Purmorphamine

Catalog No.S3042

Molecular Weight(MW): 520.62

Purmorphamine, which directly binds and activates Smoothened, blocks BODIPY-cyclopamine binding to Smo with IC50 of ~ 1.5 μM in HEK293T cell and also is an inducer of osteoblast differentiation with EC50 of 1 μM.

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a-d) LoVo cells were separately or simultaneously treated with 1 μM purmorphamine and 1 μM thiostrepton for the indicated time. a The Gli1, FoxM1, and CCNB1 protein expression levels were examined by immunoblotting after drug treatment for 48 h. b Cell viability was detected after 6 days using an MTT assay. c LoVo cells treated with indicated drugs were cultured for 2 weeks, and outgrowth colonies were stained with crystal violet. d The matched colony count of (c). Error bars represent the mean and S.D. of three independent experiments. **, p < 0.01.

J Exp Clin Cancer Res, 2017, 36(1):23. Purmorphamine purchased from Selleck.

Immunoblotting analysis illustrating that SMO agonist Purmorphamine (PUR) activated SMO expression in A549 and H1299 cells

Sci Rep, 2017, 7(1):1899. Purmorphamine purchased from Selleck.
(C) mRNA levels of Nek2A in purmorphamine treated H4 cells. Total mRNA was extracted and quantified by real-time PCR. Error bars represent the standard deviation of three independent experiments. **P<0.01 compared with the control groups.

Int J Oncol, 2016, doi: 10.3892/ijo.2016.3819. . Purmorphamine purchased from Selleck.

Purity & Quality Control

Choose Selective Hedgehog/Smoothened Inhibitors

Biological Activity

Description

Targets

Smoothened ^[1]
(HEK293T cells)

~1.5 μM

In vitro

Purmorphamine activates the Hedgehog pathway by directly binds and activates Smoothened with IC50 of ~ 1.5 μM in compete with cyclopamine, a Smo antagonist. ^[1] Purmorphamine is a potent inducer of osteogenesis in multipotent C3H10T1/2 cells. The EC50 (based on ALP expression) for Purmorphamine is 1 μM in C3H10T1/2 cells. Purmorphamine (1 μM) and BMP-4 (100 ng/mL) together increase ALP activity more than 90-fold in 3T3-L1 cells. ^[2] In contrast to BMP-4, Purmorphamine induces osteogenesis by activating Hedgehog signaling in multipotent mesenchymal progenitor cells. ^[3]

Cell Data

Cell Lines	Assay Type	Concentration	Incubation Time	Activity Description	PMID
mouse Shh Light2 cells	M3rCXGZ2dmO2aX;uJIF{e2G7		M{H5fFMxKGh?	MljxRYN1cX[jdHnvckBw\iCVaHigbY4hdW:3c3WgV4hpKEyrZ3j0NkBk\WyuczDh[pRmeiB\|MDDodpMh[nlibIXjbYZmemG\|ZTDy[ZBwenSncjDn[Y5mKGG\|c3H5MEBGSzVyPUGg{txO	M1n1fVE3PDB6MEi4
mouse C3H10T1/2 cells	Ml7oSpVv[3Srb36gZZN{[Xl?			MoPETY5lfWO2aX;uJI9nKG:\|dHXv[4Vv\XOrczDpckBud3W\|ZTDDN2gyOFRzL{KgZ4VtdHNiYYPz[ZN{\WRiYYOgbY5lfWO2aX;uJI9nKG:\|dHXvZoxie3Ric4DlZ4lncWNibXHyb4VzKGGua3HsbY5mKHCqb4PwbIF1[XOnIHL5JIludXWwb3\seY9z\XOlZX7j[UBu\XSqb3SsJGVEPTB;MTFOwG0>	NETLXYcyPjRyOECwNy=>
HEK293T cells	NVLOblNrTnWwY4Tpc44h[XO\|YYm=	MWC1JO69VQ>?	MoPrOEBp	NUHRUW11UW6qaXLpeIlwdiCxZjDCU2RKWFlvY4njcI9x[W2rbnWgZolv\GmwZzD0c{BUdW9iQz30[ZJucW6jbDDjfZRweGyjc33pZ{Bld22jaX6g[ZhxemW\|c3XkJIlvKEiHS{K5N3Qh[2WubIOgZZQhPSC3TTDh[pRmeiB2IHjyd{BjgSCobIXvdoV{[2WwY3WgcYlkem:\|Y3;wfS=>	NGjvb2QyPjRyOEC4PC=>

... Click to View More Cell Line Experimental Data

In vivo

Purmorphamine up-regulates ALP expression in human mesenchymal stem cell-based constructs on rats. ^[4]

Protocol

Kinase Assay:^[1]	+ Expand Binding assay: Smo binding assays are conducted with BODIPY-cyclopamine and Smo-overexpressing cells as previously described4,5, using CMV promoter-based, SV40 origin-containing expression constructs for Smo-Myc3, the deletion mutant SmoCRD (deletion of amino acids 68 to 182), and SmoCT (deletion of amino acids 556 to 793). HEK 293T cells are grown on poly-D-lysine-treated glass coverslips in 12-well plates until 70% confluency and then transfected with the appropriate expression construct (0.5 g/well) using FuGene 6 according the manufacturer's protocols. Two days after transfection, the HEK 293T cells are incubated with DMEM containing 0.5% bovine calf serum, 5 nM BODIPY-cyclopamine, and varying concentrations of Purmorphamine (0, 1.5, or 5 M) (1 mL/well) for 1 h at 37 ℃. The Smo-overexpressing cells are then washed with 1 × PBS buffer (1 mL/well), mounted with DAPI-containing medium, and visualized using a Leica DM4500B fluorescence microscope. For binding assays using fixed cells, the Smo-overexpressing HEK 293T cells are fixed with 3% paraformaldehyde in 1 × PBS buffer for 10 min at room temperature (1 mL/well), treated with 1 × PBS containing 10 mM glycine and 0.2% sodium azide for 5 min (1 mL/well), washed with 1 × PBS buffer (1 mL/well), and treated with the Purmorphamine-containing media described above for 4 h at room temperature.
Cell Research:^[2]	+ Expand Cell lines: C3H10T1/2 cell Concentrations: 0.5-10 μM Incubation Time: 4 days Method: C3H10T1/2 cells are expanded in T175 flasks; cells at 13th passage are detached by trypsin/EDTA and diluted in the growth media. The resulting cell suspension is then plated into black clear bottom 384-well plates with 2500 cells/well in 100 µL growth medium using a Multi-drop^TM liquid delivery system. After overnight incubation, cells attached to the bottom of the wells. A stock solution of each Purmorphamine in DMSO (500 nL) is delivered into corresponding well using a Mini Trak^TM multiposition dispenser system to make a final concentration of 5μM of Purmorphamine. Cells are then incubated at 37 ℃ with 5% CO₂ in air atmosphere. After 4 days, the medium is removed and 10 μL of passive lysis buffer is added into each well. After 5 min, 10 μL of alkaline phosphatase substrate solution is added to each well. After incubating 15 min at room temperature, the plates are read on an Acquest high-throughput plate reader following the manufacturer's protocol. (Only for Reference)

Kinase Assay:^[1]

+ Expand

Binding assay:

Smo binding assays are conducted with BODIPY-cyclopamine and Smo-overexpressing cells as previously described4,5, using CMV promoter-based, SV40 origin-containing expression constructs for Smo-Myc3, the deletion mutant SmoCRD (deletion of amino acids 68 to 182), and SmoCT (deletion of amino acids 556 to 793). HEK 293T cells are grown on poly-D-lysine-treated glass coverslips in 12-well plates until 70% confluency and then transfected with the appropriate expression construct (0.5 g/well) using FuGene 6 according the manufacturer's protocols. Two days after transfection, the HEK 293T cells are incubated with DMEM containing 0.5% bovine calf serum, 5 nM BODIPY-cyclopamine, and varying concentrations of Purmorphamine (0, 1.5, or 5 M) (1 mL/well) for 1 h at 37 ℃. The Smo-overexpressing cells are then washed with 1 × PBS buffer (1 mL/well), mounted with DAPI-containing medium, and visualized using a Leica DM4500B fluorescence microscope. For binding assays using fixed cells, the Smo-overexpressing HEK 293T cells are fixed with 3% paraformaldehyde in 1 × PBS buffer for 10 min at room temperature (1 mL/well), treated with 1 × PBS containing 10 mM glycine and 0.2% sodium azide for 5 min (1 mL/well), washed with 1 × PBS buffer (1 mL/well), and treated with the Purmorphamine-containing media described above for 4 h at room temperature.

Cell Research:^[2]

+ Expand

Cell lines: C3H10T1/2 cell
Concentrations: 0.5-10 μM
Incubation Time: 4 days
Method: C3H10T1/2 cells are expanded in T175 flasks; cells at 13th passage are detached by trypsin/EDTA and diluted in the growth media. The resulting cell suspension is then plated into black clear bottom 384-well plates with 2500 cells/well in 100 µL growth medium using a Multi-drop^TM liquid delivery system. After overnight incubation, cells attached to the bottom of the wells. A stock solution of each Purmorphamine in DMSO (500 nL) is delivered into corresponding well using a Mini Trak^TM multiposition dispenser system to make a final concentration of 5μM of Purmorphamine. Cells are then incubated at 37 ℃ with 5% CO₂ in air atmosphere. After 4 days, the medium is removed and 10 μL of passive lysis buffer is added into each well. After 5 min, 10 μL of alkaline phosphatase substrate solution is added to each well. After incubating 15 min at room temperature, the plates are read on an Acquest high-throughput plate reader following the manufacturer's protocol.
(Only for Reference)

References

[4] Faghihi F, Biomed Pharmacother, 2012, 3322(12).

+ Expand

Solubility (25°C)

In vitro	DMSO	4 mg/mL warmed (7.68 mM)
	Water	Insoluble
	Ethanol	Insoluble
In vivo	Add solvents to the product individually and in order(Data is from Selleck tests instead of citations): 2% DMSO+30% PEG 300+5% Tween 80+ddH2O For best results, use promptly after mixing.	1mg/mL

* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Information Download Purmorphamine SDF

Molecular Weight	520.62
Formula	C₃₁H₃₂N₆O₂
CAS No.	483367-10-8
Storage	3 years -20°C powder
Storage	2 years -80°C in solvent
Synonyms	N/A

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Hedgehog/Smoothened Signaling Pathway Map

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Vismodegib (GDC-0449)

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Cyclopamine

Cyclopamine is a specific Hedgehog (Hh) signaling pathway antagonist of Smoothened (Smo) with IC50 of 46 nM in TM3Hh12 cells.
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GANT61 is an inhibitor for GLI1 as well as GLI2-induced transcription, inhibits hedgehog with IC50 of 5 μM in GLI1 expressing HEK293T cell, displays selectivity over other pathways, such as TNF and glucocorticoid receptor gene transactivation.
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Taladegib (LY2940680)

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Features:Attenuates SAG stimulation of Shh-LIGHT2 cells to a greater extent relative to other antagonists.

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Purmorphamine