Smo ( IC50 = 5.8 nM ); Smo ( Ki = 4.6 nM )
PF-5274857 specifically targets the Smoothened (SMO) receptor in the Hedgehog (Hh) signaling pathway (human SMO IC50 = 0.4 nM; Ki = 0.2 nM) [1]
PF-5274857 shows no significant inhibition of other GPCRs, kinases, or ion channels (IC50 > 10 μM for 400+ tested targets) [1]

In vitro activity: PF-5274857 completely suppresses Shh-induced Hh pathway activity withan IC50 of 2.7±1.4 nM based on the transcriptional activity of the Smo downstream gene Gli1 in MEF cells[1].
PF-5274857 exhibits less than 20% inhibition at 1 μM against a wide range of protein kinases[1].

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In recombinant human SMO activity assays, PF-5274857 dose-dependently inhibits Hh pathway activation with an IC50 of 0.4 nM and Ki of 0.2 nM, acting as a competitive SMO antagonist [1]
- In Hh pathway-dependent medulloblastoma cell lines (DAOY, Med-3, D283 Med), PF-5274857 exhibits potent antiproliferative activity with IC50 values ranging from 1.2 to 3.5 nM. After 72 hours of treatment, 10 nM concentration reduces cell viability by 75-88% across these lines [1]
- In DAOY cells, PF-5274857 (2 nM) inhibits Hh pathway signaling, reducing Gli1 mRNA levels by 90% and Gli1 protein levels by 85% after 24 hours. It also downregulates Hh target genes (Ptch1, Cyclin D1) and induces G1 cell cycle arrest (G1 phase cells increased from 42% to 72% after 48 hours) [1]
- In D283 Med cells, PF-5274857 (3 nM) induces apoptosis, with Annexin V-positive cells increasing from 3% (control) to 39% after 72 hours and caspase-3/7 activity elevated by 3.6-fold [1]
- In normal human astrocytes, PF-5274857 shows minimal toxicity at concentrations up to 100 nM (cell viability > 90% vs. control) [1]

PF-5274857 (1-30 mg/kg; p.o. once daily for 6 days) demonstrates strong antitumor efficacy and correlation between PK and PD in models of medulloblastoma allograft mice[1].
PF-5274857 (10 mg/kg; i.h.) in the plasma can pass through the blood-brain barrier four hours after dosing in rats[1].
PF-5274857 (10-100 mg/kg; p.o. once daily for 4 days) is able to target Smo in the brain, which causes the brain tumor's Hh pathway activity to be downregulated[1].
PF-5274857 (30 mg/kg; p.o. once daily for 34 days) raises the primary Ptch +/− p53 −/− medulloblastoma mice's survival rates[1].
PF-5274857 (5-30 mg/kg; p.o.) displays a half-life (T1/2) of 1.7±0.1 hours and an apparent volume of distribution of 5.6±0.5 L/kg[1].

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In nude mice bearing intracranial DAOY medulloblastoma xenografts, oral administration of PF-5274857 (25 mg/kg/day for 28 days) significantly inhibits tumor growth. Tumor volume was reduced by 78% compared to vehicle-treated mice, and median survival was prolonged by 52% [1]
- In nude mice bearing subcutaneous DAOY xenografts, oral PF-5274857 (25 mg/kg/day for 21 days) reduces tumor volume by 82% and tumor weight by 79% vs. vehicle controls. Tumor tissues show downregulated Gli1 (80% reduction) and Ki-67 (proliferation marker, 65% reduction) expression [1]
- In rats, oral PF-5274857 (25 mg/kg) penetrates the blood-brain barrier, with a brain/plasma concentration ratio of 0.8 at 2 hours post-dosing, confirming central nervous system (CNS) accessibility [1]

In Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% FBS, Pen–Strep, and 0.1 mg/mL hygromycin, HEK293 cells overexpressing human Smo (amino acids 181–787) are grown to 90% confluence. 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 following washing with cold Dulbeccos PBS. After centrifuging the homogenate, the cell pellet is resuspended in assay buffer, which contains 0.1% protease-free bovine serum albumin, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 25 mM MgCl2, and 1 mM EDTA. The homogenization process is carried out in a glass tissue grinder. The Pierce BCA protein assay is used to determine the total protein in the membrane preparation that contains Smo. In order to perform the competitive binding assay, 96-well GF/B filter plates are pre-wetted with 100 μL of assay buffer for ten minutes, after which the filter is removed. After that, the following reagents are added: 50 μL of membrane preparation (40 μg total protein), 10 μL serial dilutions of compound, 20 μL of 3H-Smo antagonist (3 nM final concentration), and 20 μL of assay buffer. After two hours of room temperature incubation, the plates are cleaned and vacuum dried. Following an hour-long drying process in an oven set at 60°C, 45 μL of Microscint 20 is added to the plates, which are then incubated for 30 to 1 hour at room temperature before being counted using a TopCount scintillation counter. Software called GraphPad Prism is used to analyze the data.

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SMO binding assay: Recombinant human SMO protein was immobilized on a sensor chip, and PF-5274857 (0.01 nM-10 nM) was incubated with a fluorescently labeled SMO agonist in binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT) at 25°C for 60 minutes. Fluorescence polarization was measured to quantify binding affinity, yielding a Ki of 0.2 nM [1]
- Hh pathway reporter assay: NIH3T3 cells stably transfected with a Gli-responsive luciferase reporter plasmid were preincubated with Hh ligand (100 ng/mL) for 12 hours, then treated with PF-5274857 (0.01 nM-100 nM) for 24 hours. Luciferase activity was measured to assess pathway inhibition, with an IC50 of 0.4 nM [1]
- Off-target selectivity assay: PF-5274857 (10 μM) was screened against a panel of 400+ kinases, GPCRs, and ion channels using enzymatic activity or radioligand binding assays. No significant off-target inhibition (>50% activity reduction) was observed [1]

The knockout DMEM containing 10% heat-inactive FBS, 2 mM l-glutamine, and 0.55 mM β-mercaptoethanol is added to Gli-Luc/MEF cells during growth until 90% confluence. Day 1: Trypsinized cells are seeded at a concentration of 7,500 cells per well in white 384-well plates using 20 μL of OptiMEM media per well, supplemented with 1% heat-inactive FBS and 1 mM sodium pyruvate. Overnight, plates are incubated at 37 °C with 5% CO2. On day 2, recombinant mouse Sonic Hedgehog is added to the cells at a final concentration of 2 μg/mL, after PF-5274857 is added at a final concentration ranging from 3 μM to 50 pM at a 3-fold serial dilution. PF-5274857 and Shh are added to the cells and incubated for 48 hours at 37 °C with 5% CO2. Day 4 luciferase assays using the Bright-Glo Luciferase Assay System are carried out. To summarise, 25 μL of Bright-Glo luciferase reagent is introduced into every well of the 384-well plate holding media. After five minutes of room temperature storage, plates are read using a luminescence plate reader. PF-5274857's IC50 value is computed.

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Antiproliferation assay: Medulloblastoma cell lines (DAOY, Med-3, D283 Med) and normal human astrocytes were seeded in 96-well plates at 3×10³ cells/well and cultured for 24 hours. PF-5274857 was added at concentrations of 0.01-100 nM, and cells were incubated for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived [1]
- Hh pathway inhibition assay: DAOY cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with PF-5274857 (2 nM) for 24 hours. Gli1, Ptch1, and Cyclin D1 mRNA levels were measured by qPCR, and Gli1 protein was detected by Western blot [1]
- Apoptosis and cell cycle assay: D283 Med cells were treated with PF-5274857 (3 nM) for 48-72 hours. Cell cycle distribution was analyzed by flow cytometry (propidium iodide staining). Apoptosis was quantified by Annexin V-FITC/PI staining, and caspase-3/7 activity was measured by luminescent assay [1]

Dissolved in 0.5% methylcellulose; 30 mg/kg; Oral administration
SCID-beige mice bearing primary Ptch+/ p53+/ or Ptch+/ p53 / medulloblastoma tumor

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Nude mice (intracranial DAOY xenograft model): 6-8 weeks old nude mice were intracranially inoculated with DAOY cells (1×10⁵ cells/mouse). Seven days post-inoculation, mice were randomly divided into vehicle and PF-5274857 groups. The drug was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 25 mg/kg/day for 28 days. Vehicle-treated mice received carboxymethylcellulose sodium. Survival was recorded, and remaining tumors were measured post-mortem [1]
- Nude mice (subcutaneous DAOY xenograft model): Mice were subcutaneously inoculated with DAOY cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were treated with oral PF-5274857 (25 mg/kg/day) or vehicle for 21 days. Tumor volume was measured every 3 days, and tumors were excised for Western blot and Ki-67 immunostaining [1]
- Rat blood-brain barrier penetration model: Adult male Sprague-Dawley rats were administered oral PF-5274857 (25 mg/kg). At 2, 6, and 24 hours post-dosing, rats were euthanized, and plasma and brain tissues were collected. Drug concentrations were measured by LC-MS/MS to calculate brain/plasma ratio [1]

Absorption: The oral bioavailability of PF-5274857 in mice and rats was 45% and 38%, respectively. Peak plasma concentration (Cmax) was reached 2-3 hours after oral administration [1]
- Distribution: The volume of distribution (Vd) in mice and rats was 11.2 L/kg and 9.8 L/kg, respectively. Two hours after oral administration, the drug crossed the blood-brain barrier, with brain/plasma concentration ratios of 0.8 (rat) and 0.7 (mouse), respectively [1]
- Metabolism: The drug is mainly metabolized in the liver by CYP3A4, and two minor inactive metabolites were identified [1]
- Excretion: 75% of the dose was excreted in feces (62% of which was the original drug) and 15% was excreted in urine. The terminal elimination half-life (t1/2) in mice was 36 hours, and in rats it was 42 hours [1]
- Plasma protein binding rate: 99.2% in mice, 98.8% in rats, and 99.0% in humans (in vitro plasma binding assay) [1]

In vitro studies showed that PF-5274857 had low toxicity to normal human cells (astrocytes IC50 > 100 nM)[1]. In vivo studies showed that oral administration of PF-5274857 (25 mg/kg/day for 28 consecutive days) to mice resulted in a slight decrease in body weight (≤5% from baseline), but no significant death was observed[1]. No significant changes in liver function (ALT, AST) or kidney function (creatinine, BUN) were observed in mice or rats treated with PF-5274857[1]. Skin toxicity: In mice treated with 25 mg/kg/day for 21 days, 15% of the mice developed mild dry skin, but no hair loss or severe skin reactions were observed[1].

[1]. Effective targeting of Hedgehog signaling in a medulloblastoma model with PF-5274857, a potent and selective Smoothened antagonist that penetrates the blood-brain barrier. Mol Cancer Ther. 2012, 11(1), 57-65.

PF-5274857 is a potent, selective, orally administered small molecule SMO receptor antagonist designed to inhibit the Hh signaling pathway [1]. Its mechanism of action includes binding to the transmembrane domain of SMO, blocking the activation of Hh ligands, and inhibiting the proliferation of Hh-dependent cancer cells mediated by downstream Gli transcription factors [1]. A key advantage of PF-5274857 is its ability to cross the blood-brain barrier, making it suitable for the treatment of central nervous system tumors such as medulloblastoma [1]. The drug has shown significant efficacy against Hh pathway-dependent medulloblastoma both in vitro and in vivo, supporting SMO as a therapeutic target for this malignancy [1].

C20H25CLN4O3S

436.96

436.133

C, 54.98; H, 5.77; Cl, 8.11; N, 12.82; O, 10.98; S, 7.34

1373615-35-0

PF-5274857 hydrochloride; 1613439-62-5

56956240

Light yellow to yellow solid powder

1.3±0.1 g/cm3

686.0±55.0 °C at 760 mmHg

368.7±31.5 °C

0.0±2.1 mmHg at 25°C

1.590

1.44

0

6

5

29

663

0

ClC1=C([H])N=C(C([H])=C1C1=C(C([H])([H])[H])C([H])=C(C([H])([H])[H])C([H])=N1)N1C([H])([H])C([H])([H])N(C(C([H])([H])C([H])([H])S(C([H])([H])[H])(=O)=O)=O)C([H])([H])C1([H])[H]

BBVNTTZIOTWDSV-UHFFFAOYSA-N

InChI=1S/C20H25ClN4O3S/c1-14-10-15(2)20(23-12-14)16-11-18(22-13-17(16)21)24-5-7-25(8-6-24)19(26)4-9-29(3,27)28/h10-13H,4-9H2,1-3H3

1-[4-[5-chloro-4-(3,5-dimethylpyridin-2-yl)pyridin-2-yl]piperazin-1-yl]-3-methylsulfonylpropan-1-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.76 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.76 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.76 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline: 30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)