Purmorphamine

Kinase experiment^[1]:

Preparation method

Smo binding experiments were performed using BODIPY-Cyclopamine and cells overexpressing Smo.

Two days after transfection of HEK 293T cells, the cells were incubated at 37 ℃for 1 hour with DMEM medium containing BODIPY-Cyclopamine and varying concentrations of Purmorphamine (0, 1.5, or 5μM) (1 mL per well).

The overexpressed Smo cells were then washed with 1xPBS buffer (1 mL per well), placed in a medium containing DAPI, observed using fluorescence microscopy, and binding detection was performed using fixed cells.

Smo-overexpressing HEK293T cells were fixed at room temperature with 3% paraformaldehyde dissolved in 1 xPBS buffer (1 mL per well) for 10 minutes, then treated with 1 xPBS buffer (1 mL per well) containing 10 mm of glycerine and 0.2% sodium azide for 5 minutes with 1 xPBS buffer (1 ml per well).

Cells were washed and then treated with a medium containing Purmorphamine at room temperature for 4 hours.

Reaction Conditions

0, 1.5, or 5μM Purmorphamine

Applications

Purmorphamine directly binds and activates Smoothened, blocking BODIPY-cyclopamine binding to Smo, with an IC₅₀ of about 1.5 μM in HEK293T cells.

Cell experiment ^[2]:

Cell lines

C3H10T1/2 cells

Preparation method

Cells were treated with 2 μM of purmorphamine or 0.5% DMSO control for 4 days.

Reaction Conditions

2 μM purmorphamine;4 days

Applications

Purmorphamine induces osteogenesis in pluripotent mesenchymal progenitor cells.

Animal experiment ^[3]:

Animal models

Male C57BL/6J mouse ( HI injury)

Dosage form

10 mg/kg purmorphamine, i.p.

Applications

Purmorphamine prevents Hypoxic-Ischemic(HI)-induced brain injury.

References:

[1]. Sinha S, Chen JK. Purmorphamine activates the Hedgehog pathway by targeting Smoothened. Nat Chem Biol. 2006 Jan;2(1):29-30. doi: 10.1038/nchembio753. Epub 2005 Nov 20. PMID: 16408088.

[2].Wu X, Walker J,et,al. Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway. Chem Biol. 2004 Sep;11(9):1229-38. doi: 10.1016/j.chembiol.2004.06.010. PMID: 15380183.

[3]. Liu D, Bai X, et,al. Purmorphamine Attenuates Neuro-Inflammation and Synaptic Impairments After Hypoxic-Ischemic Injury in Neonatal Mice via Shh Signaling. Front Pharmacol. 2020 Mar 4;11:204. doi: 10.3389/fphar.2020.00204. PMID: 32194421; PMCID: PMC7064623.