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

AGI-5198 showed some anti-tumor efficacy against TS603 glioma cell line and dose-dependently inhibited R-2HG production. Under the condition of almost complete inhibition of R-2HG, AGI-5198 induced demethylation of histone H3K9me3, and induced the expression of genes related to glial gene differentiation. In genome-wide DNA methylation, AGI-5198 inhibited the growth of mIDH1-impaired IDH1 mutant, but wild-type growth was hardly affected.AGI-5198 significantly inhibited mutant IDH1 (R132H-IDH1 and R132C-IDH1), but did not inhibit the growth of wild-type IDH1 (IC50>100 μM) or IDH1 (IC50>100 μM) or wild-type IDH1 (IC50>100 μM), but very weakly inhibited any of the IDH2 isoforms (R140Q, R172K, wild-type) (IC50>100 μM).

AGI-5198 showed some anti-tumor efficacy against TS603 glioma cell line and dose-dependently inhibited R-2HG production. Under the condition of almost complete inhibition of R-2HG, AGI-5198 induced demethylation of histone H3K9me3, and induced the expression of genes related to glial gene differentiation. In genome-wide DNA methylation, AGI-5198 inhibited the growth of mIDH1-impaired IDH1 mutant, but wild-type growth was hardly affected.AGI-5198 significantly inhibited mutant IDH1 (R132H-IDH1 and R132C-IDH1), but did not inhibit the growth of wild-type IDH1 (IC50>100 μM) or IDH1 (IC50>100 μM) or wild-type IDH1 (IC50>100 μM), but very weakly inhibited any of the IDH2 isoforms (R140Q, R172K, wild-type) (IC50>100 μM).

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.