NVP-BSK805 2HCl 化学構造
分子量: 563.47

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製品説明

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製品の説明

生物活性

製品説明 NVP-BSK805 2HClは、IC50が0.5nMによりある強力で選択的なATP競争的JAK2阻害剤です。
ターゲット JAK2
IC50 0.5 nM [1]
In vitro試験 NVP-BSK805 is found to potently inhibit JAK2, whereas displaying more than 20-fold selectivity towards JAK1, JAK3, and TYK2. NVP-BSK805 causes half-maximal inhibition of full-length JAK2V617F and JAK2 wild-type enzymes at 0.5 nM. NVP-BSK805 blocks the growth of JAK2V617F cells (Ba/F3) and induces apoptosis with a GI50 at concentrations <100 nM. As constitutive STAT5 phosphorylation in dependent on JAK2, NVP-BSK805 is found to potently suppress STAT5 phophorylation at ≥ 100 nM concentrations in the JAK2 V617F -mutant cell lines, like MB-02. Incubation of SET-2 cells with 150 nM and 1µM of NVP-BSK805, which corresponds to concentration yielding 75% and 95% growth inhibition, respectively, for 24, 48, and 72 hours lead to concentration- and time- dependent induction of apoptosis. These results are evidenced by the detection of cleaved PARP, reduced Bcl-xL expression, and a strong increase in the number of cells with less than 2N DNA content. [1] NVP-BSK805 triggered cell death requires activation of caspase cascades and is overcome by caspase inhibition in both SET-2 and MB-02 cells. NVP-BSK805 modulates the post-translational modification of Bim and levels of Mcl-1 in JAK2V617F cells, SET-2 and MB-02 cells. [2]
In vivo試験 Oral bioavailability of NVP-BSK805 in mice is estimated to be 45%, while it is 50% in rats. Oral administration of NVP-BSK805 at 150 mg/kg suppresses STAT5 phosphorylation, splenomegaly, and leukemic cell spreading in a Ba/F3 JAK2V617F cell–driven mouse model. NVP-BSK805 suppresses rhEpo-induced STAT5 phosphorylation as well as rhEpo-mediated polycythemia and splenomegaly in BALB/c mice at doses of 25, 50, and 100 mg/kg orally. [1]
臨床試験
特集

プロトコル (参考用のみ)

キナーゼアッセイ: [1]

Enzymatic Assays The human JAK2 kinase domain (amino acids 840-1132) is contained in plasmid construct pAcG2TtevJAK2. The plasmid constructs for JAK3 (813-1124), TYK2 (888-1187), and JAK1(866-1154) are designed. The generation of the recombinant baculoviruses with BD BaculoGoldTM Bright linearized DNA, plaque assay, and virus amplification from single plaques is performed according to the manual (BD Biosciences Pharmingen). Janus kinase domains are expressed in Sf9 cells in 400 mL shake flasks with 100 mL ExCell420 culture medium (JRH Biosciences Ltd) with penicillin/streptomycin solution for 48 h at 27°C. Suspension culture cells are infected at a density of 1 × 106 and the multiplicity of infection (MOI) for each virus is optimized for yield of soluble protein. The kinase domain of human JAK2 and of JAK1, JAK3, and TYK2 is expressed at an MOI of 1 and 0.5, respectively. Time of expression at 27°C is 48 hours for JAK2 and 48 hours or 72 hours for JAK1, JAK3, and TYK2. Forty-eight or seventy-two hours post-infection, the cells from a 100 mL expression culture are harvested by centrifugation at 3000 x g for 5 minutes and lysed with 12 mL lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 1 mM sodium orthovanadate, 1 % Triton X-100, 10 % glycerol, 1 x EDTA-free complete protease inhibitor cocktail (Roche Diagnostics), and 12.5 U/mL Benzonase for 30 minutes at 4°C, followed by centrifugation at 14,000 × g for 45 minutes to pellet insoluble material. For GST-tag affinity purification of kinase domain proteins all steps are performed at 4°C. The cleared lysates are incubated with 0.2 mL of a 50 % slurry of washed Glutathione Sepharose 4B for 2 hours at 4°C, followed by 5 washes with 1 mL of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 % Triton X-100, 1 mM DTT, and 10 % glycerol. Bound protein is eluted in 5 aliquots each starting with a 10 minutes incubation with 0.25 mL elution buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 % Triton X-100, 1mM DTT, 10 % glycerol, 10 mM reduced L-glutathione). Eluates are concentrated about 5-fold with Amicon Ultra-4 spin columns. After addition of Brij35 to 0.1 % final concentration the protein is snap frozen in small aliquots and stored at -80 °C. In these conditions, kinase activities are stable for at least 6 months. The JAK kinasedomain enzymes are incubated for 30 minutes at room temperature in a medium containing 0.1 μM [γ33P]-ATP, 1 mM MnCl2, 5 mM MgCl2, 30 μM of synthetic peptide substrate EQEDEPEGDYFEWLE, 1 mM DTT, 1 % DMSO, 50 μg/mL BSA, 0.01 % Brij35, and 50 mM Tris-HCl pH 7.5. The ATP concentration is below the Km for all proteins. Curves are fitted by non-linearregression using the logistic equation and the global fit function of XLfit® (model 205). Expression and characterization of full-length wild type and V617F mutant JAK2 as well as kinase assay conditions have been described elsewhere. Kinase selectivity of NVP-BSK805 is assessed in an internal kinase panel: In the Caliper assays, kinase reactions are carried out with peptide substrates that migrate with different velocities in an electrical field when phosphorylated. The peptides carry a fluorescent label in order to allow the detection and quantification of the peptides in a capillary system. Peptide fluorescence intensities are quantified using the LC3000 instrument (Caliper Life Sciences, Hopkinton, MA, USA). Kinase activity is measured by quantifying the amount of ATP remaining in solution following a kinase reaction. In the LanthaScreen™ TR-FRET kinase assays, terbium is used as the lanthanide chelate combined with an antibody directed against the phosphorylated substrate.

細胞アッセイ: [1]

細胞株 Ba/F3 cells
濃度 100 nM
反応時間 72 hours
実験の流れ The anti-proliferative activity of JAK2 inhibitors is determined by incubating cells for 72 hours with an 8-point concentration range of compound and cell proliferation relative to DMSO-treated cells is measured using the colorimetric WST-1 (Roche Diagnostics GmbH) cell viability readout. Of each triplicate treatment, the mean is calculated and these data are plotted in XLfit 4 (ID Business Solutions, Ltd.) to determine the half-maximal growth inhibition (GI50) values.

動物実験: [1]

動物モデル RhEpo-induced polycythemia model in Female BALB/c mice
製剤 VP-BSK805 is freshly formulated in MP/PEG300/Solutol HS15 (5/80/15%).
投薬量 50, 75, and 100 mg/kg
投与方法 Once daily oral administration

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

SpeciesMouseRatRabbitGuinea pigHamsterDogMonkeyBaboon
Weight (kg)0.020.151.80.40.0810312
Body Surface Area (m2)0.0070.0250.150.050.020.50.240.6
Km factor361285201220
Animal A (mg/kg) = Animal B (mg/kg) multiplied by  Animal B Km
Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×  mouse Km(3)  = 11.2 mg/kg
rat Km(6)
1

参考

化学情報

Download NVP-BSK805 2HCl SDF
分子量 563.47
化学式

C27H28F2N6O.2HCl

CAS No. 1092499-93-8 (free base)
保管 2年-20℃
6月-80℃in solvent
別名 BSK805
溶解度 (25°C) * In vitro DMSO 113 mg/mL (200.54 mM)
エタノール 15 mg/mL (26.62 mM)
3 mg/mL (5.32 mM)
In vivo 30% PEG400+0.5% Tween80+5% propylene glycol 30 mg/mL
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
化学名 8-(3,5-difluoro-4-(morpholinomethyl)phenyl)-2-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)quinoxaline

カスタマーフィードバック (2)


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Source PLoS One, 2013, 8(5), e63301. NVP-BSK805 2HCl purchased from Selleck
Method Kynurenine Assay
Cell Lines HFF cells
Concentrations 0.5, 1, 2 uM
Incubation Time 72 h
Results To elucidate a possible influence of hypoxia on JAK2 we analysed the effectivity of the JAK2 inhibitor BSK805 on IFN-γ-induced IDO activity of HFF. As shown in figure the addition of BSK805 to IFN-γ stimulated HFF reduces IDO activity. However the inhibitory effect was comparable under hypoxia and normoxia and reached 60-75% of the respective positive control.

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Rating
Source Dr. Catherine Rolvering and Dr. Claude Haan of Université du Luxembour. NVP-BSK805 2HCl purchased from Selleck
Method Western Blot
Cell Lines HEL cells
Concentrations
Incubation Time 3 h
Results NVP-BSK805 inhibits Jak2-V617F mediated signal transduction at nanomolar concentrations in intact cells.

文献中の引用 (2)

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
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