AG-490

AG-490 inhibits HER-2 driven cell proliferation with IC50 of 3.5 μM. [1] Corresponding to the specific dose-dependent inhibition of constitutively activated JAK2 in pre-B acute leukemia (ALL) cells, this compound (5 μM) almost completely blocks the growth of all ALL cells by inducing programmed cell death, with no deleterious effect on normal hematopoiesis. This chemical does not inhibit the activities of Lck, Lyn, Btk, Syk, and Src. [2] It (60-100 μM) blocks the constitutive activation of Stat3sm, and inhibits spontaneous as well as interleukin 2-induced growth of mycosis fungoides (MF) tumor cells with IC50 values of 75 μM and 20 μM, respectively. [3] This compound potently inhibits IL-2-mediated human T cell growth with an IC50 of 25 μM by blocking the activities of JAK3 and STAT5a/b. [4] It significantly inhibits the constitutive activation of Stat3 in MOPC, MPC11, and S194 cells, leading to dramatic dose-dependent apoptosis. [6] This chemical (100 μM) inhibits Akt phosphorylation, inhibits the activation of nuclear factor-κB, and causes the activation of GSK-3β, leading to the reduction of c-Myc. It (50 μM) can induce apoptosis of BaF3 cells expressing T315I and E255K mutants of Bcr-Abl. [7] This compound at 30 μM inhibits not only Epo-induced phosphorylation of wild-type JAK2 but also constitutive phosphorylation of the JAK2 V617F mutant. It also potently inhibits cytokine-independent cell growth induced by the JAK2 V617F mutant in BaF3 cells. [8]

Administration of AG-490 drastically reduces the numbers of CD45⁺ and HLA-DR⁺ cells from 48 % and 46% in bone marrow of untreated mice, as well as 38% and 22% in the spleen of untreated mice to undetectale levels. [2] In vivo administration of this compound causes murine myeloma tumor cell apoptosis but does not inhibit IL-12-mediated macrophage activation and IFN-γ production by lymphocytes. [6] Consistent with the in vitro blocking of JAK2 V617F mutant activity, treatment with this chemical at 0.5 mg/day for 10 days effectively inhibits JAK2 V617F mutant-induced tumorigenesis and tumor cell invasion in nude mice. [8] Combined therapy with this agent and IL-12 induces greater antitumor effects than either agent alone in a murine myeloma tumor model. [6]

• In vitro kinase autophosphorylation

  AG-490 is dissolved in DMSO 10%-H₂O-ethanol 45%. Crude membrane extracts (0.125 μg/mL) are preactivated with EGF (20 nM) in 50 mM HEPES buffer, pH 7.6, and 125 mM NaCl, for 15 minutes at 4 °C. Autophosphorylation activity of EGFR or ErbB2 kinase is assayed at 4 °C for 30 seconds in V-shaped 96-well plates. Membrane extracts (8 μL) are added to each well containing reaction mixture (12 μL, 50mM, HEPES, pH 7.4,125 mM NaCl, 12 mM M₈Ac₂, 2 mM MnCl₂, 1 mM NaVO₃, 1 μM ATP, and 1 μCi[γ-³²P]ATP, final concentrations) and increasing concentrations of this compound (4 μL). After termination by addition of hot sample buffer, the samples are run on a 6% SDS-polyacrylamide gel electrophoresis minigel, the gels dried, and autoradiography performed during the linear exposure time period. The receptor bands are scanned densitrometrically, and the results analyzed by the Ez-Fit program. For the analysis of autophosphorylation of JAK2, JAK2 is immunoprecipitated by using anti-JAK2 antibody from lysates of G2 cells pretreated for 16 hours with increasing concentrations of this chemical (0-50 μM). Immune complexes are then immunoblotted with anti-phosphotyrosine antibody. A dose-dependent inhibition of in vitro kinase activity is demonstrated by assessing JAK2 autophosphorylation.

• 세포주

  Pre-B ALL

• 농도

  Dissolved in DMSO, final concentrations ~ 50 μM

• 배양 시간

  16 hours

• 방법

  Cells are exposed to different concentrations of AG-490 for 16 hours. For the determination of cell proliferation, [³H]tymidine (1 μCi) is added 6 hours or more before the cultures are terminated. Cells are then collected and samples counted in a liquid scintillation counter.

• 동물 모델

  SCID mice intravenously injected with ALL cells

• 용량

  0.85 mg + 0.5 mg daily

• 투여

  Continuous pump infusion supplemented with daily intraperitoneal injections