WYE-125132 (WYE-132)

U87MG, MDA361, H1975, A549, A498, and 786-O cells are injected s.c. into the right flank of CD1 nu/nu mice.

Urea, N-[4-[1-(1, 4-dioxaspiro[4.5]dec-8-yl)-4-(8-oxa-3-azabicyclo[3.2.1]oct-3-yl)-1H-pyrazolo[3, 4-d]pyrimidin-6-yl]phenyl]-N'-methyl-

WYE-125132 is a highly potent, ATP-competitive and specific mTOR inhibitor with IC50 of 0.19 nM.

WYE-125132 is a highly potent, ATP-competitive, and specific mTOR kinase inhibitor.

0.19 nM [1], 0.19 nM [1], 0.19 nM [1], 0.19 nM [1], 0.19 nM [1], 0.19 nM [1]

WYE-125132 (5 mg/kg p.o.) produces significant antitumor activity and causes dose-dependent tumor growth delay in the PI3K/mTOR- and HER2-hyperactive MDA361 tumor model. In addition, WYE-125132 also shows potent antitumor efficacy in the PTEN-null glioma U87MG, non–small cell lung cancer H1975 and A549 models. [1]

Kinase assays, mTOR enzyme assays via dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), ATP matrix assays, and mTOR immune-complex kinase assays are performed as follows. The endogenous TOR of LNCap cell lysate is immunoprecipitated by anti-FRAP/TOR (N-19). Cell lysate (1.0 mg) is mixed with 4 ug antibody coupled to protein-G/A agarose in 1 mL lysis buffer. The immune-complexes are washed sequentially with lysis buffer, lysis buffer plus 500 mM KCl, and kinase buffer wash. The immune-complexes are subjected to kinase reaction for 30 minutes at 30 C in a final volume of 50 uL containing 10 mM Hepes (pH 7.4), 50 mM NaCl, 50 mM beta-glycerophosphate, and 0.5 uM microcystin LR, 1 mM DTT, 10 mM MnCl2, 100 uM ATP, 1 ug His6-S6K or 1 ug His6-4EBP1. Kinase reactions (immune-complex and purified enzymes) are terminated by NuPAGE LDS sample buffer and resolved in a 4–12% NuPAGE Bis-Tris gel for Western blotting with anti-P(T389)-p70S6K and anti-P(T46)-4EBP1, anti-FRAP/TOR (N-19), anti-FLAG M2, and ti-His6 (Clone His-1). In the radioactive assay, 10 uCi [gamma-32P]ATP (3000 Ci/mmol) and 100 uM cold ATP are used. 32P-labeled products are resolved by SDS–PAGE and subjected to autoradiogram to Kodak X-ray films.

519, 6

DMSO 104 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL