生物活性

AGI-6780 is the first highly potent and selective small molecule inhibitor of isocitrate dehydrogenases, which binds in an allosteric manner at the dimer interface of mutant IDH2-R140Q (IC50=23 nM).

AGI-6780biochemical properties[1]

体外研究

体内研究

激酶实验

Enzymatic assay of IDH inhibitors[1]

Compoundwas prepared as 10 mM stock in DMSO and diluted to 50X final concentration inDMSO, for a 50 μl reaction mixture. IDH enzyme activity convertingalpha-ketoglutarate to 2-hydroxyglutarate was measured using a NADPH depletionassay. In the assay the remaining cofactor was measured at the end of thereaction with the addition of a catalytic excess of diaphorase and resazurin,to generate a fluorescent signal in proportion to the amount of NADPHremaining. IDH enzyme activity in the direction of isocitrate toalpha-ketoglutarate conversion was measured by direct coupling of the NADPHproduction to conversion of resazurin to resorufin by diaphorase. In bothcases, resorufin was measured fluorometrically at Ex544 Em 590

For assayof the IDH2/R140Q homodimer and the IDH2-R140Q/WT heterodimer, protein wasdiluted to 0.31 μg/ml in 40 μl Assay Buffer (150 mM NaCl, 50 mM potassiumphosphate pH 7.5, 10 mM MgCl2, 10% glycerol, 0.05% BSA, 2 mM β-mercapoethanol)containing 5 μM NADPH. 1 μl of 50X compound dilution was added and the mixtureand incubated for 16 hours at 25°C. The enzymatic reaction was started with theaddition of 10 μl of Assay Buffer containing 8 mM alpha-ketoglutarate andincubated at 25°C for 60 minutes. The reaction was stopped with the addition ofDetection Buffer (1X Assay Buffer containing 36 μg/ml diaphorase and 18 μMresazurin), incubated for five minutes at room temperature, and read on aSpectraMax 384 platereader.

细胞实验

Cellline generation and culture[1]

TF-1(human erythroleukemia) cells werecultured in IMDM/GlutaMax, 10U/ml penicillin/ streptomycin, 10%FBS and 5ng/mlhuman GM-CSF. Cells were maintained in 5% CO2at 37°C. To generateTF-1 IDH2/R140Q or TF- 1/pLVX expressing cells, TF1 cells were infected withlenti-virus containing full-length IDH2/R140Q or vector control pLVX. Forinfection, cells were plated with the viral supernatant supplemented with 8 μg/mlpolybrene (5.0 104 cells/ml viral supernatant) and incubated in 5% CO2at 32°C for 24h. After infection, transduced cells were selected using G418 (1mg/ml) for 2 weeks to generate stably expressing cells. Subclones were generatedby limited dilution cultures (0.5 cells/well) in a 96-well plate. Primary AML cellswere incubated with SYTOX blue to label dead cells prior to flow cytometrysorting. Cells were treated with DMSO or AGI-6780 (0.2 μM, 1 μM, 5 μM) andcultured in serum-free conditions in the presence of recombinant humancytokines (IL-3, IL-6, SCF, TPO, EPO, FLT3L, GM-CSF, G-CSF).

Differentiationassay

TF1/pLVX and TF1 IDH2/R140Q cells werepretreated for 7 days with 1 μM AGI-6780 (medium changed every 2 days) andwashed with PBS to remove residual GM-CSF. Cells were then induced todifferentiate using EPO (2 unit/ml) in the presence or absence of AGI-6780.Induction continued for 7 days and the cell pellets were collected andsubjected to real-time qPCR to detect fatal hemoglobin (HBG) and KLF-1 geneexpression.

动物实验

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Wang F TJ, DeLaBarre B, Penard-Lacronique V, Schalm S, Hansen E, Straley K, Kernytsky A, Liu W, Gliser C, Yang H, Gross S, Artin E, Saada V, Mylonas E, Quivoron C, Popovici-Muller J, Saunders JO, Salituro FG, Yan S, Murray S, Wei W, Gao Y, Dang L, Dorsch M, Agresta S, Schenkein DP, Biller SA, Su SM, de Botton S, Yen KE. Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation. Science. 2013;340(6132):622-666.

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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