PKC412(Midostaurin (PKC412); CGP41231; CGP41251) is a broad spectrum protein kinase inhibitor. Midostaurin inhibits protein kinase C alpha (PKCalpha), vascular endothelial growth factor receptor 2 (VEGFR2), c-kit, platelet-derived growth factor receptor (PDGFR) and FMS-like tyrosine kinase 3 (FLT3) tyrosine kinases, which may result in disruption of the cell cycle, inhibition of proliferation, apoptosis, and inhibition of angiogenesis in susceptible tumors.

PKCα:22 nM, PKCβ2:31 nM, PKCγ:24 nM, PKCβ1:30 nM, PPK:38 nM

Midostaurin(pkc412)是一种广谱蛋白激酶抑制剂。它与常见的PKC-α、-β、-γ、PDGFRβ、VEGF-R2、VEGF-R1以及细胞周期依赖性激酶1-细胞周期蛋白B复合体的ATP结合位点有强烈的相互作用。Midostaurin(pkc412)能够在亚微摩尔浓度下抑制各种人类和动物细胞系体外生长。此外，Midostaurin(pkc412)有效抑制了胶质母细胞瘤体外增殖，并诱导细胞在G2/M期积累及巨核形成，伴随广泛的碎片化和凋亡体生成。Midostaurin(pkc412)还能逆转肿瘤细胞体外p-糖蛋白介导的多药耐药性。[1]

Midostaurin(pkc412)通过抑制肿瘤血管生成（通过其对VEGF受体酪氨酸激酶的影响）以及直接抑制肿瘤细胞增殖（通过其对PKCs的影响），可能抑制肿瘤生长。这种抗血管生成作用可能有助于midostaurin(pkc412)所展示的抗转移和广泛的抗肿瘤活性，以及与包括多柔比星、环磷酰胺、顺铂和吉西他滨在内的细胞毒性化合物的协同作用。口服时，midostaurin(pkc412)的最大耐受剂量为>300 mg/kg。[1]

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)