AG490 (Tyrphostin B42) is an inhibitor of EGFR (IC50: 0.1 μM). It is 135-fold more selective for EGFR than ErbB2, also inhibits JAK2 with no effect to Lyn, Lck, Syk, Btk, and Src.

EGFR:0.1 μM

In vivo, treatment with AG-490 facilitates apoptosis in mouse myeloma cells without impairing the activation of macrophages or the production of IFN-γ induced by IL-12. When administered at 0.5 mg/day for 10 days to nude mice, AG-490 significantly impedes tumor formation and invasion driven by the JAK2 V617F mutation. Additionally, AG-490 markedly reduces the numbers of CD45+ and HLA-DR+ cells; in the bone marrow, it diminishes their levels from 48% and 46% to undetectable, respectively, and in untreated mouse spleens, from 38% and 22% to undetectable levels.

AG-490 induces programmed cell death, almost completely inhibiting the growth of all ALL cells at 5 μM, without affecting normal hematopoietic functions. At 30 μM, it inhibits phosphorylation in both Epo-induced wild-type JAK2 and the constitutively active JAK2 V617F mutant. AG-490 at 50 μM induces apoptosis in Imatinib-resistant BaF3 cells expressing Bcr-Abl mutations E255K and T315I. Additionally, at 60-100 μM, it suppresses constitutive activation of Stat3sm and inhibits spontaneous (IC50: 75 μM) or IL-2 induced (IC50: 20 μM) mycelial tumor cell growth. At 100 μM, AG-490 inhibits Akt phosphorylation and NF-κB activation, activates GSK-3β, and results in a reduction of c-Myc. It also inhibits the proliferation of EGF-dependent HER 14 cells (IC50: 3.5 μM) by blocking JAK3 and STAT5a/b activities, effectively suppressing IL-2 regulated human T cell growth (IC50: 25 μM). While AG-490 does not affect FDrv210H cell proliferation alone at 5 μM, it enhances the inhibitory effect of STI571 on p210bcr-abl, thus promoting antiproliferative activity.

In vitro kinase autophosphorylation: AG-490 is dissolved in DMSO 10%-Water-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, 50 mM, 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 AG-490 (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 AG-490 (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.

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. (Only for Reference)