Home Stem Cells & Wnt Hedgehog/Smoothened agonist - Autophagy inhibitor Purmorphamine

Purmorphamine

Catalog No.S3042 Synonyms: Shh Signaling Antagonist VI

For research use only.

Purmorphamine (Shh Signaling Antagonist VI), 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. Purmorphamine can reduce both basal and induced autophagy.

Purmorphamine Chemical Structure

CAS No. 483367-10-8

Selleck's Purmorphamine has been cited by 36 publications

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Hedgehog/Smoothened Signaling Pathways

Hedgehog/Smoothened Signaling Pathway Map

Biological Activity

Description Purmorphamine (Shh Signaling Antagonist VI), 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. Purmorphamine can reduce both basal and induced autophagy.
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 Formulation Activity Description PMID
C3H10T1/2 M2SwNWZ2dmO2aX;uJIF{e2G7 Mn7aOkBl[Xm| MWDBZ5Rqfmm2eTDheEBUdW9iaX6gcY92e2ViQ{PINVBVOS9{IHPlcIx{KGG|c3Xzd4VlKGG|IHnu[JVkfGmxbjDv[kBk\WyuIHTp[oZmemWwdHnheIlwdiCrboTvJI9{fGWxYnzhd5QhcW6ldXLheIVlKG[xcjC2JIRigXNiYomgZYxs[WyrbnWgdIhwe3CqYYThd4Uh[XO|YYmsJGVEPTBiPTCwMlgh|ryPLh?= M1\xNFxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzJ5NEK5NlU2Lz5{N{SyPVI2PTxxYU6=
Shh Light2 M2fRbmZ2dmO2aX;uJIF{e2G7 NXfQdpREOzBiaILz MkDzRYN1cX[jdHnvckBw\iCVaHigbY4hdW:3c3WgV4hpKEyrZ3j0NkBk\WyuczDh[pRmeiB|MDDodpMh[nlibIXjbYZmemG|ZTDy[ZBwenSncjDn[Y5mKGG|c3H5MEBGSzVyIE2gNUDPxE1w MnPGQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xcIXicYVlNm6lYnmucoxuNm6raD7nc5YwOTZ2MEiwPFgoRjF4NEC4NFg5RC:jPh?=
C3H10T1/2 MojYSpVv[3Srb36gZZN{[Xl? MorITY5lfWO2aX;uJI9nKG:|dHXv[4Vv\XOrczDpckBud3W|ZTDDN2gyOFRzL{KgZ4VtdHNiYYPz[ZN{\WRiYYOgbY5lfWO2aX;uJI9nKG:|dHXvZoxie3Ric4DlZ4lncWNibXHyb4VzKGGua3HsbY5mKHCqb4PwbIF1[XOnIHL5JIludXWwb3\seY9z\XOlZX7j[UBu\XSqb3SsJGVEPTBiPTCxJO69VS5? M4TFV|xiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzF4NEC4NFA{Lz5zNkSwPFAxOzxxYU6=
HEK293T Ml7HSpVv[3Srb36gZZN{[Xl? NH;EboIyKGi{ MmnRTY5pcWKrdHnvckBw\iCET1TJVHku[3mlbH;wZY1qdmViYnnu[Ilv\yC2bzDTcY8h\XiycnXzd4VlKGmwIFjFT|I6O1RiY3XscJMh[W[2ZYKgNUBpeiCkeTDmcJVwemW|Y3XuZ4UhdWmlcn;zZ49xgSxiSVO1NEA:KDFwNTFOwG0v NXL0V3FIRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMU[0NFgxQDhpPkG2OFA5ODh6PD;hQi=>
Shh Light2 MoHRSpVv[3Srb36gZZN{[Xl? MYCzNEBpenN? MVrBZ5RqfmG2aX;uJI9nKFOqaDDpckBud3W|ZTDTbIghVGmpaISyJINmdGy|IHHzd4V{e2WmIHHzJIJmfGFvZ3HsZYN1d3OrZHHz[UBi[3Srdnn0fUBi\nSncjCzNEBpenNiYomgcJVkcW[ncnHz[UBz\XCxcoTldkBo\W6nIHHzd4F6KGmwIIDy[ZNmdmOnIH;mJFExOCCwTTCzMYtmfG9vTj3hcYlvd2W2aInsMW4oNWGvaX7vZ4Fxem:7bHTpbJllem:laX7uZY1wgWxiY4njcI9x[W2rbnW= MWK8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8yPjRyOEC4PEc,OTZ2MEiwPFg9N2F-
HEK293T MYjGeY5kfGmxbjDhd5NigQ>? NFPqOlc2KHWP NY\YS|lnPCCqcoO= MkjmTY5pcWKrdHnvckBw\iCET1TJVHku[3mlbH;wZY1qdmViYnnu[Ilv\yC2bzDTcY8hVi22ZYLtbY5idCCleYP0[Ylv\SCmb33hbY4h\XiycnXzd4VlKGmwIFjFT|I6O1RiY3XscJMh[XRiNTD1UUBi\nSncjC0JIhzeyCkeTDmcJVwemW|Y3XuZ4UhdWmlcn;zZ49xgQ>? NXzLT5hORGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:vdJVjdWWmLn7jZokvdmyvLn7pbE5od3ZxMU[0NFgxQDhpPkG2OFA5ODh6PD;hQi=>
HEK293T M1z0cWZ2dmO2aX;uJIF{e2G7 NIHjbpc2KHWP MVi0JIhzew>? MnznTY5pcWKrdHnvckBw\iCET1TJVHku[3mlbH;wZY1qdmViYnnu[Ilv\yC2bzDTcY8hSy22ZYLtbY5idCCleYTvdIxie22rYzDkc41icW5iZYjwdoV{e2WmIHnuJGhGUzJ7M2SgZ4VtdHNiYYSgOUB2VSCjZoTldkA1KGi{czDifUBndHWxcnXzZ4Vv[2VibXnjdo9{[2:yeR?= M1uwVlxiKHSjcnfleF0oZ2KuYX7rK{BpemWoPTfoeJRxezpxL4D1Zo1m\C6wY3LpMo5tdS6waXiu[493NzF4NEC4NFg5Lz5zNkSwPFA5QDxxYU6=
SK-N-MC MnPHdWhVWyCjc4PhfS=> M{jQZ5FJXFNib3[gdIVlcWG2cnnjJINidmOncjDj[YxtKGyrbnXzJJRwKGmmZX70bYZ6KG23bITpdIxmKG:ycH;yeJVvcXSrZYOg[o9zKGS{dXegdoVxfXKyb4Ppcoc7KFC{aX3hdpkhe2O{ZXXuJIZweiCVSz3OMW1EKGOnbHzz MUG8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zQTR|NUGzPUc,Ojl2M{WxN|k9N2F-
Click to View More Cell Line Experimental Data
Assay
Methods Test Index PMID
Western blot Patch1 / Gli1 / LC3 / p62 26609469
Immunofluorescence SOX18 26588701
Growth inhibition assay Cell viability 26588701
In vivo Purmorphamine up-regulates ALP expression in human mesenchymal stem cell-based constructs on rats. [4]

Protocol (from reference)

Kinase Assay:[1]
  • 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]
  • 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-dropTM 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 TrakTM 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.

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

Chemical Information

Molecular Weight 520.62
Formula

C31H32N6O2
CAS No. 483367-10-8
Storage 3 years -20°C powder
2 years -80°C in solvent
Smiles C1CCC(CC1)N2C=NC3=C(N=C(N=C32)OC4=CC=CC5=CC=CC=C54)NC6=CC=C(C=C6)N7CCOCC7

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Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

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