WAY 316606 is an inhibitor of the secreted protein sFRP-1, an endogenous antagonist of the secreted glycoprotein Wnt.

SFRP1:0.5 μM

WAY-316606对U2-OS细胞中Wnt-Luciferase活性的EC50值为0.65 μM[1]。WAY-316606以0.08 μM的KD结合到分泌型frizzled相关蛋白(sFRP)-1抑制剂，并以0.65 μM的EC50抑制sFRP-1。同时，WAY-316606也能与sFRP-2结合，但结合能力弱于sFRP-1，其KD值为1 μM。使用一种含有荧光探针化合物的荧光偏振结合实验，并采用竞争结合方式对纯化的人sFRP-1蛋白进行测试，得出WAY-316606的IC50为0.5 μM [2]。

WAY-316606在新生小鼠颅骨实验中能促进骨骼形成。该化合物以剂量依赖的方式显著增加总骨骼面积，高达60%，其中EC50约为1 nM。WAY-316606具有良好的水溶性、对细胞色素p450酶系（3A4、2D6、2C9）的中度至低度抑制作用，并在大鼠与人类肝微粒体中表现出良好稳定性（每种生物体中半衰期均>60分钟）。在雌性Sprague-Dawley大鼠中，经单次静脉注射给药（2 mg/kg）后，WAY-316606展现出高等于肝脏血流量的血浆清除率（77 mL/min/kg），导致无论给药途径如何，血浆中化合物暴露水平迅速下降[2]。

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 [2].

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 a luciferase substrate.