GNF-2 is a highly selective non-ATP competitive inhibitor of Bcr-Abl.

BCR-ABL (K562 cell lines):273 nM, BCR-ABL (SUP-B15 cell lines):268 nM

1 μM GNF-2诱导 Ba/F3.p210 细胞和Ba/F3.p210E255V 细胞的凋亡。GNF-2以剂量依赖性方式抑制Bcr-abl的细胞酪氨酸磷酸化,IC5??0为267 nM。1 μM GNF-2诱导Ba/F3.p210细胞中磷酸化Stat5水平的显著降低。10 μM GNF-2需要BCR和/或 c-Abl SH3 和/或 SH2 域抑制BCR-Abl依赖性细胞增殖。10 μM GNF-2以剂量依赖性方式明显抑制CrkII的酪氨酸磷酸化。GNF-2作用于表达 c-AblG2A 的细胞,抑制CrkII 磷酸化,IC50为 0.051 μM。GNF-2抑制表达p210Bcr-Abl和p210Bcr-Abl突变体的BafF3细胞的自体磷酸化和增殖。与GNF-5（20 nM）组合的GNF-2（8 nM）在抑制Abl64-515激酶活性方面产生累加效应。GNF-2抑制Bcr-abl阳性细胞生长,IC50值为273 nM (K562) 和 268 nM (SUP-B15),这种作用具有剂量依赖性。GNF-2抑制Ba/F3.p210E255V Ba/F3.p185Y253H细胞生长,IC50分别为268 nM和194 nM。

Binding assay: Recombinant proteins (100 nM for each construct) or immunoprecipitated proteins are diluted in kinase buffer (20 mM HEPES (pH 7.4), 50 mM KCl, 0.1% CHAPS, 30 mM MgCl2, 2 mM MnCl2, 1 mM DTT, and 1% glycerol). Aliquots of the diluted proteins are preincubated with either DMSO or compounds for 30 min at room temperature and then added to K-LISA PTK EAY reaction plates. The kinase reaction is initiated by adding 0.1 mM ATP and is allowed to proceed for 30 min at room temperature. The phosphorylation of GST-Abltide is monitored by SDS-PAGE and phosphorimaging analysis or autoradiography.

Cells (0.3-0.6 × 106 per mL) are plated in duplicate or triplicate in 96-well plates containing increasing GNF-2 concentrations (5 nM–10 μM). After incubation at 37 ℃ in 5% CO2 for 48 hours, the effect of GNF-2 on cell viability is determined by the MTT colorimetric dye reduction method. Inhibition of cell proliferation is calculated as a percentage of growth of DMSO-treated cells, and IC50 values are determined with Microsoft Excel XLfit3.(Only for Reference)