OSI-027

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Biological Activity

Description	OSI-027 is a selective and potent dual inhibitor of mTORC1 and mTORC2 with IC50 of 22 nM and 65 nM, and more than 100-fold selectivity observed for mTOR than PI3Kα, PI3Kβ, PI3Kγ or DNA-PK.
Targets	mTORC1	mTORC2
IC50	22 nM [1]	65 nM [1]
In vitro	OSI-027 shows the selective and ATP competitive inhibition activities against mTORC1 and mTORC2 with IC50 of 22 nM and 65 nM, respectively. In addition, OSI-027 inhibits mTOR signaling of phospho-4E-BP1 with an IC50 of 1 μM in cell-based assays. [1] OSI-027 exhibits anti-proliferative activities against several acute leukemia cell lines of myeloid/megakaryocytic origin in a dose-dependent manner, including U937, KG-1, KBM-3B, ML-1, HL-60, and MEG-01 cells. [2] A recent study shows that inhibition of mTORC1/2 by OSI-027 effectively suppresses phosphorylation of Akt (S473) and cell proliferation in breast cancer cells. [3]
In vivo	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]
Features

Protocol(Only for Reference)

Kinase Assay: 

^[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 μg of mTOR antibody in 15 μL of buffer A for 1 hour at 4 °C. To each well, 40 μg of HeLa cell lysate in 15 μL 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 μM 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 μM ATP to each well in 25 μL of freshly prepared kinase buffer [Buffer C: 20 mM HEPES (pH 7.5), 10 mM MgCl2, 4 mM MnCl2, 10 mM β-mercaptoethanol, and 200 μM vanadate] and incubated at room temperature (RT) for 30 minutes. The reaction is stopped by transferring 25 μL 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 μL 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 μL 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 μL of chemiluminescent reagents A+B is added and chemiluminescence is measured using an Analyst plate reader.

Cell Assay: 

^[2]

Cell lines	U937, KG-1, KBM-3B, ML-1, HL-60, and MEG-01
Concentrations	0-10 μM
Incubation Time	72 hours
Method	Inhibition of proliferation is measured using the Cell Titer Glo Assay , as noted in figure legends. To generate dose–response curves, cell lines are seeded at a density of 5,000 cells per well in a 96-well plate. After 24 hours of plating, cells are dosed with varying concentrations of either OSI-027 or rapamycin. The signal for Cell Titer Glo Assay is determined 72 hours after dosing and normalized to that of vehicle-treated controls. Inhibition of proliferation, relative to vehicle-treated controls, is expressed as a fraction of 1 and graphed using PRISM software.

Animal Study: 

^[1]

Animal Models	GEO colorectal cells are injected s.c. into the right flank of nu/nu CD-1 mice.
Dosages	≤65 mg/kg
Administration	Administered via gavage.
Solubility	1% DMSO/30% polyethylene glycol/1% Tween 80, 30 mg/mL

1

References

Clinical Trial Information( data from http://clinicaltrials.gov)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT00698243	Completed	Any Solid Tumor or Lymphoma	Astellas Pharma Inc	2008-06	Phase 1

Chemical Information

Download OSI-027 SDF

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

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.40644	4.0644	8.1288	12.1932

Customer Reviews (1)

Click to enlarge	Rating
	Source	Urol Oncol. 2013 Sep 17. pii: S1078-1439(13)00251-2 . OSI-027 purchased from Selleck
	Method	Cell proliferation assays
	Cell Lines	T24, HT1376, and UM-UC-3
	Concentrations	0.1-25 uM
	Incubation Time	72 h
	Results	Dual mTOR inhibitor OSI-027 inhibit bladder cancer cell growth in a dose-dependent manner.

Product Citations (3)

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  Benzofuran derivatives as a novel class of inhibitors of mTOR signaling [Christophe Salom? et al. European Journal of Medicinal Chemistry 2013;10.1016/j.ejmech.2013.12.020]

  
  


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  PI 3-kinase independent role for AKT in F-actin regulation during outer segment phagocytosis by RPE cells. [Bulloj A, et al. Exp Eye Res 2013;113:9-18]

  
  PubMed: 23669303


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  The combination of an mTORc1/TORc2 inhibitor with lapatinib is synergistic in bladder cancer in vitro. [Becker MN, et al. Urol Oncol 2013;10.1016/j.urolonc.2013.06.002]

  
  PubMed: 24054871