AGI-5198 (IDH-C35) is a potent and selective inhibitor of IDH1 R132H and R132C mutants, with IC50 values of 0.07 μM and 0.16 μM, respectively.

R132C-IDH1:0.16 μM, R132H-IDH1:70 nM

在带有Ki-67蛋白抗体染色的小鼠体内,AGI-5198可使其肿瘤减少.但空白对照组和AGI-5198处理组的小鼠肿瘤中裂解的caspase-3无显著差异.AGI-5198（450 mg/kg/day）治疗R132H-IDH1胶质瘤移植瘤三周,可抑制50-60％的生长,但不影响IDH1野生型胶质瘤移植瘤的生长.

AGI-5198对TS603胶质瘤细胞系有一定抗肿瘤疗效,且剂量依赖性抑制R-2HG产生。在R-2HG几乎被完全抑制的条件下,AGI-5198对组蛋白H3K9me3去甲基化有诱导作用,且诱导与胶质基因分化相关的基因表达。在全基因组DNA甲基化中,AGI-5198对mIDH1受损的IDH1突变型生长具有抑制作用,但野生型生长几乎没有受到影响。AGI-5198对突变型IDH1(R132H-IDH1 和 R132C-IDH1)有明显抑制作用,但对野生型IDH1 (IC50>100 μM) 或任何IDH2亚型(R140Q, R172K, 野生型) (IC50>100 μM)抑制作用非常弱。

IDH enzyme activity: Compound is prepared as 10 mM stock in DMSO and diluted to 50X final concentration in DMSO, for a 50 μL reaction mixture. IDH enzyme activity converting alpha-ketogluta rate to 2-hydroxyglutarate is measured using a NADPH depletion assay. In the assay the remaining cofactor is measured at the end of the reaction with the addition of a catalytic excess of diaphorase and resazurin, to generate a fluorescent signal in proportion to the amount of NADPH remaining. IDH enzyme activity in the direction of isocitrate to alpha-ketoglutarate conversion is measured by direct coupling of the NADPH production to conversion of resazurin to resorufin by diaphorase. In both cases, resorufin is measured fluorometrically at Ex544 Em 590.

AGI-5198 is dissolved in DMSO.TS603 cells are grown in medium containing either AGI-5198 (1.5 μM) or DMSO vehicle control.One week prior to harvest cells are ransferred to differentiation medium (DMEM F12; 15 mM HEPES; 0.06% glucose; B27 without vitamin A; N2; Insulin/transferrin; 1% FBS) containing freshly added retinoic acid (1 μM).ChIP of non-crosslinked cells is then carried out using established ChIP methods.350 μg of lysate is immunoprecipitated-using anti-H3K9Me3,H3K27me3 or Rabbit Control IgG.After washing,ChIP DNA is eluted from protein G beads and analyzed by RT-PCR using SYBR green.Relative occupancy is calculated using the standard curve method and fold enrichment versus IgG.Enrichment in AGI- 5198-treated cells is normalized to vehicle control.Means and standard deviation are calculated from 4 technical replicates.