Pharmacological characterization of TQ05310, a potent inhibitor of isocitrate dehydrogenase 2 R140Q and R172K mutants

Abstract

Isocitrate dehydrogenase 2 (IDH2), an important mitochondrial metabolic enzyme involved in the tricarboxylic acid cycle, is mutated in a variety of cancers. AG-221, an inhibitor primarily targeting the IDH2-R140Q mutant, has shown remarkable clinical benefits in the treatment of relapsed or refractory acute myeloid leukemia patients. However, AG-221 has weak inhibitory activity toward IDH2-R172K, a mutant form of IDH2 with more severe clinical manifestations. Herein, we report TQ05310 as the first mutant IDH2 inhibitor that potently targets both IDH2-R140Q and IDH2-R172K mutants. TQ05310 inhibited mutant IDH2 enzymatic activity, suppressed (R)-2-hydroxyglutarate (2-HG) production and induced differentiation in cells expressing IDH2-R140Q and IDH2-R172K, but not in cells expressing wild-type IDH1/2 or mutant IDH1. TQ05310 bound to both IDH2-R140Q and IDH2-R172K, with Q316 being the critical residue mediating the binding of TQ05310 with IDH2-R140Q, but not with IDH2-R172K. TQ05310 also had favorable pharmacokinetic characteristics and profoundly inhibited 2-HG production in a tumor xenografts model. The results of the current study establish a solid foundation for further clinical investigation of TQ05310, and provide new insight into the development of novel mutant IDH2 inhibitors.

Keywords: AG-221; IDH2; R140Q; R172K; leukemia.

Figure 1

Chemical structure of TQ05310

Figure 2

Establishment of cell models with exogenous mutant isocitrate dehydrogenase (mIDH) genes. Schematic depiction of the strategy for generating inducible (A) and constitutive (B) plasmids. C,D, Expression levels of exogenously expressed IDH were detected by western blotting. E, (R)‐2‐hydroxyglutarate (2‐HG) production was detected by liquid chromatography coupled with tandem mass spectrometry. Data shown represent means ± SD of three independent experiments. DOX, doxycycline

Figure 3

Effects of TQ05310 on mutant isocitrate dehydrogenase (mIDH) enzymatic activity and (R)‐2‐hydroxyglutarate (2‐HG) production. A, Lysates of U‐87 MG cells exogenously expressing mIDH genes were incubated with TQ05310 and tested for enzymatic activity. U‐87 MG (B) and TF‐1 (C) cells expressing IDH2‐R140Q or IDH2‐R172K were treated with TQ05310 for 3 d. 2‐HG was detected by liquid chromatography coupled with tandem mass spectrometry. Data shown represent means ± SD of three independent experiments. *P < .05

Figure 4

Effects of TQ05310 on cell differentiation and proliferation. TF‐1 cells expressing IDH2‐R140Q or IDH2‐R172K were treated with TQ05310 for 7 d. A, Cells were induced to differentiate by treating with erythropoietin (EPO) for 7 d in the presence of TQ05310, and mRNA levels of hemoglobin (HBG) were analyzed by qRT‐PCR. B, Cells were treated with TQ05310 for another 7 d, and cell proliferation was measured using MTT assays. Data shown represent means ± SD (error bars) from triplicates. DOX, doxycycline; IDH, isocitrate dehydrogenase

Figure 5

Structural basis for the inhibition of IDH2‐R140Q and IDH2‐R172K by TQ05310. A,D, U‐87 MG cells exogenously expressing mutant isocitrate dehydrogenase 2 (mIDH2) genes were treated with TQ05310 for 1 h. Cellular thermal shift assay was carried out to evaluate drug‐target interactions. B, Molecular modeling of the IDH2‐R140Q‐AG221/TQ05310 complex. C, (R)‐2‐hydroxyglutarate (2‐HG) production in U‐87 MG cells exogenously expressing mIDH2 genes was detected by liquid chromatography coupled with tandem mass spectrometry. Data shown represent means ± SD of three independent experiments

Figure 6

Pharmacokinetic/pharmacodynamic characteristics of TQ05310. Mice bearing U‐87 MG/IDH2‐140Q xenografts were given a single oral dose of TQ05310 (18.05 or 54.14 mg/kg) and were killed at the indicated times. Concentrations of (A) TQ05310 and (B) (R)‐2‐hydroxyglutarate (2‐HG) production in plasma and tumor were determined