| Protocol | In Vitro | In vitro activity: GNF-5, when used in combination with imatinib or nilotinib, suppresses the emergence of resistance mutations in vitro, and displays additive inhibitory activity in biochemical and cellular assays against Bcr-Abl T315I mutant. GNF-5 shows potent antiproliferative activity with EC50 of 430 nM and 580 nM against wt-Bcr-Abl and E255K mutant Bcr-Abl transformed cells, respectively. Kinase Assay: The ATP/NADH-coupled assay system in a 96-well format is used to determine the initial velocity of Abl tyrosine kinase catalyzed peptide phosphorylation. The reaction mixture contained 20 mM Tris-HCl, (pH 8.0), 50 mM NaCl, 10 mM MgCl2, 2 mM PEP [2-(Phosphonooxy)- 2-propenoic acid) and 20 μg Abl peptide substrate (EAIYAAPFAKKK), fixed or varied (to determine inhibitor kinetic parameters) concentration of inhibitor applied, 1/50 of the final reaction mixture volume of PK/LDH enzyme (pyruvate kinase/lactic dehydrogenase enzymes from rabbit muscle), 160 μM NADH, 0.16 μM Abl, and ATP added last to start the reaction. Absorbance data are collected every 20s at 340 nm using a SpectraMax M5 Microplate Reader. Cell Assay: Ba/F3.p210 cells are obtained by transfecting the IL-3-dependent murine hematopoietic Ba/F3 cell line with a pEYK vector containing p210BCR-ABL and Bcr-Abl mutations. All cell lines are cultured with 5% CO2 at 37 °C in RPMI 1640 with 10% fetal bovine serum (FBS) and supplemented with 1% l-glutamine. Parental Ba/F3 cells are similarly cultured with 10% WEHI-conditioned medium as a source of IL-3. Transfected cell lines are cultured in media supplemented with 25 μg/mL zeocin. The 48 h cell proliferation studies are obtained using the CellTiter-Glo assay. |
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| In Vivo | GNF-5 (100 mg/kg ) displays efficacy on wild-type and T315I Bcr-Abl dependent proliferation in xenograft and bone marrow transplantation models. Moreover, a combination of GNF-5 (75 mg/kg) with nilotinib (50 mg/kg) results in improved overall survival in a T315I Bcr-Abl BMT model. |
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| Animal model | Ba/F3.p210 xenograft mouse model |
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| Formulation | Dissolved in PEG400/saline (1:1) |
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| Dosages | 100 mg/kg ; p.o. |
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These protocols are for reference only. InvivoChem does not
independently validate these methods.
Preparing Stock Solutions
| Solvent volume to be added |
Mass (the weight of a compound) |
| Mother liquor concentration |
1mg | 5mg | 10mg | 20mg |
| 1mM | 2.3902 mL | 11.9511 mL | 23.9023 mL | 47.8046 mL |
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| 5mM | 0.4780 mL | 2.3902 mL | 4.7805 mL | 9.5609 mL |
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| 10mM | 0.2390 mL | 1.1951 mL | 2.3902 mL | 4.7805 mL |
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| 20mM | 0.1195 mL | 0.5976 mL | 1.1951 mL | 2.3902 mL |
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Quality Control Documentation
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