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-autofosforylering

  AG-490 wordt opgelost in DMSO 10%-H₂O-ethanol 45%. Ruwe membraanextracten (0,125 μg/mL) worden vooractief gemaakt met EGF (20 nM) in 50 mM HEPES-buffer, pH 7,6, en 125 mM NaCl, gedurende 15 minuten bij 4 °C. Autofosforyleringsactiviteit van EGFR of ErbB2 kinase wordt gedurende 30 seconden bij 4 °C bepaald in V-vormige 96-well platen. Membraanextracten (8 μL) worden toegevoegd aan elk putje met reactiemengsel (12 μL, 50 mM, HEPES, pH 7,4, 125 mM NaCl, 12 mM M₈Ac₂, 2 mM MnCl₂, 1 mM NaVO₃, 1 μM ATP, en 1 μCi[γ-³²P]ATP, eindconcentraties) en toenemende concentraties van deze verbinding (4 μL). Na beëindiging door toevoeging van hete monsterbuffer, worden de monsters gescheiden op een 6% SDS-polyacrylamide gelelektroforese minigel, de gels gedroogd en autoradiografie uitgevoerd gedurende de lineaire belichtingstijd. De receptorbanden worden densitometrisch gescand en de resultaten geanalyseerd met het Ez-Fit programma. Voor de analyse van autofosforylering van JAK2, wordt JAK2 geïmmunoprecipiteerd met behulp van anti-JAK2 antilichaam uit lysaten van G2-cellen die gedurende 16 uur zijn voorbehandeld met toenemende concentraties van deze chemische stof (0-50 μM). Immuuncomplexen worden vervolgens geïmmunoblot met anti-fosfotyrosine antilichaam. Een dosisafhankelijke remming van in vitro kinase-activiteit wordt aangetoond door JAK2 autofosforylering te beoordelen.

• Cellijnen

  Pre-B ALL

• Concentraties

  Dissolved in DMSO, final concentrations ~ 50 μM

• Incubatietijd

  16 hours

• Methode

  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.

• Dierlijke modellen

  SCID mice intravenously injected with ALL cells

• Doseringen

  0.85 mg + 0.5 mg daily

• Toediening

  Continuous pump infusion supplemented with daily intraperitoneal injections