WAY 316606

Kinase experiment:

WAY-316606 binding to purified sFRP is determined by spectroscopy methods. The sFRP-1 or -2 stock solutions are diluted to 1 μM in a buffered solution and the initial fluorescence is measured. Increasing concentrations of WAY-316606 (0 to 50 μM) are added to the protein in the cuvette and incubated for 5 min prior to assessing fluorescence intensity using a Fluoromax-2 fluorometer. In control experiments, the DMSO (vehicle control)-matched buffer solution is used. Fluorescence spectra are scanned in the ratio mode (S/R, signal/reference) to compensate for variations in lamp output as a function of wavelength. Fluorescence changes are fitted to a quadratic equation to obtain apparent dissociation constants[2].

Cell experiment:

U2OS bone cells are infected with recombinant adenovirus 5 (Ad5)−WNT3 at a multiplicity of infection (MOI) of 2, followed by infection with Ad5-sFRP-1 and Ad5-16xTCF-luciferase, each at an MOI of 10. Four hours after infection, the cells are frozen in sterile cryogenic vials at a cell density of 9×106 cells/mL and stored in a −150°C freezer. For the assay, a vial of frozen cells is thawed, and the cells are resuspended in plating medium [phenol red-free RPMI 1640 medium containing 5% fetal calf serum, 2 mM GlutaMAX-l, and 1% (v/v) penicillin-streptomycin] to a final cell density of 1.5×105 cells/mL. The resuspended cells are then plated in 96-well tissue culture treated plates at a volume of 100 μL of cell suspension/well (i.e., 1.5×104 cells/well). The plates are incubated at 37°C inside a 5% CO2/ 95% humidified air incubator for 5 h or until the cells have attached and started to spread. Prior to the addition of WAY-316606, the medium is replaced with 50 μL/well of phenol red-free RPMI 1640 containing 10% fetal calf serum, 2 mM GlutaMAX-l, and 1% (v/v) penicillin-streptomycin. WAY-316606, or vehicle (typically DMSO), diluted in phenol red-free RPMI 1640 containing 2 mM GlutaMAX-l, and l % (v/v) penicillin-streptomycin are then added to the wells in replicates of 4 wells/dilution and the plates are incubated at 37°C overnight. Dose−response experiments are performed with the compounds in 2-fold serial dilutions from 10000−4.9 nM. After the overnight incubation, the cells are washed twice with 150 uL/well of PBS w/o calcium or magnesium and lysed with 50 μL/well of 1× cell culture lysis reagent on a shaker at room temperature for 30 min. Aliquots of the cell lysates (30 μL) are transferred to 96-well luminometer plates, and the luciferase activity is measured in a MicroLumat PLUS luminometer using 100 μL/well of luciferase substrate.

References:

[1]. Moore, WJ, et al. Modulation of Wnt Signaling Through Inhibition of Secreted Frizzled-Related Protein I (sFRP-1) with N-Substituted Piperidinyl Diphenylsulfonyl Sulfonamides. Journal of Medicinal Chemistry (2009), 52(1), 105-116.
[2]. Bodine PV, et al. A small molecule inhibitor of the Wnt antagonist secreted frizzled-related protein-1 stimulates bone formation. Bone. 2009 Jun;44(6):1063-8.