Binding assays

Kinases were activated by preincubating the enzyme (2.7 μmol/L final concentration) in 50 mmol/L HEPES (pH 7.5), 10 mmol/L MgCl2, 0.1 mg/mL bovine serum albumin, and 2 mmol/L ATP. Compounds were diluted in DMSO and dispensed into the assay plates (100 nL/well). Kinase reactions contained (in 10 μL volume) 50 mmol/L HEPES (pH 7.5), 3 mmol/L DTT, 0.1 mg/mL bovine serum albumin, 1 mmol/L CHAPS, 10 mmol/L MgCl2, 10 μmol/L ATP, 500 nmol/L substrate peptide (biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2; QPC), and 0.5 nmol/L activated enzyme. Reactions were stopped after 1 h at room temperature with 33 μmol/L EDTA. Peptide phosphorylation was measured by time-resolved fluorescence resonance energy transfer with 7 nmol/L streptavidin-Surelight allophycocyanin and 1 nmol/L europium-conjugated phosphotyrosine antibodies. Plates were read in a Victor X5 or ViewLux 1430 ultra HTS microplate imager.

Cell lines

NIH-3T3 cells that overexpress human IGF-IR [NIH-3T3-hIGF-IR (NIH-3T3/LISN)] and IR (NIH-3T3-hIR)

Preparation method

The solubility of this compound in DMSO is >42.6mg/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.

Reacting condition

72 h

Applications

In the NIH-3T3/LISN and NIH-3T3-hIR cells, GSK1904529A potently inhibited phosphorylation of IGF-IR and IR with the IC50 of 22 ± 8 nmol/L and 19 ± 8 nmol/L, respectively. GSK1904529A suppressed cell proliferation in a variety of tumor cells with the IC50 values of 81, 35, 43, 124, 137 and 68 nM for NCI-H929, TC-71, SK-N-MC, COLO 205, MCF7 and PREC, respectively. In COLO 205, MCF-7, and NCI-H929 cells, GSK1904529A resulted in cell accumulation in G1 and decrease in S and G2-M phases.

Animal models

Female athymic nu/nu CD-1 mice bearing NIH-3T3/LISN, COLO 205, HT29, and BxPC3 cells

Dosage form

Oral administration, 30 mg/kg, once or twice daily for 21 d

Application

In NIH-3T3/LISN tumor-bearing mice, GSK1904529A (oral, 30 mg/kg, twice-daily) inhibited 98% tumor growth. In COLO 205 xenografts mice, GSK1904529A (oral, 30 mg/kg, once daily) inhibited 75% tumor growth. Among HT29 and BxPC3 xenografts, GSK1904529A produced moderate tumor growth inhibition with no side effects at a dose of 30 mg/kg.

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.

GSK1904529A is a small-molecule inhibitor of the insulin-like growth factor-I receptor (IGF-IR) with IC50 value of 27 nM¹.

GSK1904529A is a reversible and ATP-competitive inhibitor with Ki value of 1.6 nM. In NIH-3T3/LISN cells, GSK1904529A potently inhibited phosphorylation of IGF-IR with IC50 value of 22 nM. It also demonstrated to be a selective inhibitor since it showed poor inhibitory activity against 45 other serine/threonine and tyrosine kinases. When treated with whole-cell extracts, GSK1904529A significantly inhibited the ligand-induced phosphorylation of IGF-IR and decreased phosphorylation of downstream signaling including AKT, IRS-1 and ERK at concentrations >0.01μM. GSK1904529A suppressed cell proliferation in a variety of tumor cells. The IC50 values for NCI-H929, TC-71, SK-N-MC, COLO 205, MCF7 and PREC are 81, 35, 43, 124, 137 and 68 nM, respectively. In COLO 205, MCF-7, and NCI-H929 cells, GSK1904529A treatment resulted in cell accumulation in G1 and decrease in S and G2-M phases. Moreover, in NIH-3T3/LISN xenograft model, once daily administration of GSK1904529A at 30 mg/kg inhibited 56% of tumor growth¹.

References:
1. Sabbatini P, Rowand J L, Groy A, et al. Antitumor activity of GSK1904529A, a small-molecule inhibitor of the insulin-like growth factor-I receptor tyrosine kinase. Clinical Cancer Research, 2009, 15(9): 3058-3067.