In the DELFIA measuring His6-S6K1 T389 phosphorylation, WYE-354 inhibits recombinant mTOR enzyme with an IC ₅₀ of 5 nM. Cell viability is analyzed by MTS assay. G-415 and TGBC-2TKB cell lines are treated with increasing concentrations of WYE-354 (0.1, 1, 5 and 10 μM) for 24, 48, and 72 hours. WYE-354 significantly reduces cell viability starting at a 1 μM concentration after a 24 hours exposure, in both studied cell lines (P<0.001). A decrease in cell viability is not observed at a dose of 100 nM, except for the TGBC-2TKB cell line after 72 hours of treatment.

The effect of Rapamycin and WYE-354 on tumor growth is evaluated in xenograft GBC tumor models. 2×10 ⁶ or 5×10 ⁶ cells of G-415 or TGBC2TKB, respectively, are xenotransplanted into NOD-SCID mice subcutaneously. When tumors reach an average volume of 100 mm ³ , the mice are treated either with Rapamycin or WYE354. Rapamycin is administered i.p. at a concentration of 10 mg/kg, daily for 5 days per week for 3 weeks, while WYE-354 is administrated at a daily i.p. dose of 50 mg/kg for 5 days. Mice are sacrificed 30 days after the initiation of the treatments and an autopsy is performed that include removal of the entire tumor area. Mice treated with WYE-354 exhibit 68.6% and 52.4% reduction in average tumor size (P<0.01; P<0.01), as well as 82.9% and 45.5% (P<0.01; ns) reduction in tumor weight, respectively.