GSK1904529A (GSK 4529) is a specific inhibitor of IGF-1R (IC50=27 nM) and IR(IC50=25 nM) .

IGF-1R:27 nM, Insulin receptor:25 nM

GSK1904529A是一种可逆的、与ATP竞争的抑制剂，对IGF-1R和IR有很强的酶-抑制剂结合活性，其Ki值分别为1.6 nM和1.3 nM。在0.01 μM以上浓度时，能强效抑制IGF-1R和IR的配体诱导的磷酸化，进而阻断下游信号传导（AKT, IRS-1, 和 ERK）。GSK1904529A对NIH-3T3/LISN、TC-71、SK-N-MC、SK-ES RD-ES细胞具有强效抑制作用，其IC50值分别为60 nM、35 nM、43 nM、61 nM和62 nM。此外，GSK1904529A还能抑制其他多种骨髓瘤和尤因肉瘤细胞系，包括NCI-H929、MOLP-8、LP-1和KMS-12-BM等。GSK1904529A能在对GK1904529A敏感的细胞系COLO 205、MCF-7和NCI-H929中诱导细胞周期在G1期阻滞。[1]

GSK1904529A在以30 mg/kg剂量（口服，每日两次）治疗的NIH-3T3/LISN肿瘤携带的小鼠中显示出98%的肿瘤生长抑制效果，在COLO 205异种移植小鼠中（每日一次）显示出75%的效果。在HT29和BxPC3异种移植小鼠中，以30 mg/kg剂量的GSK1904529A产生了中等程度的肿瘤生长抑制效果，且没有副作用。同时，GSK1904529A对血糖水平的影响最小。血中约3.5 μM浓度的GSK1904529A能完全抑制IGF-1R的磷酸化。GSK1904529A已被暗示用于治疗包括前列腺癌、结肠癌、乳腺癌、胰腺癌、卵巢癌和肉瘤在内的多种IGF-1R依赖型肿瘤。[1]

Kinase assays: GSK1904529A is dissolved in DMSO as stock solution at 10 mM. Baculovirus-expressed glutathione S-transferase-tagged proteins encoding the intracellular domain of IGF-1R (amino acids 957-1367) and IR (amino acids 979-1382) are used for determination of IC50. Kinases are activated by preincubating the enzyme (2.7 μM final concentration) in 50 mM HEPES (pH 7.5), 10 mM MgCl2, 0.1 mg/mL bovine serum albumin, and 2 mM ATP. GSK1904529A is diluted in DMSO and dispensed into the assay plates (100 nL/well). Kinase reactions contained (in 10 μL) 50 mM HEPES (pH 7.5), 3 mM DTT, 0.1 mg/mL bovine serum albumin, 1 mM CHAPS, 10 mM MgCl2, 10 μM ATP, 500 nM substrate peptide (biotin-aminohexylAEEEEYMMMMAKKKK-NH2; QPC), and 0.5 nM activated enzyme. Reactions are stopped after 1 hour at room temperature with 33 μM EDTA. Peptide phosphorylation is measured by time-resolved fluorescence resonance energy transfer with 7 nM streptavidin Surelight allophycocyanin and 1 nM europium-conjugated phosphotyrosine antibodies. Plates are read in a multilabel reader.

Cells are seeded in 96-well plates and incubated overnight at 37 °C, and treated with various concentrations of GSK1904529A for 72 hours. For the NIH-3T3/LISN, cells are seeded on collagen-coated 96-well tissue culture plates and allowed to adhere for 24 hours. The tissue culture medium is replaced with serum-free medium and the cells are treated with DMSO or GSK1904529A for 2 hours. IGF-I (30 ng/mL) is added and the cells are incubated at 37 °C for 72 hours. Cell proliferation is quantified using the CellTiter-Glo Luminescent Cell Viability Assay. IC50 values are determined.