Midostaurin

Midostaurin (PKC412) is a multi-targeted tyrosine kinase inhibitor with antitumor activity, exhibiting IC50 values ranging from 22 to 500 nM against PKCα/β/γ, Syk, Flk-1, Akt, PKA, c-Kit, c-Fgr, c-Src, FLT3, PDFRβ, and VEGFR1/2. Midostaurin is indicated for the treatment of acute myeloid leukemia (AML) and systemic mastocytosis (SM).

PKCβ2:31 nM, PKCβ1:30 nM, PPK:38 nM, p70 S6K:5000 nM, APKCζ:465 μM, FLK1:3900 nM, MLCK:1900 nM, nPKC-δ:33 nM, EGFR:1100 nM, PKCα:22 nM, CSK:8000 nM, PKA:570 nM, c-Src:800 nM, c-Lyn:4300 nM, Myosin light chain kinase:1900 nM, KDR:86 nM, nPKC-η:160 nM, mTOR (p70S6K):5000 nM, FLT1:912 nM, c-Syk:95 nM, CDK1-CyclinB:570 nM, PKCγ:24 nM, Protein kinase A:570 nM, nPKC-ε:1250 nM, c-Fgr:790 nM, CDK1-cyclinB:570 nM

Methods: HMC-1.1 mast cell leukemia cells were treated with midostaurin (1 μM) for 24–48 hours, followed by TUNEL staining to detect apoptosis.
Results: Midostaurin significantly induced apoptosis. [1]
Methods: K562-FLT3 cells (K562 cells overexpressing FLT3) were treated with a concentration gradient of midostaurin (0.001–10 μM) for 72 hours.
Results: Midostaurin exhibited significant inhibitory effects on K562-FLT3 cells. [2]

Methods: K562-FLT3 cells were subcutaneously implanted into immunodeficient mice. Once tumors became palpable, mice were randomly assigned to treatment groups. Midostaurin 50 mg/kg was administered orally via gavage once daily. Imatinib 50 mg/kg was administered orally via gavage twice daily. The combination therapy group received both midostaurin and imatinib at the aforementioned doses concurrently.
Results: Tumor growth was significantly suppressed in mice in the combination therapy group.[2]

Cells are plated overnight and treated with 100 nM AZD3965 or vehicle for 24 hours. The cells are then washed, once in PBS and twice with lysis buffer (50 mM Mops, 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.1 mM DTT, 1 mM PMSF supplemented with 1× mini complete protease inhibitor cocktail tablets. The cells are harvested by scraping and centrifugation, and the pellet snap frozen without buffer in liquid nitrogen. Frozen aliquots of cells are thawed on ice and re-suspended in lysis buffer. Cells are lysed by 3 rounds of freezing in liquid nitrogen and thawing at 37°C for 2 minutes each. Lysates are subsequently centrifuged (13000 g, 10min, 4°C) until clear and then stored on ice. Enzyme activity in the cell lysates is determined using a micro-plate reader to measure either production or depletion of NADH/NADPH, through its absorbance at 340/10 nm, occurring as a result of direct or coupled enzyme reactions. The 96 well plates used for the assays are pre-heated to 37°C for 10 minutes prior to starting reactions, initiated by the addition of 5 μL cell lysate to 95 μL of reaction buffer (50 mM Mops pH 7.4, 100 mM KCl, 5 mM free magnesium). The standard reaction buffer is supplemented to assay the kinetics of the different enzymes. Absorbance values for controls are subtracted from absorbance of corresponding reactions. Graphpad prism 6 is used to plot V0 values against substrate concentration and determine Vmax and Km values. The Vmax is then normalised to the protein concentration in the cell lysate[1].

Each well is added with 5 mM WST-1 and 0.2 mM 1-methoxy PMS and the absorbance at 450 nm is measured by a Microplate Reader.(Only for Reference)

DMSO: 247.5 mg/mL (433.72 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 5 mg/mL (8.76 mM), Sonication is recommended.