Kinase assays

The assay buffer contained 50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 0.1 mg/ml BSA, 5mM DTT, pH 7.8. For HTS 0.8 μL of 5 mM of the test compounds dissolved in DMSO were dotted on 384-well plates. To measure IC50s the compound plate was prepared by 3-fold and 11-point serial dilutions. 0.8 μL of the compound in DMSO was transferred from the compound plate to the assay plate. Solutions of 8 nM unphosphorylated c-Met or 0.5 nM phosphorylated c-Met were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide dissolved in DMSO was diluted to 1 μM in assay buffer containing 400 μM ATP (unphosphorylated c-Met) or 160 uM ATP (phosphorylated c-Met). A 20 μL volume of enzyme solution was added to the appropriate wells in each plate and then 20 μL/well of substrate solution to initiate the reaction. The plate was protected from light and incubated at 25 °C for 90 min. The reaction was stopped by adding 20 μL of a solution containing 45 mM EDTA, 50 mM Tris-HCl, 50 mM NaCl, 0.4 mg/ml BSA, 200 nM SA-APC and 3 nM EU-Py20. The plate was incubated for 15-30 min at room temperature and HTRF (homogenous time resolved fluorescence) was measured on a Perkin Elmer Fusion α-FP instrument.

Cell lines

Human cancer cell lines (SNU-5, SNU-1, U-87MG, 786-O, A549, H441, H596, H1437, H1993, BT474, A549, and HT-29), MKN-45 cell line, S114 cell line, Blood samples obtained from healthy volunteers and cancer patients

Preparation method

Limited solubility in DMSO. 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

IC50: 0.13 nM (c-MET), 72-hour

Applications

INCB28060 (IC50: 1 nM) inhibited human c-MET phosphorylation and c-MET–mediated signaling in the SNU-5 human gastric cancer cell line. INCB28060 inhibited SNU-5 viability or proliferation with an average IC50 value of 1.2 nM. INCB28060 (2 nM) prevented HGF-stimulated H441 cell migration. INCB28060 exhibited strong antitumor activity in c-MET-dependent tumor models. INCB28060 exhibited picomolar enzymatic potency and was highly specific for c-MET with more than 10,000-fold selectivity over a large panel of human kinases. INCB28060 (24 hours) potently inhibited c-MET-dependent tumor cell proliferation and migration and effectively induced apoptosis.

Animal models

Female Balb/c nu/nu mice (Charles River) subcutaneously injected with S114 tumor cells or U-87MG glioblastoma tumor cells

Dosage form

0.03, 0.1, 0.3, 1, 3, or 10 mg/kg

Application

Oral dosing of INCB28060 resulted in time- and dose-dependent inhibition of c-MET phosphorylation and tumor growth in c-MET-driven mouse tumor models. INCB28060 was well tolerated at doses that achieve complete tumor inhibition. Once daily dosing of 10 mg/kg INCB28060 resulted in partial regressions in 6 of 10 U-87MG tumor-bearing mice.

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]. Behshad E, Klabe R M, Margulis A, et al. Phosphorylation state-dependent high throughput screening of the c-MET kinase[J]. Current chemical genomics, 2010, 4: 27.

[2]. Liu X, Wang Q, Yang G, et al. A novel kinase inhibitor INCB28060 blocks c-MET-dependent signaling, neoplastic activities, and crosstalk with EGFR and HER-3[J]. Clinical Cancer Research, 2011: clincanres. 1157.2011.