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    PF-5274857
    PF-5274857

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    This product is for research use only, not for human use. We do not sell to patients.
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    InvivoChem Cat #: V1336
    CAS #: 1373615-35-0Purity ≥98%

    Description: PF-5274857 (PF5274857; PF 5274857) is a novel, potent, orally bioavailable and selective Smoothened (Smo) antagonist with potential antineoplastic activity. It inhibits Hedgehog (Hh) signaling pathway with IC50s and Ki of 5.8 nM and 4.6 nM, respectively. 

    References: Mol Cancer Ther. 2012 Jan;11(1):57-65.

    Related CAS#613439-62-5 (HCl)1373615-35-0 (free base)

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    Molecular Weight (MW)436.96
    FormulaC20H25ClN4O3S 
    CAS No.1373615-35-0 (free base);
    Storage-20℃ for 3 years in powder form
    -80℃ for 2 years in solvent
    Solubility (In vitro)DMSO: 93 mg/mL (212.8 mM)
    Water:<1 mg/mL
    Ethanol: 93 mg/mL (212.8 mM)
    Solubility (In vivo)Saline: 30 mg/mL
    SynonymsPF-5274857; PF 5274857; PF5274857


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    In Vitro

    In vitro activity: PF-5274857 completely inhibits Shh-induced Hh pathway activity with IC50 of 2.7 nM measured by the transcriptional activity of Smo downstream gene Gli1 in MEF cells. The μ-opioid receptor is weakly inhibited by PF-5274857 with a dissociation constant of 36 μM subsequently determined in a functional assay.


    Kinase Assay: 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 μL 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 μL of assay buffer, 10 μL serial dilutions of compound, 20 μL of 3H-Smo antagonist (3 nM final concentration), and 50 μL of membrane preparation (40 μg 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 60°C oven before the addition of 45 μL 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.  


    Cell Assay: Gli-Luc/MEF cells are grown in the knockout DMEM supplemented with 10% heat-inactive FBS, 2 mM l-glutamine, and 0.55 mM β-mercaptoethanol until 90% confluence. On day 1, cells are trypsinized and seeded into white 384-well plates in 20 μL per well of OptiMEM media that is supplemented with 1% heat-inactive FBS and 1 mM sodium pyruvate at a concentration of 7,500 cells per well. Plates are incubated at 37 °C and 5% CO2 overnight. On day 2, PF-5274857 is added to the cells at a final concentration ranging from 3 μM to 50 pM at a 3-fold serial dilution followed by addition of recombinant mouse Sonic Hedgehog to a final concentration of 2 μg/mL. The cells are incubated with PF-5274857 and Shh for 48 hours at 37 °C and 5% CO2. Luciferase assays are conducted on day 4 using the Bright-Glo Luciferase Assay System. Briefly, Bright-Glo luciferase reagent (25 μL) is added to each well of the 384-well plate containing media. Plates are kept at room temperature for 5 minutes and then read on a Luminescence plate reader. The IC50 value of PF-5274857 is calculated.

    In VivoPF-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 within 4 hours post-dose.
    Animal modelSCID-beige mice bearing primary Ptch+/−p53+/− or Ptch+/−p53−/− medulloblastoma tumor
    Formulation & DosageDissolved in 0.5% methylcellulose; 30 mg/kg; Oral administration
    References

    Mol Cancer Ther. 2012 Jan;11(1):57-65.


    These protocols are for reference only. InvivoChem does not independently validate these methods.

    PF-5274857


    Inhibition of smoothened by PF-5274857.  2012 Jan;11(1):57-65.

    PF-5274857

    Imaging of Hh pathway in brain of Gli-luc transgenic mice.  2012 Jan;11(1):57-65.

     PF-5274857


    Pharmacokinetics and biomarker response in single dose study.  2012 Jan;11(1):57-65.

    PF-5274857

    Modulation of Hh pathway genes in tumor (A) and skin (B) in Ptch+/−p53−/− medulloblastoma allograft mice treated with a single dose of 30 mg/kg PF-5274857.  2012 Jan;11(1):57-65.

     

     PF-5274857


    PF-5274857 penetrates blood–brain barrier in primary Ptch+/−p53−/− medulloblastoma mice.

    PF-5274857

    In vivo effects of PF-5274857 in Ptch+/−p53+/− medulloblastoma allograft mice.  2012 Jan;11(1):57-65.


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