, Pen–Strep, and 0.1 mg/mL hygromycin to 90% confluence. After washing with cold Dulbecco's PBS, the cell pellet is resuspended in membrane preparation buffer (50 mM Tris-HCl, pH 7.5, 250 mM sucrose with Roche complete protease cocktail) and homogenized. The homogenate is centrifuged and the cell pellet is resuspended in assay buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 25 mM MgCl2, 1 mM EDTA, and 0.1% protease-free bovine serum albumin) and homogenized in a glass tissue grinder. Total protein in the membrane preparation containing Smo is determined using the Pierce BCA protein assay. For the competitive binding assay, 100 uL of assay buffer is added to a 96-well GF/B filter plate for 10 minutes to pre-wet the filter and then removed. The following reagents are then added: 20 uL of assay buffer, 10 uL serial dilutions of compound, 20 uL of 3H-Smo aagonist (3 nM final concentration), and 50 uL of membrane preparation (40 ug total protein). The plates are incubated at room temperature for 2 hours, then washed and vacuum dried. The plates are then dried for 1 hour in a 60C oven before the addition of 45 uL of Microscint 20 and incubated at room temperature for 30 minutes to 1 hour, then counted in a TopCount scintillation counter. The data are analyzed using GraphPad Prism software.

PF-5274857 is a potent and selective Smoothened (Smo) antagonist, inhibits Hedgehog (Hh) signaling with IC50 and Ki of 5.8 nM and 4.6 nM, respectively, and can penetrate the blood–brain barrier.