An inhibitor of c-Kit
Masitinib is an inhibitor of the receptor tyrosine kinase c-Kit (IC50 = 200 nM for the recombinant human enzyme).1 It is also an inhibitor of PDGFRα, PDGFRβ, and Lyn B (IC50s = 540, 800, and 510 nM, respectively). Masitinib inhibits stem cell factor-induced proliferation of Ba/F3 cells expressing wild-type KIT (IC50 = 150 nM), as well as those expressing the KIT mutants KITV559D and KITΔ27 (IC50s = 3 and 5 nM, respectively). It reduces tumor growth in a KITΔ27-expressing Ba/F3 murine model of cancer. Masitinib is also an inhibitor of the severe acute respiratory coronavirus 2 (SARS-CoV-2) main protease (Mpro; Ki = 2.6 ?M), also known as 3C-like protease (3CLpro), and inhibits replication of SARS-CoV-2 in infected A549 cells (EC50 = 3.2 ?M).2 It decreases lung and nose viral titers, as well as reduces lung inflammation and increases survival, in ACE2-humanized mice infected with SARS-CoV-2 when administered at a dose of 25 mg/kg.
1.Dubreuil, P., Letard, S., Ciufolini, M., et al.Masitinib (AB1010), a potent and selective tyrosine kinase inhibitor targeting KITPLoS One4(9)e7258(2009) 2.Drayman, N., DeMarco, J.K., Jones, K.A., et al.Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2Science373(6557)931-936(2021)
Kinase experiment: | A 96-well microtitre plateis coated overnight with 0.25 mg/mL poly(Glu,Tyr 4:1), rinsed twice with 250 μL of washing buffer (10 mM phosphate-buffered saline [pH 7.4] and 0.05% Tween 20) and dried for 2 hours at room temperature. Assays are performed at room temperature with a final volume of 50 μL in kinase buffer (10 mM MgCl2, 1 mM MnCl2, 1 mM sodium orthovanadate, 20 mM HEPES, pH 7.8) containing ATP at a concentration of at least twice the Km for each enzyme and an appropriate amount of recombinant enzyme to ensure a linear reaction rate. Reactions are initiated upon introduction of the enzyme and terminated with the addition of one reaction volume (50 μL) of 100 mM EDTA per 5mol/Lurea mix. Plates are washed three times and incubated with 1:30,000 horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody, then washed three times and incubated with tetramethylbenzidine. The final reaction product is quantified by spectrophotometry at 450 nm. |
Cell experiment: | For the assay of Ba/F3 cell proliferation, microtitre plates are seeded with a total of 104 cells/well in 100 μL of RPMI 1640 medium with 10% foetal bovine serum at 37°C. These are supplemented, or not, with either 0.1% conditioned medium from X63-IL-3 cells or 250 ng/mL murine SCF. The murine SCF, which activates Kit, is purified from the conditioned medium of SCF-producing CHO cells. Cells are grown for 48 hours at 37°C with masitinib and then incubated with 10 μL/well of WST-1 reagent for 3 hours at 37°C. The amount of formazan dye formed is quantified by its absorbance at 450 nm using a scanning multiwell spectrophotometer. A blank well without cells is used as a background control for the spectrophotometer. |
Animal experiment: | Male Nog-SCID mice (7 weeks old) are under specific pathogen-free conditions at 20±1°C in a 12-hour light/12-hour dark cycle and ad libitum access to food and filtered water. Mia Paca-2 cells are cultured as described above. At day 0 (D0), mice are injected with 107 Mia Paca-2 cells in 200 μL PBS into the right flank. Tumours are allowed to grow for 1.5 to 4 weeks until the desired tumour size is reached (appr 200 mm3). At day 28, animals are allocated into four treatment groups (n=7 to 8 per group), ensuring that each group's mean body weight and tumour volume are well matched. Treatment is then administered for up to 4 weeks, after which time the animals are sacrificed. Treatments consisted of either: a) daily sterile water for the control group, b) an intraperitoneal (i.p.) injection of 50 mg/kg gemcitabine twice a week, c) daily gavage with 100 mg/kg masitinib, or d) combined i.p injection of 50 mg/kg gemcitabine twice a week and daily gavage with 100 mg/kg masitinib. Tumour size is measured with callipers and tumour volume is estimated using the formula: volume=(length×width2)/2. The tumour growth inhibition ratio is calculated as (100)×(median tumour volume of treated group)/(median tumour volume of control group). |
参考文献: [1]. Dubreuil P, et al. Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT. PLoS One, 2009, 4(9), e7258. | |
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