Home PI3K/Akt/mTOR mTOR inhibitor - Autophagy activator OSI-027 For research use only.

OSI-027

Synonyms: ASP4786, CERC 006, AEVI-006

OSI-027 (ASP4786, CERC 006, AEVI-006) is a selective and potent dual inhibitor of mTORC1 and mTORC2 with IC50 of 22 nM and 65 nM in cell-free assays, and more than 100-fold selectivity observed for mTOR than PI3Kα, PI3Kβ, PI3Kγ or DNA-PK. OSI-027 induces autophagy in cancer cells.

OSI-027 Chemical Structure

OSI-027 Chemical Structure

CAS No. 936890-98-1

Purity & Quality Control

OSI-027 Related Products

Signaling Pathway

mTOR Signaling Pathways

mTOR Signaling Pathway

Cell Data

Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
human SQ20B cells Cytotoxicity assay 72 h Cytotoxicity against human SQ20B cells after 72 hrs by MTT assay, IC50=1.3 μM 24445311
human Caki1 cells Cytotoxicity assay 72 h Cytotoxicity against human Caki1 cells after 72 hrs by MTT assay, IC50=1.9 μM 24445311
human SKHEP1 cells Cytotoxicity assay 72 h Cytotoxicity against human SKHEP1 cells after 72 hrs by MTT assay, IC50=3.2 μM 24445311
human DU145 cells Cytotoxicity assay 72 h Cytotoxicity against human DU145 cells after 72 hrs by MTT assay, IC50=4.1 μM 24445311
human HCT116 cells Cytotoxicity assay 72 h Cytotoxicity against human HCT116 cells after 72 hrs by MTT assay, IC50=5.6 μM 24445311
human ColoR cells Cytotoxicity assay 72 h Cytotoxicity against human ColoR cells after 72 hrs by MTT assay, IC50=5.8 μM 24445311
human COLO205 cells Cytotoxicity assay 72 h Cytotoxicity against human COLO205 cells after 72 hrs by MTT assay, IC50=5.8 μM 24445311
human OVCAR3 cells Cytotoxicity assay 72 h Cytotoxicity against human OVCAR3 cells after 72 hrs by MTT assay, IC50=8.1 μM 24445311
human HOP62 cells Cytotoxicity assay 72 h Cytotoxicity against human HOP62 cells after 72 hrs by MTT assay, IC50=41 μM 24445311
human COLO205 cells Cytotoxicity assay 72 h Cytotoxicity against human COLO205 cells after 72 hrs by MTT assay 24836070
human HOP62 cells Cytotoxicity assay 72 h Cytotoxicity against human HOP62 cells after 72 hrs by MTT assay 24836070
Click to View More Cell Line Experimental Data

Biological Activity

Description OSI-027 (ASP4786, CERC 006, AEVI-006) is a selective and potent dual inhibitor of mTORC1 and mTORC2 with IC50 of 22 nM and 65 nM in cell-free assays, and more than 100-fold selectivity observed for mTOR than PI3Kα, PI3Kβ, PI3Kγ or DNA-PK. OSI-027 induces autophagy in cancer cells.
Targets
mTOR [4]
(Cell-free assay)		 mTORC1 [1]
(Cell-free assay)		 mTORC2 [1]
(Cell-free assay)		 PI3Kγ [4]
(Cell-free assay)
4 nM 22 nM 65 nM 0.42 μM
In vitro
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]
Kinase Assay 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 Research 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.
Experimental Result Images Methods Biomarkers Images PMID
Western blot p-mTOR / mTOR / p-AKT / AKT / p-P70S6K / P70S6K / p-4EBP1 / 4EBP1 26026051
Immunofluorescence LC3 24610825
Growth inhibition assay Cell viability 25823922
In Vivo
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]
Animal Research 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.
NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT00698243 Completed
Any Solid Tumor or Lymphoma
Astellas Pharma Inc
 June 2008 Phase 1

Chemical Information & Solubility

Molecular Weight 406.44 Formula

C21H22N6O3
CAS No. 936890-98-1 SDF Download OSI-027 SDF
Smiles COC1=CC=CC2=C1NC(=C2)C3=C4C(=NC=NN4C(=N3)C5CCC(CC5)C(=O)O)N
Storage (From the date of receipt)

In vitro
Batch:

DMSO : 18 mg/mL ( (44.28 mM) Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Water : Insoluble

Ethanol : Insoluble


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Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

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