614.2 TAE684 is a potent and selective ALK Inhibitor with IC50 of 3 nM, 100-fold more sensitive for ALK than InsR.
TAE684 does not exhibit significant cross-reactivity against other
kinases. TAE684 potently inhibits the proliferation of Ba/F3 NPM-ALK
cells with IC50 of 3 nM, without affecting the survival of Ba/F3 cells
even at 1 μM. TAE684 also inhibits proliferation of NPM-ALK-expressing
human ALCL cell lines including Karpas-299 and SU-DHL-1 with IC50 of 2–5
nM. Molecular modeling reveals that L258 may be one of the major
kinase-selectivity determinants for TAE684.
TAE684 treatment results in a
rapid and sustained inhibition of phosphorylation of NPM-ALK. TAE684
induces Apoptosis and G1 phase arrest in NPM-ALK-expressing Ba/F3 cells
and ALCL patient cell lines. TAE684 markedly overcomes
Crizotinib-resistance in H3122 CR cells, harboring the fusion oncogene
EML4-ALK, decreasing cell growth, suppressing ALK phosphorylation and
inducing apoptosis. Neurite outgrowth induced by expression of the mALK R1279Q mutant could be completely inhibited by TAE684 at 30 nM.
After 4 weeks of treatment with TAE684 at 3 and 10 mg/kg, there is a
significant delay in lymphoma development and 100- to 1,000-fold
reduction in luminescence signal, without any signs of compound- or
disease-related toxicity in Karpas-299 lymphoma model. TAE684 treatment
also induces disease regression in established Karpas-299 lymphomas and
down-regulates CD30 expression.
TAE684 also shows impressive antitumor activity against H3122 CR xenograft tumors. Furthermore, treatment with TAE684 improves the rough eye phenotype of
both ALK mutants, especially that seen with ALKR1275Q, whereas
Crizotinib has little effect on either phenotype.
In vitro Enzyme Assays
All in vitro enzyme assays are done at Upstate Biotechnology with the
exception of InsR and IGF-1R. To determine the IC50 of TAE684 against
InsR and IGF-1R a homogeneous time-resolved fluorescence assay is
performed. ATP (10 mM) and 20 mg/ml biotinylated PolyEY (Glu, Tyr 4:1)
are combined with 50 nL of serial dilutions of TAE684 (10-500 nM) and 4
ng of InsR enzyme in the presence of the kinase reaction buffer (20 mM
TrisCl, pH 7.5/10 mM MgCl2/3 mM MnCl2/1 mM DTT/10 mM NaVO4/0.1
mg/ml of BSA).
Assays are incubated for 1 hour at ambient temperature.
Reactions are terminated by adding 10 mL of the detection solution
containing 50 mM EDTA, 500 mM KF, 0.5 mg/ml of BSA, 5 mg/mL Eu3+
cryptate-labeled anti-phosphotyrosine antibody Mab PT66-K, and 5 mg/mL
Streptavidin-XLent. The reaction is incubated for half an hour, and
fluorescence signals are read on Analyst GT.
Cells are seeded in 384-well plates (2.5×104 cells per well)
and incubated with serial dilutions of TAE684 or DMSO for 2–3 days.
Luciferase expression is used as a measure of cell
proliferation/survival and is evaluated with the Bright-Glo Luciferase
Assay System. IC50 values are generated by using XLFit software.
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