Edited by John M. Denu Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP؉ -dependent conversion of isocitrate (ICT) to ␣-ketoglutarate (␣KG) in the cytosol and peroxisomes. Mutations in IDH1 have been implicated in >80% of lower grade gliomas and secondary glioblastomas and primarily affect residue 132, which helps coordinate substrate binding. However, other mutations found in the active site have also been identified in tumors. IDH1 mutations typically result in a loss of catalytic activity, but many also can catalyze a new reaction, the NADPH-dependent reduction of ␣KG to D-2-hydroxyglutarate (D2HG). D2HG is a proposed oncometabolite that can competitively inhibit ␣KG-dependent enzymes. Some kinetic parameters have been reported for several IDH1 mutations, and there is evidence that mutant IDH1 enzymes vary widely in their ability to produce D2HG. We report that most IDH1 mutations identified in tumors are severely deficient in catalyzing the normal oxidation reaction, but that D2HG production efficiency varies among mutant enzymes up to ϳ640-fold. Common IDH1 mutations have moderate catalytic efficiencies for D2HG production, whereas rarer mutations exhibit either very low or very high efficiencies. We then designed a series of experimental IDH1 mutants to understand the features that support D2HG production. We show that this new catalytic activity observed in tumors is supported by mutations at residue 132 that have a smaller van der Waals volume and are more hydrophobic. We report that one mutation can support both the normal and neomorphic reactions. These studies illuminate catalytic features of mutations found in the majority of patients with lower grade gliomas.Metabolic changes in tumors have been described for nearly a century (1-3), but only relatively recently have enzymes involved in metabolic processes been established as tumor suppressors or oncoproteins. One of the more striking examples of metabolic enzymes playing a role in tumorigenesis includes isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2).3 These homodimeric enzymes are responsible for the reversible NADP ϩ -and Mg 2ϩ -dependent conversion of ICT to ␣KG (Fig. 1A) in the cytosol and peroxisomes (IDH1), or mitochondria (IDH2). IDH3 is responsible for the same reaction within the context of the TCA cycle, although the oxidative decarboxylation catalyzed by this enzyme is non-reversible and NAD ϩ -dependent. Mutations in IDH1 and IDH2 were identified in glioblastoma multiforme in a large sequencing effort (4), and soon Ͼ80% of adult grade II/III gliomas and secondary glioblastomas were found to have IDH1 mutations, commonly R132H or R132C IDH1 (5, 6) (reviewed in Refs. 7-9). Subsequently ϳ10 -20% of acute myeloid leukemias were shown to have primarily IDH2 mutations, typically R140Q or R172K IDH2 (10). Early mechanisms of tumorigenesis focused on deficient conversion of ICT to ␣KG (11), suggesting that IDH serves as a tumor suppressor, in part through altering levels of hypoxia-inducible transcription factor-1␣ (12). However,...

Mutations in isocitrate dehydrogenase 1 (IDH1) drive most low-grade gliomas and secondary glioblastomas and many chondrosarcomas and acute myeloid leukemia cases. Most tumor-relevant IDH1 mutations are deficient in the normal oxidization of isocitrate to α-ketoglutarate (αKG), but gain the neomorphic activity of reducing αKG to D-2-hydroxyglutarate (D2HG), which drives tumorigenesis. We found previously that IDH1 mutants exhibit one of two reactivities: deficient αKG and moderate D2HG production (including commonly observed R132H and R132C) or moderate αKG and high D2HG production (R132Q). Here, we identify a third type of reactivity, deficient αKG and high D2HG production (R132L). We show that R132Q IDH1 has unique structural features and distinct reactivities towards mutant IDH1 inhibitors. Biochemical and cell-based assays demonstrate that while most tumor-relevant mutations were effectively inhibited by mutant IDH1 inhibitors, R132Q IDH1 had up to a 16 300-fold increase in IC50 versus R132H IDH1. Only compounds that inhibited wild-type (WT) IDH1 were effective against R132Q. This suggests that patients with a R132Q mutation may have a poor response to mutant IDH1 therapies. Molecular dynamics simulations revealed that near the NADP+/NADPH-binding site in R132Q IDH1, a pair of α-helices switches between conformations that are more wild-type-like or more mutant-like, highlighting mechanisms for preserved WT activity. Dihedral angle changes in the dimer interface and buried surface area charges highlight possible mechanisms for loss of inhibitor affinity against R132Q. This work provides a platform for predicting a patient’s therapeutic response and identifies a potential resistance mutation that may arise upon treatment with mutant IDH inhibitors.

Human isocitrate dehydrogenase 1 (IDH1) is a highly conserved metabolic enzyme that catalyzes the interconversion of isocitrate and α-ketoglutarate. Kinetic and structural studies with IDH1 have revealed evidence of striking conformational changes that occur upon binding of its substrates, isocitrate and NADP+, and its catalytic metal cation. Here, we used hydrogen–deuterium exchange mass spectrometry (HDX-MS) to build a comprehensive map of the dynamic conformational changes experienced by IDH1 upon ligand binding. IDH1 proved well-suited for HDX-MS analysis, allowing us to capture profound changes in solvent accessibility at substrate binding sites and at a known regulatory region, as well as at more distant local subdomains that appear to support closure of this protein into its active conformation. HDX-MS analysis suggested that IDH1 is primarily purified with NADP­(H) bound in the absence of its metal cation. Subsequent metal cation binding, even in the absence of isocitrate, was critical for driving large conformational changes. WT IDH1 folded into its fully closed conformation only when the full complement of substrates and metal was present. Finally, we show evidence supporting a previously hypothesized partially open conformation that forms prior to the catalytically active state, and we propose this conformation is driven by isocitrate binding in the absence of metal.

Point mutations in human isocitrate dehydrogenase 1 (IDH1) can drive malignancies, including lower-grade gliomas and secondary glioblastomas, chondrosarcomas, and acute myeloid leukemias. These mutations, which usually affect residue R132, ablate the normal activity of catalyzing the NADP+-dependent oxidation of isocitrate to α-ketoglutarate (αKG) while also acquiring a neomorphic activity of reducing αKG to d-2-hydroxyglutarate (D2HG). Mutant IDH1 can be selectively therapeutically targeted due to structural differences that occur in the wild type (WT) versus mutant form of the enzyme, though the full mechanisms of this selectivity are still under investigation. Here we probe the mechanistic features of the neomorphic activity and selective small molecule inhibition through a new lens, employing WaterMap and molecular dynamics simulations. These tools identified a high-energy path of water molecules connecting the inhibitor binding site with the αKG and NADP+ binding sites in mutant IDH1. This water path aligns spatially with the α10 helix from WT IDH1 crystal structures. Mutating residues at the termini of this water path specifically disrupted inhibitor binding and/or D2HG production, revealing additional key residues to consider in optimizing druglike molecules against mutant IDH1. Taken together, our findings from molecular simulations and mutant enzyme kinetic assays provide insight into how disrupting water paths through enzyme active sites can impact not only inhibitor potency but also substrate recognition and activity.

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme responsible for catalysis of isocitrate to α‐ketoglutarate using NADP+ as a cofactor. Cancerous mutations of IDH1 yield the neomorphic conversion of α‐ketoglutarate to D‐2‐hydroxyglutarate, a proposed oncometabolite. Various cancers such as gliomas, glioblastomas, acute myeloid leukemias, and bone cancers, are driven by IDH1 mutations. Although it has been established that IDH1 is an enzymatic driver of disease, more research is necessary to elucidate the molecular mechanisms of its regulation. Post‐translational modifications (PTMs), such as acetylation, serve as effective methods of protein regulation. Here, we hypothesize that acetylation may have a significant impact on IDH1 efficiency. To investigate this, we treated wild type IDH1 with acetyl group donors and generated wild type IDH1 mutants (K81Q, K224Q, K321Q) to mimic acetylation in a physiologically relevant setting. We then employed steady state enzyme kinetics to measure the rate of the normal reaction yielding α‐ketoglutarate. We report that acetylation has a notable effect on IDH1 catalytic efficiency in both the acetylation mimics and acetyl‐treated IDH1. Our data suggests that acetylation may be an essential post‐translational modification for regulating IDH1 catalytic efficiency, thus helping us understand pathways relevant to IDH1 activity. Support or Funding Information This work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).