生物活性

AGI-5198, a potent and selectivecell-permeable R132H-IDH1 inhibitor, abrogates the ability of mutant IDH1 toproduce the oncometabolite D-2 hydroxyglutarate (D-2HG) in gliomas. The ability of the mutant enzyme (mIDH1) to produce R-2-hydroxyglutarate (R-2HG). The recent discovery of mutations in metabolic enzymes has rekindled interest in harnessing the altered metabolism of cancer cells for cancer therapy.

Enzymeactivities of AGI-5198[1]

Anti-proliferation of AGI-5198

体外研究

体内研究

0.5% methylcellulose+0.2% Tween 80

激酶实验

Determination of compound inhibition potencyagainst the R132H[1]

In the primary reaction, the reduction ofα-KG acid to 2-HG is accompanied by a concomitant oxidation of NADPH to NADP.The amount of NADPH remaining at the end of the reaction time is measured in asecondary diaphorase/resazurin reaction in which the NADPH is consumed in a 1:1molar ratio with the conversion of resazurin to the highly fluorescentresorufin. Uninhibited reactions exhibit a low fluorescence at the end of theassay, while reactions in which the consumption of NADPH by R132H IDH1 has beeninhibited by a small molecule show a high fluorescence. The primary reactionwas performed in a volume of 50μL 1X Buffer (150mM NaCl, 20mM Tris 7.5, 10mM MgCl₂,0.05% (w/v) bovine serum albumin), contained 2nM R132H IDH1, 1mM alphaketoglutarate,and 4μM NADPH, and was conducted for sixty minutes at 25°C. To perform the secondaryreaction, 25μL of 1X buffer containing 36μg/mL diaphorase and 30mM resazurinwas added to the primary reaction and incubated for a further 10 minutes at25°C. Florescence was read on a Spectramax platereader at Ex 544 Em 590.Compounds or compound dilutions were prepared in 100% DMSO concentration anddiluted 1:100 into the final reaction. R132C IDH1 was assayed under similarconditions, with the exception that the 1X Buffer was 50mM K2HP04, pH 6.5; 40mMNaHCO3; 5mM MgCl₂; 10% glycerol; 0.03% (w/v) bovine serum albumin.

细胞实验

Compounds[2]

The specific IDH1 mutant inhibitor AGI-5198was dissolved in DMSO. D-2-hydroxyglutarate and L-2-hydroxyglutarate weredissolved in PBS.

Celllines and culturing

The chondrosarcoma cell lines CH2879, NDCS-1,OUMS27, JJ012, SW1353, L3252, L2975, and L835 were used. In addition, we usedHT1080 as it was originally reported as a fibrosarcoma of bone, and is nowknown to harbor a mutation in IDH1. Cells were cultured in RPMI1640supplemented with 1% penicillin/ streptomycin (P/S) (100 U/ml), and 10%heat-inactivated fetal bovine serum. Cells were grown at 37°C in a humidifiedincubator with 95% air and 5% CO₂. Cell lines were tested formycoplasma once a month via PCR. STR typing was performed on all cell linesusing the PowerPlex 1.2 system to confirm their identity.

Viabilityassay

Cells were seeded in 96 well plates andallowed to adhere overnight. For SW1353 and JJ012 5000 cells per well wereseeded, for HT1080 and L835 10 000 cells and 15 000 cells, respectively. After72 hours incubation with AGI-5198, a WST-1 assay was performed according to themanufacturer’s instruction and analyzed with the Victor3V, 1420 Multilabelplate reader. Assays were performed in triplicate for three independentexperiments. For long term treatment with AGI-5198 and D-2-HG, cells weretreated for 10 or 20 passages with 1.5μM AGI-5198. Medium with compounds orsolvent controls were refreshed twice a week.

Realtime proliferation and migration assays

The RTCA xCelligence system was used tostudy either proliferation or migration in real time. Proliferation wasmonitored for 72 hours in E-plates. For the migration assay, SIM plates were usedwith the lower compartment filled with RPMI1640 with 20% FCS as achemoattractant, cells were plated in the upper compartment in RPMI1640 only.

Colonyforming assay

HT1080, JJ012, L835 and SW1353 cells were trypsinizedand 100 and 1000 cells were seeded in a 6-well format. Cells were allowed toform colonies over 10 days and were either treated with DMSO or 10μM AGI-5198 onday 0. Afterwards, cells were fixed and stained with 6.0% glutaraldehyde and0.5% crystal violet.

Quantitativereal time PCR

PCR reactions were carried out and relativegene expression levels were normalized for DNA input using the housekeepinggenes PPIA, CPSF6 and GPR108. We used Real Time-PCR to evaluate the effect ofD-2-HG and mutant IDH1 inhibition on Indian hedgehog signaling (IHH, SMO, PTCH1,GLI1, GLI2), HIF1α signaling (GLUT1, BNIP3, EGLN3, ENO1, VEGF), anddifferentiation (COL1a1, COL2a1, RUNX2, BGLAP, OPN, SPARC). In addition, weevaluated the expression of COL1A2 and DIO2. PCRprimers are shown inSupplementary Table 1. Bio-Rad CFX Manager software was used to analyze datafrom the PCR experiments. As normal controls for Hedgehog signalling andcartilaginous differentiation we included RNA isolated from normal articularcartilage (n=3) and normal growth plate cartilage (n=3). For genes involved inosteogenic differentiation we included RNA derived from osteoblastoma (n=3). Togive an overview of the expression levels of each of the genes and samples, a heatmapwas made using the Gene-E tool. 

动物实验

In-vivo experiments[1]

SCID mice were injected subcutaneously with10⁶ glioma cells, which were suspended in 100μL of a 50:50 mixtureof growth media and Matrige. Once tumors had reached a measurable size, micewere randomized into the indicated treatment groups. 

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] Rohle D P-MJ, Palaskas N, Turcan S, Grommes C, Campos C, Tsoi J, Clark O, Oldrini B, Komisopoulou E, Kunii K, Pedraza A, Schalm S, Silverman L, Miller A, Wang F, Yang H, Chen Y, Kernytsky A, Rosenblum MK, Liu W, Biller SA, Su SM, Brennan CW, Chan TA, Graeber TG, Yen KE, Mellinghoff IK. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science. 2013 340(6132):626-630.
[2] Suijker J OJ, Koornneef A, Struys EA, Salomons GS, Schaap FG, Waaijer CJ, Wijers-Koster PM, Briaire-de Bruijn IH, Haazen L, Riester SM, Dudakovic A, Danen E, Cleton-Jansen AM, van Wijnen AJ, Bovée JV. Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines. Oncotarget. 2015;6(14):12505-12519.

体内溶解度
约40 mg/mL

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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