Identification of a potent kinase inhibitor targeting EML4-ALK fusion protein in non-small cell lung cancer

Abstract

ALK-fusion proteins play a fundamental role in the development of about 5% of non-small cell lung cancers. Herein, we identified the compound 5067-0952 as a potent ALK inhibitor, which inhibited cell growth, induced apoptosis, and suppressed the phosphorylation of ALK, subsequently blocking its downstream signaling pathway.

Fig. 1. (A) The structure of 5067-0952. (B) The binding mode of 5067-0952 with ALK at the ATP-binding site. ALK is depicted as the green cartoon. 5067-0952 is depicted as the yellow sticks. Red dotted lines represent hydrogen bonds.

Fig. 2. 5067-0952 as a potent ALK inhibitor. (A) Enzymatic assay for an ALK domain was conducted using the LanthaScreen® Eu kinase binding assay. (B) Cell lines were seeded in 96-well plates and treated with various concentrations of crizotinib and 5067-0952 for 72 h. Cell survival was analyzed using the MTT assay.

Fig. 3. 5067-0952 inhibits proliferation and induces apoptosis in H2228 cells. (A) Colony formation of H2228 cells after 5067-0952 (2.5, 5, and 10 μM) treatment for 10 days; colony numbers are plotted and photographs of violet-stained colonies are shown. (B) H2228 cells were treated with the indicated drug concentrations (2.5, 5, and 10 μM) for 48 h; the apoptotic cells were measured by the Annexin V/PI staining method. **P < 0.01 vs. vehicle; ***P < 0.001 vs. vehicle.

Fig. 4. Effects of 5067-0952 on the expression and phosphorylation of ALK as well as its downstream signaling pathway in H2228. H2228 cells were treated with 5067-0952 at the indicated concentrations for 48 h, and the levels of p-ALK, ALK, p-STAT3, STAT3, p-AKT, AKT, p-ERK, and ERK were evaluated by western blot analysis of cell lysates using specific antibodies. GAPDH was used as a loading control.