GNF-2

Catalog No.S2899 Batch:S289901

Technical Data

Formula	C₁₈H₁₃F₃N₄O₂
Molecular Weight	374.32	CAS No.	778270-11-4
Solubility (25°C)*	In vitro	DMSO	75 mg/mL (200.36 mM)
		Water	Insoluble
		Ethanol	Insoluble
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description

GNF-2 is a highly selective non-ATP competitive inhibitor of Bcr-Abl, shows no activity to Flt3-ITD, Tel-PDGFR, TPR-MET and Tel-JAK1 transformed tumor cells.

Targets

Bcr-Abl (SUP-B15 cell line) ^[1]	Bcr-Abl (K562 cell line) ^[1]
268 nM	273 nM

In vitro

GNF-2 causes a dose-dependent growth inhibition of the Bcr-abl–positive cell lines with IC50 values of 273 nM (K562) and 268 nM (SUP-B15). GNF-2 inhibits the growth of Ba/F3.p210^E255V and Ba/F3.p185^Y253H cells with IC50 values of 268 nM and 194 nM respectively. GNF-2 (1 μM) induces apoptosis of Ba/F3.p210 cells as well as Ba/F3.p210^E255V cells. GNF-2 inhibits the cellular tyrosine phosphorylation of Bcr-abl in a dose-dependent manner with IC50 of 267 nM. GNF-2 (1 μM) induces a significant decrease in the levels of phospho-Stat5 in Ba/F3.p210 cells. GNF-2 binds to the myristic binding pocket of Bcr-abl. ^[1] GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. GNF-2 (10 μM) requires BCR and/or the c-Abl SH3 and/or SH2 domains to inhibit BCR-Abl-dependent cell proliferation. GNF-2, but not a methylated GNF-2 analog, binds c-Abl in cellular extracts derived from 3T3 fibroblasts. GNF-2 (10 μM), in a dose-dependent manner, clearly inhibits tyrosine phosphorylation of CrkII. GNF-2 inhibits the phosphorylation of CrkII in c-AblG2A-expressing cells with IC50 of 0.051 μM. ^[2] GNF-2 binds in an extended conformation in the myristate pocket with the CF3-group buried at the same depth as the final two carbons of the myristate ligand. GNF-2 (10 µM) combined with imatinib (1 µM) reduces the number of resistant clones to 1 µM imatinib by at least 90%. ^[3] GNF-2 inhibits the auto-phosphorylation and proliferation of BafF3 cells expressing p210Bcr–Abl and p210Bcr–Abl mutants. GNF-2 (8 nM) in combination with GNF-5 (20 nM) results in additive effects with respect to inhibition of the Abl64–515 kinase activity. ^[4]

Protocol (from reference)

Kinase Assay:^{^[2]}	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.
Cell Assay:^{^[1]}	Cell lines Ba/F3.p210, Ba/F3.p210^E255V and Ba/F3.p185^Y253H cells Concentrations ~10 μM Incubation Time 48 hours Method 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.

Kinase Assay:^{^[2]}

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.

Cell Assay:^{^[1]}

Cell lines
Ba/F3.p210, Ba/F3.p210^E255V and Ba/F3.p185^Y253H cells
Concentrations
~10 μM
Incubation Time
48 hours
Method
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.

References

[4] Fabbro D, et al. Biochim Biophys Acta, 2010, 1804(3), 454-462.

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Customer Product Validation

: Data from [Data independently produced by , , Oncotarget, 2017, 8(24):38717-38730]

Selleck's GNF-2 has been cited by 9 publications

A multiplexed siRNA screen identifies key kinase signaling networks of brain glia [ Life Sci Alliance, 2023, 6(5)e202201605]	PubMed: 36878638
Imatinib protects against human beta-cell death via inhibition of mitochondrial respiration and activation of AMPK [ Clin Sci (Lond), 2021, 135(19):2243-2263]	PubMed: 34569605
Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement [Vasta JD Cell Chem Biol, 2018, 25(2):206-214]	PubMed: 29174542
Abl and Arg mediate cysteine cathepsin secretion to facilitate melanoma invasion and metastasis [ Sci Signal, 2018, 11(518)eaao0422]	PubMed: 29463776
Abl and Arg mediate cysteine cathepsin secretion to facilitate melanoma invasion and metastasis [ Sci Signal, 2018, 11(518)eaao0422]	PubMed: 29463776
Coronavirus S protein-induced fusion is blocked prior to hemifusion by Abl kinase inhibitors. [ J Gen Virol, 2018, 99(5):619-630]	PubMed: 29557770
Comparative Characterization of Osteoclasts Derived From Murine Bone Marrow Macrophages and RAW 264.7 Cells Using Quantitative Proteomics. [ JBMR Plus, 2018, 2(6):328-340]	PubMed: 30460336
Abl kinase regulation by BRAF/ERK and cooperation with Akt in melanoma. [ Oncogene, 2017, 36(32):4585-4596]	PubMed: 28368422
Contributions of MET activation to BCR-ABL1 tyrosine kinase inhibitor resistance in chronic myeloid leukemia cells. [Tsubaki M, et al. Oncotarget, 2017, 8(24):38717-38730]	PubMed: 28418880

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SHIPPING AND STORAGE
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