Molecule

Description

Cancer

Description

TAE684 is a potent and selective ALK inhibitor with IC50 of 3 nM.

Targets

IC₅₀

In Vitro

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]

In Vivo

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]

Clinical Trials

Features

In vitro Enzyme Assays.

All in vitro enzyme assays are done at Upstate Biotechnology with the exception of InsR and IGF1R. To determine the IC50 of TAE684 against InsR and IGF1R 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×HCl, 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

Methods

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.

Formulation

Resuspended in 10% 1-methyl-2-pyrrolidinone/90% polyethylene glycol 300 solution

Doses

1, 3, and 10 mg/kg

Administration

Once daily by oral gavage for 3 weeks