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Research Area
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Description
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Cancer |
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Biological Activity
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Description
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AS703026 is a highly selective and potent non-competitive inhibitor of MEK1/2 with IC50 of 5-11 nM. |
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Targets
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MEK1/2 |
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IC50 |
5-11 nM [1] |
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In Vitro
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AS703026 is a novel, selective, orally bioavailable MEK1/2 inhibitor that binds to the distinctive MEK allosteric site and therefore exhibits exquisite kinase selectivity. AS703026 inhibits growth and survival of human multiple myeloma (MM) cells, including U266 and INA-6, with IC50 of 5 nM and 11 nM, respectively. Such an inhibitory effect by AS703026 is mediated by G0-G1 cell cycle arrest and is accompanied by reduced expresson of the MAF oncogene. AS703026 further induces apoptosis via caspase-3 and PARP cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). [1]AS703026 may be an effective therapy in colorectal cancer caused by K-Ras mutation. AS703026 (10 μM) effectively inhibits the ERK pathway, proliferation, and transformation in human DLD-1 colorectal cancer cells what carry a mutant allele of K-Ras (D-MUT). [2] |
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In Vivo
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AS703026 (15 and 30 mg/kg) significantly inhibits tumor growth in a human plasmacytoma xenograft model of H929 MM cells. This can be correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels. [1]AS703026 (10 mg/kg) inhibits tumor growth, and markedly decreases p-ERK level in a xenograft mouse model of human K-Ras mutated (D-MUT) colorectal tumor. [2] |
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Clinical Trials
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AS703026 is currently under Phase II clinical trial for acute myeloid leukemia. |
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Features
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AS703026 is a novel, highly selective and potent allosteric inhibitor of MEK1/2. |
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Protocol
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Kinase Assay
[3]
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| MEK1 enzyme assays |
AS703026 is dissolved in 2.5% DMSO. Activated diphosphorylated MEK (pp-MEK) assays contained 40 μM 33P-γATP (AppKm 8.5 μM), 0.5 nM human-activated MEK1 or MEK2, 1 μM kinase-dead ERK2 (AppKm 0.73 μM). All assays are done in buffer containing 20 mM HEPES (pH 7.2), 5 mM 2-mercaptoethanol, 0.15 mg/mL BSA, and 10 mM MgCl2. The final concentration of 33P- ATP is 0.02 μCi/μL for all the assays. pp-MEK kinase reactions are stopped after 40 min by transferring 30 μL of reaction mixture to Durapore 0.45-μm filters plates containing 12.5% TCA. Filters are dried and read with liquid scintilant on a TopCount. Concentration response data are analyzed for IC50. 0.2 nM recombinant human MEK1 or MEK2 is preincubated with vehicle or with AS703026 for 40 minutes in reaction buffer to determine IC50 of initially unphosphorylated MEK (u-MEK). Phosphorylation/activation is initiated by the addition of a final concentration of 20 nM final B-RafV600E and 30 μM final ATP for 10 min. B-Raf activity is then quenched by addition of the B-Raf inhibitor SB590885 (final concentration 100 nM), and MEK kinase activity is assayed by the addition of 1 μM KD-ERK2 and 0.02 μCi/μL 33P-ATP in reaction buffer. The kinase reactions are stopped after 90 min by transferring 30μL of reaction mixture to a Durapore filter plate, and read as above. |
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Cell Assay
[1]
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| Cell Lines |
U266 and INA-6 cells |
| Concentrations |
2 nM - 20 μM (stock: 10 mM in DMSO) |
| Incubation Time |
48 hours |
| Methods |
Cytotoxicity assays for AS703026 are assessed by measuring both [3H]thymidine incorporation and MTT dye absorbance. Cells (1 × 104 per well) are cultured in 96-well plates for 3 days. For the [3H]thymidine incorporation assay, cells are pulsed with 18.5 kBq/well [3H]thymidine for 6 hours, harvested onto glass fibre filters, and counted in a β-scintillation counter.Cell cycle analysis is assessed by propidium iodide (PI) staining using flow cytometry. AS703026-induced apoptosis is determined by annexin-V/PI staining and flow cytometric data analysis. |
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Animal Study
[1]
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| Animal Models |
H929 MM xenografts are established in CB17 (SCID) mice |
| Formulation |
10 mg/mL, dissolved in 0.5% carboxymethylcellulose / 0.25% Tween20 |
| Doses |
15 or 30 mg/kg |
| Administration |
Oral gavage twice daily |
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References |
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[1] Kim K, et al. Br J Haematol, 2010, 149(4), 537-549.
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[2] Yoon J, et al. Cancer Res, 2011, 71(2), 445-453
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[3] Gilmartin AG, Clin Cancer Res, 2011, 17(5), 989-1000.
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