AZ 960 905586-69-8

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Biological Activity


AZ960 also inhibits many kinases less than 50% at a concentration of 0.1
μM such as JAK3 (IC50=9 nM), TrkA, Aurora, and ARK5. In cells, AZ 960
inhibits the phosphorylation of STAT5 in TEL-JAK2 cells with an average
IC50 of 15 nM and exhibits 15-30 -fold sensitivity for TEL-JAK2-driven
STAT5 phosphorylation compared with cell lines driven by other JAK
kinase family members (TEL-JAK1, -JAK3, and -TYK2).


AZ 960 shows potent
activity in inhibiting the proliferation of the TEL-JAK2, -JAK1, -JAK3,
and -Tyk2 cell lines with GI50 values of 25 nM, 230 nM, 279 nM, and 214
nM, respectively. Moreover, AZ 960 also potently inhibits SET-2 cell
proliferation with an average GI50 of 33 nM by reducing both STAT3 and
STAT5 phosphorylation levels.


AZ 960 causes growth arrest
and Apoptosis of human T-cell lymphotropic virus type 1, HTLV-1pe 1,
HTLV-1LV-1osis of human T-cSET-2 cell proliferation witBcl-xL by small
interfering RNA potentiates anti-proliferative effects of AZ 960 in MT-1
cells. A recent study shows that AZ 960 leads to
significant inhibition of the clonogenic growth and induction of
apoptosis of freshly isolated AML cells from patients.






Enzyme Biochemical Assay and Kinase Profiling


Inhibition studies of AZ 960 are performed using a recombinant JAK2
kinase (Amino Acids 808–1132) at a peptide (Tyk2 peptide) concentration
of 100 nM and an ATP concentration of 15 μM. Concentrations of AZ 960
ranging from 0.003 μM to 30 μM are used. The mode of inhibition and
inhibition constant (Ki) of AZ960 against JAK2 kinase are
further evaluated by inhibition kinetics.


Specifically, a series of
JAK2-catalyzed reactions are set up in HEPES buffer (75 mM, pH 7.3) with
a fixed concentration of peptide (FL-Ahx-IPTSPITTTYFFFKKK-COOH), and
varied concentrations of ATP and AZ 960. The progress of each reaction
is subsequently monitored by the Caliper LC3000 system, and the initial
velocity of each reaction is extracted from the corresponding reaction
time course.


To define the mode of inhibition, initial velocities are
plotted against corresponding ATP concentrations using Lineweaver-Burk
plots and the characteristic convergence of the lines on the y axis
demonstrates the competitiveness of AZ 960 to ATP. Initial inspection of
Ki using the Michealis-Menten equation revealed that AZ960
is a tight-binding inhibitor of JAK2. AZ960 is profiled against 83
kinases at three inhibitor concentrations (0.01 μM, 0.10 μM, and 1.0
μM).

Method

Cellular proliferation is evaluated using the fluorometric/colorimetric
BIOSOURCE AlamarBlue Assay and read in the Spectra Max Gemini EM
microplate reader. SET-2 cells are plated at 20,000 cells/well, TEL-JAK2
Ba/F3 cells at 2000 cells/well, and all Other TEL-JAKs at 5000
cells/well in 96-well plates. Cells are treated with AZ 960 24 hours
after plating and grown for 72 hours for SET-2 and 48 hours for TEL-JAK
Ba/F3 cells.


Following the indicated growth period Alamar Blue (10
μL/well) is added, cells are incubated at 37 °C in 5% CO2 for
2 hours, and fluorescence is measured at 545 (excitation) and 600 nm
(emission). Data are normalized to percent of the control, and GI50
values (the concentration that causes 50% growth inhibition) are
calculated using Xlfit4 version 4.2.2 for Microsoft Excel.

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