Fig. 3: Isocitrate–Mg²⁺ binding to wt IDH1 is not fully diminished by R132H IDH1 inhibitors.

Non-denaturing MS analysis of EDTA-treated wt IDH1 with inhibitors. Data for the IDH1 dimer m/z = 20⁺ charge state are shown. Cone voltage: 100 V. Buffer: 200 mM ammonium acetate, pH 7.5. See ‘Methods’ for details. a wt IDH1 (50 µM) with predominantly two NADPHs bound. b wt IDH1 + d-isocitrate + Mg²⁺ (50:200:200 µM) showing two isocitrate–Mg²⁺ bound to wt IDH1 dimer. c wt IDH1 + AG-120 (50:200 µM) showing AG-120 bound to wt IDH1 with a predominant stoichiometry of one inhibitor/IDH1 dimer. Note that excess AG-120 does not promote the binding of a second AG-120. d wt IDH1 + GSK864 (50:400 µM) showing GSK864 bound to wt IDH1 with a predominant stoichiometry of two inhibitors/IDH1 dimer. The GSK864 concentration (400 µM) was chosen to saturate its binding to wt IDH1 without inducing precipitation. e wt IDH1 + d-isocitrate + Mg²⁺ + AG-120 (50:200:200:200 µM) reflecting apparent superposition of b and c. f wt IDH1 + d-isocitrate + Mg²⁺ + GSK864 (50:200:200:400 µM) reflecting apparent superposition of b and d. In e, f, wt IDH1 complexed with both isocitrate–Mg²⁺ and R132H IDH1 inhibitor is not observed, indicating wt IDH1 binds to either the isocitrate–Mg²⁺ complex or inhibitor, but not both simultaneously. Note, Mg²⁺ addition does not affect inhibitor binding (Supplementary Fig. S16).