Fig 2: Production of D-2HG in vitro is pH sensitive only with WT:R132H heterodimers. Colorimetric analysis of enzymatic reactions prepared with the IDH1-WT homodimer (WT:WT), IDH1-R132H homodimer (R132H:R132H), and heterodimer (WT:R132H) (see Methods for details). Reaction mixtures were prepared at 250 nM total protein (WT:WT, R132H:R132H, or 1:1 WT:R132H). Under these conditions, we measured production of (A) AKG and (B) D-2HG. In all cases, four experimental replicates (each result is the average of two technical replicates) across two protein preparations are shown, with significance determined by two-way analysis of variance with Tukey multiple-comparison correction (*p < 0.05, **p < 0.01, and ***p < 0.001).

Fig 3: Characterization of IDH1 R132H/D279N.a Immunoblot analysis of IDH1 expression in TF-1 cells infected with lentivirus expressing the indicated IDH1 variants and treated with vehicle-control (DMSO), 500 nM ivosidenib (IVO), or 500 nM LY3410738 (LY), as indicated. b, c Quantification of (R)-2HG (b) and fold-change in (R)-2HG (c) in the TF-1 cell lines shown in a, as indicated. Shown are mean values (±SD) of duplicate experiments. d, e Proliferation under cytokine-poor conditions (d) and fold-change in day 15 cell counts (e) of the TF-1 cells shown in (a) The mean (±SD) cell counts of three replicates is shown. f Fold-change in intracellular (R)-2HG in TF-1 cells expressing IDH1 R132H (left) or IDH1 R132H/D279N (right) treated overnight with the indicated concentrations of LY3410738. Shown are mean values (±SD) of duplicate experiments. In all cases, representative results from at least two independent experiments are shown.

Fig 4: IDH1R132H promotes malignant transformation of prostate benign epithelial cell RWPE-1. (A, B) Western blot and qRT-PCR analysis of protein and mRNA expression levels of wild-type IDH1 and IDH1R132H in RWPE-1cells, following stably expressing empty vector, wild-type, or mutant IDH1R132H. (C) IDH1R132H enhances the cytokine-independent growth of benign prostatic epithelial cells. The growth of stable RWPE-1 cells expressing IDH1R132H, IDH1WT, and VECTOR was examined by MTS assay. Cells were cultured under cytokine-poor conditions (1/4 normal dose EGF and BPE). (D, E) IDH1R132H promotes migration of benign prostatic epithelial cells. The migration of IDH1R132H on RWPE-1 cells was examined by wound-healing assay and Transwell migration assay. (F, G) The growth and migration of RWPE-1 cells. Cells were treated with 20 µM AGI-5198 or DMSO and analyzed using MTS and Transwell migration assay. All data represent mean ± SD of at least three independent replicates. *P < .05, **P < .01.

Fig 5: In silico models of mutant IDH1 inhibitors inside the allosteric sites of IDH1 R132C and IDH1 R132C/D279N.a Predicted binding mode of ivosidenib (IVO, carbons in yellow) in the allosteric site of IDH1 R132C (carbons in blue). IVO is recognized by IDH1 R132C principally by donating a hydrogen bond to the IDH1 D279 side chain and accepting a hydrogen bond from the IDH1 A111 backbone. Further specific intermolecular interactions involve aromatic contacts with IDH1 W267, IDH1 W124, and IDH1 Y285. b IVO is predicted to have impaired binding to IDH1 R132C/D279N (carbons in red) because the chlorophenyl ring clashes with the mutated asparagine side chain (orange X). c Predicted binding mode of LY3410738 (LY, carbons in green) in the allosteric site of IDH1 R132C (carbons in blue). LY3410738 covalently binds to IDH1 C269 and displays many favorable electrostatic interactions with the protein: the protonated piperazine makes an ionic interaction with the IDH1 D279 side chain and the inhibitor additionally makes two hydrogen bonds with the IDH1 I128 backbone and accepts a hydrogen bond from the IDH1 L120 backbone. d LY3410738 is predicted to adopt a similar covalent binding mode to IDH1 R132C/D279N as it does to IDH1 R132C by keeping all of its numerous anchor points and by replacing the ionic interaction with aspartate with a hydrogen bond to the oxygen of the asparagine-mutated side chain at position 279.