OSI-027

MTOR

406, 44

GEO colorectal cells are injected s.c. into the right flank of nu/nu CD-1 mice.

OSI-027 is currently in Phase I clinical trials in patients with Advanced Solid Tumors or Lymphoma.

<=65 mg/kg

22 nM [1], 22 nM [1], 22 nM [1], 22 nM [1], 22 nM [1], 22 nM [1]

In GEO colorectal xenograft, OSI-027 (65 mg/kg) inhibits both mTORC1 and mTORC2 effectors, including 4E-BP1, Akt, and S6 phosphorylation. Furthermore, mTORC1 and mTORC2 inhibition together by OSI-027 potently inhibits tumor growth more than mTORC1 inhibition by rapamycin. [1]

Biochemical assays, mTORC1 and mTORC2 inhibition is assayed using native enzyme complex immunoprecipitated from HeLa lysates at 1 mM ATP. To prepare whole cell lysates from HeLa cells, 25 g cell pellet is lysed in 60 mL of ice-cold buffer A [40 mM HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate, and EDTA-free protease inhibitors, containing 0.3% CHAPS] for 30 minutes on a magnetic stirrer in a cold room. After clearing of the lysates by centrifugation at 13, 000 g for 10 minutes, Protein G-coated 384-well plates are incubated with 0.25 ug of mTOR antibody in 15 uL of buffer A for 1 hour at 4 C. To each well, 40 ug of HeLa cell lysate in 15 uL of buffer A is added and incubated overnight at 4 C to immunoprecipitate mTOR complexes. Plates are washed 3 times with buffer A and twice with immunoprecipitation wash buffer [Buffer B: 50 mM HEPES (pH 7.5) and 150 mM NaCl]. OSI-027 is added at 10 uM concentration to each well and DMSO is added to the control wells. The reaction is started by adding 150 ng of His-tagged 4E-BP1 as a substrate in the presence or absence of 100 uM ATP to each well in 25 uL of freshly prepared kinase buffer [Buffer C: 20 mM HEPES (pH 7.5), 10 mM MgCl2, 4 mM MnCl2, 10 mM beta-mercaptoethanol, and 200 uM vanadate] and incubated at room temperature (RT) for 30 minutes. The reaction is stopped by transferring 25 uL of reaction mixture from each well to corresponding wells of fresh Ni-chelate-coated plates and incubated overnight at 4 C followed by 2 hours at 37 C. To detect phosphorylation of 4E-BP1, the plates are washed once with TBST (Tris-buffered saline containing 0.1% Tween-20) containing 5% skim milk powder. To each well, 25 uL of 1:1, 000 diluted phospho-4E-BP1 antibodies in TBST containing 5% skim milk are added and incubated for 1 hour at RT. The plates are washed once with TBST and then 25 uL of anti-rabbit HRP (diluted 1:10, 000) in TBST containing 5% skim milk is added. The plates are incubated for 1 hour at RT and washed 5 times with TBST. For detection of phospho-4E-BP1, 25 uL of chemiluminescent reagents A+B is added and chemiluminescence is measured using an Analyst plate reader.

DMSO 81 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL

(1r, 4r)-4-(4-amino-5-(7-methoxy-1H-indol-2-yl)imidazo[1, 5-f][1, 2, 4]triazin-7-yl)cyclohexanecarboxylic acid