TAE684 (NVP-TAE684)

TAE684 does not exhibit significant cross-reactivity against other kinases. TAE684 potently inhibits the proliferation of Ba/F3 NPM-ALK cells with IC50 of 3 nM, without affecting the survival of Ba/F3 cells even at 1 μM. TAE684 also inhibits proliferation of NPM-ALK-expressing human ALCL cell lines including Karpas-299 and SU-DHL-1 with IC50 of 2–5 nM. Molecular modeling reveals that L258 may be one of the major kinase-selectivity determinants for TAE684. TAE684 treatment results in a rapid and sustained inhibition of phosphorylation of NPM-ALK. TAE684 induces apoptosis and G1 phase arrest in NPM-ALK-expressing Ba/F3 cells and ALCL patient cell lines. ^[1] TAE684 markedly overcomes Crizotinib-resistance in H3122 CR cells, harboring the fusion oncogene EML4-ALK, decreasing cell growth, suppressing ALK phosphorylation and inducing apoptosis.^[2] Neurite outgrowth induced by expression of the mALK R1279Q mutant could be completely inhibited by TAE684 at 30 nM. ^[3]

After 4 weeks of treatment with TAE684 at 3 and 10 mg/kg, there is a significant delay in lymphoma development and 100- to 1,000-fold reduction in luminescence signal, without any signs of compound- or disease-related toxicity in Karpas-299 lymphoma model. TAE684 treatment also induces disease regression in established Karpas-299 lymphomas and down-regulates CD30 expression. ^[1] TAE684 also shows impressive antitumor activity against H3122 CR xenograft tumors. ^[2] Furthermore, treatment with TAE684 improves the rough eye phenotype of both ALK mutants, especially that seen with ALKR1275Q, whereas Crizotinib has little effect on either phenotype. ^[3]

• In vitro Enzyme Assays.

  All in vitro enzyme assays are done at Upstate Biotechnology with the exception of InsR and IGF-1R. To determine the IC50 of TAE684 against InsR and IGF-1R a homogeneous time-resolved fluorescence assay is performed. ATP (10 mM) and 20 mg/ml biotinylated PolyEY (Glu, Tyr 4:1) are combined with 50 nL of serial dilutions of TAE684 (10-500 nM) and 4 ng of InsR enzyme in the presence of the kinase reaction buffer (20 mM Tris譎Cl, pH 7.5/10 mM MgCl₂/3 mM MnCl₂/1 mM DTT/10 mM NaVO₄/0.1 mg/ml of BSA). Assays are incubated for 1 hour at ambient temperature. Reactions are terminated by adding 10 mL of the detection solution containing 50 mM EDTA, 500 mM KF, 0.5 mg/ml of BSA, 5 mg/mL Eu3+ cryptate-labeled anti-phosphotyrosine antibody Mab PT66-K, and 5 mg/mL Streptavidin-XLent. The reaction is incubated for half an hour, and fluorescence signals are read on Analyst GT.

• Cell lines

  Luciferase-expressing Karpas-299, SU-DHL-1, and Ba/F3 cells and transformed Ba/F3 stably expressing NPM-ALK, Bcr-Abl, or TEL-kinase fusion constructs.

• Concentrations

  1 nM-10 μM

• Incubation Time

  2–3 days

• Method

  Cells are seeded in 384-well plates (2.5×10⁴ cells per well) and incubated with serial dilutions of TAE684 or DMSO for 2–3 days. Luciferase expression is used as a measure of cell proliferation/survival and is evaluated with the Bright-Glo Luciferase Assay System. IC50 values are generated by using XLFit software.

• Animal Models

  Karpas-299 xenografts are established in 4- to 6-week old female Fox Chase SCIDBeige mice.

• Dosages

  1, 3, and 10 mg/kg

• Administration

  Once daily by oral gavage for 3 weeks