AZ 960

Biological Activity

AZ960 is a novel and specific inhibitor of the JAK2 kinase with a Ki of 0.45nM in vitro.  AZ 960 effectively induced growth arrest and apoptosis of human T-cell lymphotropic virus type 1, HTLV-1-infected T cells (MT-1 and MT-2) in parallel with downregulation of the phosphorylated forms of Jak2 and Bcl-2 family proteins including Bcl-2 and Mcl-1.  Importantly, genetic inhibition of Bcl-xL by a small interfering RNA potentiated antiproliferative effects of AZ960 in MT-1 cells. Taken together, Jak2 is an attractive molecular target for treatment of ATL.

Product Citations

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  Bioorg Med Chem. 2016 Oct 1;24(19):4647-51.

  Discovery and antiparasitic activity of AZ960 as a Trypanosoma brucei ERK8 inhibitor
  AZ 960 purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Bioorganic & Medicinal Chemistry (2016) . Figure 6. AZ960 was purchased from Abmole (Catalog No. M1660; Houston, TX)
	Method	Kinase assays
	Cell Lines	T. brucei, primary mouse left ventricle smooth muscle cells (vSMC), and rat pancreatic b cells (ins-1)
	Concentrations	0.5 and 1 µM
	Incubation Time	30 min
	Results	As shown in Figure 6, treatment of these cells with inhibitor concentrations ranging from 100 nM to 2 mM resulted in dosedependent inhibition of cell growth. The IC50 of AZ960 for mouse ventricular smooth muscle cells was determined to be 25 lM ± 3 and 10 lM ± 1 for rat pancreatic b cells.

	Source	Bioorganic & Medicinal Chemistry (2016) . Figure 5. AZ960 was purchased from Abmole (Catalog No. M1660; Houston, TX)
	Method	Kinase assays
	Cell Lines
	Concentrations	0.5 and 1 µM
	Incubation Time	30 min
	Results	We calculated the Michaelis–Menten constant (Km) for TbERK8 to be 3.4 lM ± 0.12 (Fig. 5A). The inhibition constant (Ki) of AZ960 for TbERK8 was calculated to be 1.25 lM ± 0.35 (Fig. 5B).

	Source	Bioorganic & Medicinal Chemistry (2016) . Figure 4. AZ960 was purchased from Abmole (Catalog No. M1660; Houston, TX)
	Method	Kinase assays
	Cell Lines
	Concentrations	1 µM
	Incubation Time	30 min
	Results	As shown in Figure 4, compounds that inhibited TbERK8 kinase activity to 20% or less included AZD5438, PF-03814735, Milciclib, and AZ960. Our studies suggest that SNS-032, NVP-BGT226, GSK2126458, Dinaciclib, and Torin 2 have anti-T. brucei activities with mechanisms that likely do not involve inhibition of TbERK8.

	Source	Bioorganic & Medicinal Chemistry (2016) . Figure 3. AZ960 was purchased from Abmole (Catalog No. M1660; Houston, TX)
	Method	Cell Titer-Glo Promega assay
	Cell Lines	T. brucei strain WT-221
	Concentrations	80 nM to 1 µM
	Incubation Time	48 h
	Results	The inhibitor activities ranged from 80 nM to 1 µM. Dinaciclib was the most potent inhibitor with an IC50 of 80 nM, but SNS-032, GSK2126458, and AZ960 were almost equally as potent with IC50 values of 120 nM, 111 nM, 120 nM, respectively. A representative dose–response curve for AZ960 is shown in Figure 3.

	Source	Bioorganic & Medicinal Chemistry (2016) . Figure 2. AZ960 was purchased from Abmole (Catalog No. M1660; Houston, TX)
	Method	luciferase-based phenotypic screen
	Cell Lines	T. brucei strain WT-221
	Concentrations	1 µM
	Incubation Time	48 h
	Results	A rescreen that tested the select hits demonstrated a dramatic decrease in the mean RLU values by about 2 log10 orders, confirming the antiparasitic activity of the compounds (Fig. 2).

Protocol

Cell Experiment
Cell lines	SET-2 and TEL-JAK2 Ba/F3 cells
Preparation method	Proliferation Assay Cellular proliferation was evaluated using the fluorometric/colorimetric BIOSOURCE AlamarBlue Assay (Invitrogen) and read in the Spectra Max Gemini EM microplate reader (Molecular Devices, Sunnyvale, CA). SET-2 cells were 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 were treated with compound 24 h after plating and grown for 72 h for SET-2 and 48 h for TEL-JAK Ba/F3 cells. Following the indicated growth period Alamar Blue (10 μl/well) was added, cells were incubated at 37 °C in 5% CO2 for 2 h, and fluorescence was 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) were calculated using Xlfit4 version 4.2.2 for Microsoft Excel.
Concentrations	0~100 μM
Incubation time	72 h for SET-2 and 48 h for TEL-JAK Ba/F3 cells

Animal Experiment
Animal models
Formulation
Dosages
Administration

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

Chemical Information

References

[1] Ikezoe et al. Int J Cancer. Expression of p-JAK2 predicts clinical outcome and is a potential molecular target of acute myelogenous leukemia.

[2] Yang et al. Mol Cancer Ther. AZ960, a novel Jak2 inhibitor, induces growth arrest and apoptosis in adult T-cell leukemia cells.

[3] Gozgit JM, et al. J Biol Chem. Effects of the JAK2 inhibitor, AZ960, on Pim/BAD/BCL-xL survival signaling in the human JAK2 V617F cell line SET-2.