Inhibitory activities

IC50 values for the inhibition of RTKs by CHIR-258 were determined in a time-resolved fluorescence (TRF) or radioactive format, measuring the inhibition by CHIR-258 of phosphate transfer to a substrate by the respective enzyme. The kinase domains of FGFR3, FGFR1, PDGFR-β and VEGFR1-3 were assayed in 50 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid), pH 7.0, 2 mM MgCl2, 10 mM MnCl2, 1 mM NaF, 1 mM dithiothreitol (DTT), 1 mg/mL bovine serum albumin (BSA), 0.25 μM biotinylated peptide substrate (GGGGQDGKDYIVLPI), and 1 to 30 μM adenosine triphosphate (ATP) depending on the Km for the respective enzyme. ATP concentrations were at or just below Km. For c-KIT and FLT3 reactions the pH was raised to 7.5 with 0.2 to 8 μM ATP in the presence of 0.25 to 1 μM biotinylated peptide substrate (GGLFDDPSYVNVQNL). Reactions were incubated at room temperature for 1 to 4 hours and the phosphorylated peptide captured on streptavidin-coated microtiter plates containing stop reaction buffer (25 mM EDTA, 50mM HEPES, pH 7.5).

Cell lines

Human multiple myeloma (MM) cell lines and B9 cells

Preparation method

The solubility of this compound in DMSO is >36.4mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

100 nM CHIR-258; 48-96 h.

Applications

Dovitinib is a receptor tyrosine kinases inhibitor. Dovitinib selectively inhibits the growth of human myeloma cell lines and B9 cells expressing wild-type (WT) or activated mutant FGFR3. Dovitinib also causes cytostatic and cytotoxic effects and inhibits downstream extracellular signal-regulated kinase (ERK) 1/2 phosphorylation.

Animal models

6- to 8-week-old female BNX mice bearing 3 ×107 KMS11 cells.

Dosage form

10, 30, or 60 mg/kg for 21 days by gavage.

Preparation method

Dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 20 mM. For animal experiments: formulated in 5 mM citrate buffer.

Applications

Dovitinib causes antitumor effect and inhibits tumor growths by 48%, 78.5%, and 94% in the 10 mg/kg, 30 mg/kg, and 60 mg/kg treatment arms, respectively. Weight loss, as a marker of significant toxicity, is not observed in any of the treatment groups. Dovitinib completely inhibits FGFR3 at the 60 mg/kg dose. CHIR-258 induces both cytostatic and cytotoxic responses.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Trudel S, Li ZH, Wei E, et al. CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for the potential treatment of t(4;14) multiple myeloma. Blood, 2005, 105(7): 2941-2948.