Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1

Article Cite This: J. Med. Chem. 2018, 61, 6647−6657

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Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315 Clarissa G. Jakob,† Anup K. Upadhyay,∥ Pamela L. Donner,† Emily Nicholl,§ Sadiya N. Addo,‡ Wei Qiu,† Christopher Ling,† Sujatha M. Gopalakrishnan,§ Maricel Torrent,† Steven P. Cepa,† Jason Shanley,† Alexander R. Shoemaker,‡ Chaohong C. Sun,∥ Anil Vasudevan,† Kevin R. Woller,† J. Brad Shotwell,*,† Bailin Shaw,‡ Zhiguo Bian,† and Jessica E. Hutti‡ Discovery Chemistry and Technology, ‡Oncology Discovery, §Targeting Enabling Science and Technology, and ∥Global Protein Sciences, AbbVie, 1 North Waukegan Road, North Chicago, Illinois 60064, United States

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S Supporting Information *

ABSTRACT: IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsaturated enone 1 makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design.

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INTRODUCTION Isocitrate dehydrogenase 1 (IDH1) is one of three isozymes (IDH1, IDH2, IDH3) that convert isocitrate into αketoglutarate (α-KG). While IDH1−3 perform similar enzymatic reactions, they have disparate cellular functions and localization. IDH1 and IDH2 exist as homodimers which reduce NADP+, while IDH3 is a structurally distinct enzyme that utilizes NAD+ as a cofactor.1 IDH2 and IDH3 are mitochondrial enzymes that play critical roles in generating mitochondrial NADPH and driving TCA cycle flux, respectively.1 In contrast, IDH1 is localized primarily in the cytosol where it plays an important role in maintaining cytosolic NADPH levels. Interest in IDH1 as a drug target has grown during the past few years following a series of studies demonstrating that IDH1 is an oncogene that is mutated in acute myeloid leukemia (AML), secondary glioblastomas (GBMs), and several other tumor types.2−7 In response to these findings, a number of compounds have been generated that specifically target the mutant isoform of IDH1, several of which are currently being evaluated in clinical trials.8−12 IDH2 is also mutated in AML, and the first inhibitor of mutant IDH2, enasidenib (Idhifa, Celgene/Agios) was recently approved by the FDA for the treatment of relapsed/refractory AML with IDH2 mutation. Most of the reported inhibitors of mutant IDH1 have exquisite selectivity against wild-type IDH1. However, in addition to its role as an oncogene, the normal functions of wild-type IDH1 are important for a number of cellular © 2018 American Chemical Society

processes. As one of the primary sources of cytosolic NADPH, IDH1 is thought to play an important role in maintaining reducing power within the cytosol, and hepatocytes from IDH1 knockout mice have both increased steady-state levels of reactive oxygen species (ROS) and increased oxidative stress following treatment with lipopolysaccharide (LPS).13 A later study showed that IDH1-null mice have decreased blood glucose levels and increased levels of amino acids in plasma, suggesting that IDH1 may play additional roles in maintaining metabolic homeostasis in vivo.14 Interestingly, IDH1 is a highly reversible enzyme, and IDH1-driven reductive carboxylation of α-KG derived from anaplerotic glutamine occurs at a low level in many cells under normal growth conditions.15 Under some conditions of cell stress, such as hypoxia, during anchorageindependent growth, or in the presence of mitochondrial defects, however, many cells dramatically upregulate flux though IDH1-dependent reductive glutaminolysis.15−18 Consistent with this, the pseudohypoxic state created following the loss of the tumor suppressor VHL also causes a dramatic increase in flux through reductive glutaminolysis.15,18 In spite of the important roles of wild-type IDH1 in cellular metabolism, to our knowledge no tool inhibitors of wild-type IDH1 have been described. Here we describe a highthroughput screen performed to identify inhibitors of WT IDH1. In addition, we disclose (i) three unique modes of Received: February 22, 2018 Published: July 13, 2018 6647

DOI: 10.1021/acs.jmedchem.8b00305 J. Med. Chem. 2018, 61, 6647−6657

Journal of Medicinal Chemistry

Article

IDH1 inhibition exemplified by the first reported crystallographic characterization of inhibitor-bound (1−3, 11, 13, 15) IDH1 WT structures, (ii) a unique susceptibility of H315 within the IDH1 NADPH binding pocket to be covalently modified by small molecule inhibitors, (iii) SAR and structural studies related to the optimization of 1, and (iv) inhibition of cellular reductive glutaminolysis flux by small molecule engagement at both an allosteric site at the dimer interface and the NADPH binding site of IDH1. For the remainder of the manuscript, “IDH1” will refer to wild-type IDH1 unless otherwise indicated.

ketoglutarate is complete and the isocitrate-R132 hydrogen bonds are lost. This is in contrast to reports of R132H mutants of IDH1, where the protein has only been observed in the open or semiopen conformation in the presence of both NADP and isocitrate, having only two ordered central helices accompanied by two disordered loops at the dimer interface.20 Loss of the arginine residue in the R132H mutation prevents isocitrate from bridging the two molecules as observed in the wild-type structure. To date, crystal structures of IDH1 containing the R132H mutation with small molecules can be generally characterized as having an open or semiopen tertiary structure (see Table SI-5, Supporting Information). Because high affinity hits were identified directly from screening, IDH1:ligand crystal structures for 1−3 (Figure 2) were solved prior to initiating their chemical optimization. Notably, three distinct binding sites are observed for 1−3 (Figure 2A−C). We found enone 1 bound to a symmetrical fully closed conformation of IDH1 (Figure 2A,D) in a pocket that overlaps with a known NADPH binding site (PDB code 1T0L). Hydrogen bonding of D375 to the aminolinker NH, nitrile engagement of the S326 backbone carbonyl, and interaction of the carbonyl directly with K374 is accompanied by direct covalent attachment of the ligand through H315 (Figure 2D). This result was surprising, as during our initial hit triage we expected a variety of exposed cysteine residues to be the likely target(s) of 1. Covalent engagement by histidine has been previously described but is rarely exploited in reversible inhibitor design.21−23 For example, covalent inhibitor− histidine engagement has been reported for fumagillin,21 4hydroxynonenal,22 and prostaglandin J2.23 The histidine− ligand engagement of fumagillin and 4-hydroxynonenal are unsurprisingly irreversible, with fumagillin engagement resulting in irreversible epoxide opening and 4-hydroxynonenal engagement affording a stabilized hemiacetal arising from attack of the 4 hydroxy group on the aliphatic aldehyde generated from initial histidine addition. In contrast, the prostaglandin J2−human serum albumin interaction as characterized by surface plasmon resonance studies is readily reversible and is most structurally analogous to the 1:IDH1 structure reported here.23 Imidazolone 2 is observed to bind in an allosteric pocket at the dimer interface between the four central helices of the two monomers, with a single copy of 2 for each IDH1 dimer (1:2 stoichiometry). This site is close to a location recently reported by Bayer for R132H mutant IDH1 inhibitor BAY1436032.8 BAY1436032 does not engage both monomers in a symmetric fashion nor does it bind with significant potency to WT IDH1. The BAY1436032 structure is an open R132H IDH1 conformation. In contrast, the complex of 2 with WT IDH1, which crystallizes in the presence of 0.15 M malate, results in an asymmetrical fully closed dimer, where malate is bound in the more tightly closed monomer (Figure 2B). The fact that compound 2 results in an asymmetrical closed dimer is interesting as the compound itself is symmetrical and binds in what can be described as an allosteric pocket that lies on the 2fold axis of the protein dimer making equivalent interactions with both IDH1 molecules. The imidazolone directly engages both copies of Q277 from the corresponding monomers, and the bromines sit adjacent to M259 filling the hydrophobic space (Figure 2E). In contrast to 1 and 2, 3 binds in an allosteric pocket with both subunits in the semiopen form of the enzyme (Figure 2C). This is distinct from the reported structure of its isomer

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RESULTS AND DISCUSSION High-Throughput Screen To Identify Inhibitors of IDH1. In an effort to identify IDH1 inhibitor tool molecules, 750 000 AbbVie proprietary molecules were screened at 30 μM with an IDH1 fluorescence activity assay (see Figure SI-2 and Supporting Information) for an ability to inhibit NADPH production accompanying the IDH1 catalyzed conversion of isocitrate to α-ketoglutarate. Analysis of the hit set revealed a large cluster of α,β-unsaturated enones centered around 1 (IC50 = 410 nM) with structures suggestive of a covalent modification of IDH1 (Figure 1). In addition to 1, singleton 2

Figure 1. WT IDH1 inhibitors identified by HTS (1 and 2) and 3.

(IC50 = 270 nM) was identified with similar potency to 3 (IC50 = 120 nM), an isomer of GSK321 (IC50 = 970 nM) reported by GlaxoSmithKline as a mutant R132H IDH1 inhibitor with some wild-type cross reactivity (Figure 1).10 IDH1 Structural Biology and IDH1:Inhibitor Cocrystallization Studies for 1−3. The crystal structure of IDH1 is an asymmetric open homodimer consisting of one open and one semiopen subunit when bound to NADP (PDB code 1T09).19 Upon binding substrate (isocitrate), the protein transitions to a symmetric closed conformation in which isocitrate bridges both molecules of the dimer via a direct hydrogen bond with R132 (PDB code 1T0L).19 The fully closed structure contains four central α-helices (two from each monomer), wherein the bridging isocitrates of each monomer hydrogen-bond to the opposing IDH1 molecules, preserving the closed form until the conversion of isocitrate to α6648



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Figure 2. Crystal structures of 1, 2, and 3 bound to WT IDH1. (A) Structure of WT IDH1 complexed with 1 in the fully closed form with IDH1 shown in gray as ribbons, 1 shown in teal spheres, and isocitrate shown as yellow sticks (PDB code 6BKX). (B) Structure of WT IDH1 complexed with 2 in an asymmetric fully closed form of IDH1 with 2 in yellow spheres and malate shown as blue sticks (PDB code 6BKY). (C) Structure of WT IDH1 complexed with 3 in the semiopen form with 3 in orange spheres and NADP as fuchsia sticks (PDB code 6BKZ). (D) Binding site of 1 with H-bonds shown in red. Superposition with IDH1 with NADPH and citrate (PDB code 1T0L) showing that 1 would be competitive with NADPH shown in gray sticks. (E) Allosteric, symmetric binding site of 2 with H-bonds shown in red. (F) Nonequivalent allosteric binding sites of 3 with H-bonds shown in red. All proteins are shown as ribbons, key interacting residues are displayed as sticks and colored by atom type, and all ligands are colored by atom type.

IDH1 engaging R119 in one monomer and L120 in the other while hydrogen bonds to backbone carbonyls of I128 and V281 are conserved in both copies (Figure 2F). Our observations from the structures with 2 and 3 lead us to

(GSK321) in R132H mutant IDH1 (PDB code 5DE1, Table SI-5).10 Nonequivalent IDH1 monomers are observed with two copies of 3 bound nonsymmetrically within each copy of the dimer. The two copies of 3 make disparate contacts with 6649



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Scheme 1. Synthesis of IDH1 Inhibitors 10 and 11a

a Conditions: (i) bromobenzene, t-AmOH/dioxane, K3PO4, Pd(OAc)2, 2-(di-tert-butylphospino)-2′-methylbiphenyl, reflux, 24 h; (ii) ethyl vinyl ketone, TEA, acetonitrile, 75 °C, 24 h; (iii) L-phenylalamine, pyridinium p-toluenesulfonate, DMSO, 45 °C, 66 h; (iv) ethylene glycol, ptoluenesulfonic acid, toluene, reflux, Dean−Stark; (v) NaBH4, EtOH, −5 °C, 4 h; AcOH, rt, 24 h; (vi) 5% Pd/C, THF, pyridine, 60−100 psi H2, DBU, rt, 24 h; (vii) pyridinium dichromate, magnesium sulfate, DCM, reflux, 24 h; (viii) ethyl formate, 1 M potassium tert-butoxide, −5 °C to rt, 1 h; (ix) hydrazine, EtOH, rt, 24 h; (x) 1 N HCl, THF, rt, 24 h; (xi) ethyl formate, 25% NaOMe, MeOH, rt, 24 h; (xii) hydroxylamine hydrochloride, EtOH, 50 °C 24 h; (xiii) 25% NaOMe, MeOH, rt, 2 h; (xiv) DDQ, THF, rt, 10 min; (xv) TEA, benzoyl chloride, DCM, rt.

Scheme 2. Synthesis of IDH1 Inhibitors 13 and 11a

support the prior hypotheses that though the IDH1 dimer is made up of identical subunits, their concerted dynamics lead to enzymatic differences between them.19 Synthetic Chemistry. Acylated pyrazole 11 was accessed in the sequence outlined in Scheme 1.24 Arylation of diketone 4 followed by treatment with ethyl vinyl ketone and subsequent Robinson annulation and then protection of ketone via ketal afforded 6 in 55% overall yield for four steps. Reduction of the C(5) ketone to the corresponding alcohol, olefin hydrogenation, and treatment with DBU to give the preferred epimer at C(1) and then reoxidation at C(5) afforded ketal 7 in 27% overall yield for four steps. Formylation of 7 with ethyl formate and sodium methoxide afforded versatile oxocarboxaldehyde 8 which was converted to the fused pyrazole by condensation with hydrazine and deprotection to provide 9. Subsequent α,β-unsaturated enone installation proceeded via a five-step process beginning with deprotection of the ketone followed by formylation at C(3) and condensation with hydroxylamine hydrochloride to give the corresponding isoxazole which was ring opened under basic conditions to give the C(3) nitrile. Bromination/ elimination with 1,3-dibromo-5,5-dimethylimidazolidine-2,4dione and then heating in pyridine or oxidation with DDQ afforded 10.24 Direct acylation with benzoyl chloride afforded 11 in 15 steps and 2.8% overall yield. Aminopyrimidine 13 was accessed by condensation of phenylguanidine with key intermediate 8 and subsequent conversion to the α,βunsaturated enone (i.e., 12 → 13, Scheme 2, six steps, 32% yield) in a manner analogous to that described for conversion of 9 → 10. Similarly, pyridine 16 (Table 1) was accessed via substitution of (pyridine-3-yl)guanidine for phenylguanidine. C(10) phenyl benzoate substituted 19 and 15 (Table 1) were generated by substitution of 3-iodobenzoic acid ethyl ester for bromobenzene in the initial transformation illustrated in Scheme 1.

a

Conditions. (i) hydrazine, EtOH, rt, 24 h; (ii) 1 N HCl, THF, rt, 24 h; (iii) ethyl formate, 25% NaOMe, MeOH, rt, 24 h; (iv) hydroxylamine hydrochloride, EtOH, 50 °C, 24 h; (v) 25% NaOMe, MeOH, rt, 2 h; (vi) 1,2-dibromo-5,5-dimethylhydantoin, pyridine, 0 °C → 50 °C, 3 h.

Structure−Activity Relationships and Binding Mode Comparison for 11, 13, and 15. While development of structure−activity relationships for 2 proved intractable, with dozens of differentially substituted imidazolones failing to inhibit IDH1 in vitro, the binding mode and diversity of optimization trajectories afforded by 1 proved more fruitful (Table 1). Specifically, direct engagement of the side chain of D375 by the aminopyrimidine NH of 1 was established as critical, with 13 showing a ∼100-fold improved IC50 for IDH1 relative to 14. Improved potency (3-fold) via addition of an aromatic ring at R1 (i.e., 1 vs 13) is consistent with the filling of a lipophilic groove between K374/L383/A378 observed in 6650



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Table 1. IDH1 Potency for 1, 10−11, and 13−19a

a

See Supporting Information Table SI-2 for experimental protocol, replicates, standard deviation, and compound purity/identify confirmation.

IDH1 cocrystal structures obtained for 13 and suggested a clear trajectory to the K260 residue of the opposing monomer (Figure 3C). Placement of a carboxylic acid (15) with trajectory appropriate to target K260 afforded a modest 3fold improvement in potency, while other acidic isosteres were unsuccessful (not shown). Structural studies with 15 revealed it maintains the critical engagement of D375 in addition to the other key interactions observed for 1 and 13 while engaging K260 directly as designed (Figure 3C). Consistent with covalent engagement of H315 observed in structures for 1, 13, and 15, reduction to the corresponding saturated ketone 17 affords a ∼10-fold reduction in potency (13 vs 17). Elimination of the α-nitrile (18) abrogated target affinity completely (Table 1), consistent with H315 attack at the βcarbon and a direct hydrogen bond between the nitrile and S326. Incorporation of a fused pyrazole was well-tolerated, wherein incorporation of a carboxylic acid (19) directed toward K260 was found to afford a ≥10-fold improvement in potency relative to 10 in analogy to the pyrimidine series. Acylation of 10 afforded potent acyl pyrazole inhibitor 11. Structural studies demonstrated that 11 maintained the critical covalent link to H315 but shifted slightly within the NADPH binding site to maximize water-mediated engagement of S326

and direct hydrogen bonding to N328 as surrogates for the D375 interaction observed for 1, 13, and 15 (Figure 3A). Additionally, although the nitrile−S326 interaction is maintained for 11, the observed carbonyl−K374 distance is beyond optimal relative to those for 1, 13, and 15 (Figure 3A−C). The binding modes of compounds 11 and 13 to IDH1 were further confirmed by competition binding assays performed in the presence and absence of NADP and isocitrate. As shown in Figure 4, 11 and 13 did not bind to IDH1 precomplexed with NADP but bound tightly in the presence of isocitrate (see Figure SI-5 for ΔTm shifts). These orthogonal biophysical data corroborate the binding mode observed for these compounds in their respective crystal structures. These findings contrast with those made with 3, wherein as measured by isothermal titration calorimetry (ITC, see Figure SI-6) 3 has a similar affinity in the presence or absence of NADP+ but shows no binding to the protein in the presence of isocitrate. All TSA and ITC studies are consistent with the binding modes presented in the X-ray structures in Figure 2 and Figure 3. Thermal stability (Tm) of the complexes formed by compounds 11 and 13 decreases upon washing these samples with the protein buffer (see Figure SI-5), and IDH1 enzyme activity recovers in enzymatic jump dilution experiments 6651



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SI-3). In contrast, 11 showed a high propensity toward pyrazole amide hydrolysis under mild conditions to afford less active pyrazole 10 in typical cellular buffers (see Table SI-3, Supporting Information). Carboxylic acid 15 was found to have poor permeability (see Figure SI-1). As such, 13 was advanced into cell-based assays in addition to control inhibitor 3. Inhibition of Cellular Flux through Reductive Glutaminolysis. It has been previously shown that VHL mutant ccRCC cells demonstrate significant IDH1-dependent flux through reductive glutaminolysis.15,18 Therefore, VHL mutant A-498 cells were used to develop a cellular pharmacodynamic (PD) assay for IDH1. Briefly, A-498 cells were treated with growth medium containing 1-13C-glutamine, and incorporation of the 13C label into citrate was measured, as described previously.15 Determining the ratio of 13C-citrate/12C-citrate within cell lysates allows one to then determine the amount of reductive, IDH1-dependent glutamine metabolism relative to the amount of oxidative glutamine metabolism. As expected, under basal growth conditions, A-498 cells show significant flux through reductive glutaminolysis (Figure 5). In contrast, when treated with 3 or 13, A-498 cells demonstrate a dosedependent decrease in reductive glutaminolysis (Figure 5) consistent with dose-dependent intracellular inhibition of IDH1 by 3 and 13.

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CONCLUSIONS While a number of inhibitors of mutant IDH1 have been wellcharacterized, to our knowledge no tool molecules for WT IDH1 have been reported. Screening efforts in our laboratories have identified two novel inhibitors of IDH1 with biochemical activity. Structural characterization of these inhibitors along with previously reported 3 provides some interesting structural observations for the IDH1 dimer. Three distinct IDH1 binding sites have been fully characterized. Notably, covalent inhibitor 1 binds in the active site and results in the fully closed form of IDH1. This and additional reversible covalent chemotypes exemplified by 11 and 13 are competitive with NADPH binding within the IDH1 active site. Importantly, their potency benefits from a unique covalent adduct formed via the reversible trapping of H315, a mechanism that is not frequently exploited in the design of reversible inhibitors. In contrast, compounds 2 and 3 bind in allosteric sites and lead to nonsymmetric forms of the IDH1 dimer with stoichiometries of 1:2 and 1:1 respectively. These observations are likely due to the dynamic nature of the protein and are consistent with the previous proposal of a dynamic influence of one subunit on the other.19 The recently described mutant IDH1 inhibitor BAY1436032 also binds near the dimer interface.8 However, BAY1436032 does not have significant activity against WT IDH1 and, in contrast to 2, does not bind in the closed conformation. All three of the binding sites described here allow robust inhibition of recombinant IDH1 in biochemical assays. Compounds 13 and 3 also show dose-dependent inhibition of cellular IDH1 activity, as measured by tracking incorporation of 13C from 1-13C-glutamine to citrate. IDH1 plays a key role in regulating cellular metabolism, especially under conditions of mitochondrial stress or hypoxia.15−18 In addition, IDH1-driven reductive glutaminolysis is dramatically upregulated in the absence of the tumor suppressor VHL, which is the most commonly mutated gene in clear-cell renal cell carcinoma (ccRCC).25 However, studies of the role of wild-type IDH1 in metabolism or cancer have been

Figure 3. X-ray crystal structures of 11, 13, and 15 with WT IDH1. (A−C) The binding site of one monomer of the WT IDH1 complex is shown with key protein residues shown in sticks. H-bonds are illustrated by dashed red lines. (C) K260 of the adjacent subunit is shown in dark teal (PDB codes 6BL0, 6BL1, and 6BL2, respectively).

performed with compound 13 (see Figure SI-7). These observations are consistent with the absence of any stable covalent adduct formation with these compounds using mass spectrometry assays (see Figure SI-4) and corroborate the reversibility of enone engagement of histidines reported in the literature for similar systems.23 All leads were judged as high fidelity and showed no response in a counterscreen designed to identify artifacts as a result of diaphorase inhibition (see Figure SI-2). It was found that 13 demonstrated high stability and permeability in cellular experiments (see Figure SI-1 and Table 6652



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Figure 4. Competition studies for 11 and 13 with ±250 μM NADP or isocitrate. Compounds 11 and 13 did not show binding in the presence of NADP (A, C) but bound in the presence of isocitrate (B, D). This is consistent with the observed binding mode in Figure 2. Compound 2 is both commercially available and previously described.26 Compound 3 was prepared according to protocols previously desribed.20 Chemical syntheses and purities for final compounds 10, 11, 14, and 19 have been previously described and appropriate links to protocols and annotation of final purities are included in the Supporting Information. 2-Phenylcyclohexane-1,3-dione (5). A 5 L round-bottomed flask was charged with bromobenzene (0.109 L, 1038 mmol), tertamyl alcohol (1 L), and dioxane (2 L), and the contents were purged with N2 for 45 min. A 5 L round-bottomed flask was charged with potassium phosphate tribasic (459 g, 2163 mmol), 1,3-cyclohexanedione (100 g, 865 mmol), palladium(II) acetate (3.59 g, 16.00 mmol), and 2-(di-tert-butylphosphino)-2′-methylbiphenyl (10 g, 32.0 mmol), and the contents were purged with N2 for 45 min. The bromobenzene solution was then transferred to 1,3-cyclohexanedione containing vessel via canula, and the reaction was heated to reflux overnight. After cooling to room temperature, the reaction mixture was partitioned between ethyl acetate (2 L) and 10% HCl (3 L) with mixing. The lower aqueous layer was separated and extracted with ethyl acetate (2 L). The combined organic layers were washed with brine (1 L) and concentrated. The residue was taken up in toluene (1 L) and again concentrated. The residue was taken up in toluene (500 mL) and warmed to 50 °C. After cooling to room temperature, the solids were collected by filtration, washed with toluene (2 × 100 mL), and dried in a vacuum oven at 50 °C to provide 5 (149.2 g, 90% yield). 1H NMR (400 MHz, CDCl3) δ ppm 7.48−7.40 (m, 2H), 7.38−7.29 (m, 1H), 7.18 (dt, J = 8.0, 1.7 Hz, 2H), 6.10 (s, 1H), 2.65−2.39 (m, 4H), 2.15−2.04 (m, 2H). (8aR)-5-Methyl-8a-phenyl-3,4,8,8a-tetrahydronaphthalene1,6(2H,7H)-dione (6). A 5 L round-bottomed flask was charged with 5 (163 g, 903 mmol) in acetonitrile (1.5 L). Triethylamine (252 mL, 1805 mmol) and ethyl vinyl ketone (135 mL, 1.35 mol) were added, and the contents were warmed to 75 °C and stirred overnight. After cooling, the reaction mixture was concentrated. The residue was taken

previously hindered by the lack of high-quality tool compounds for this enzyme. Here we describe the activity and structural characterization for two novel IDH1 inhibitors which may now be used to further elaborate the important and complex cellular roles of IDH1.

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EXPERIMENTAL SECTION

General Procedures and Materials. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. Anhydrous solvents were obtained from Aldrich (Milwaukee, WI) and used directly. All reactions involving air- or moisture-sensitive reagents were performed under a nitrogen or argon atmosphere. NMR spectra were collected on either a Varian Inova 400 or 500 MHz spectrometer as specified with chemical shifts given in ppm (δ) and are referenced to an internal standard of tetramethylsilane (δ 0.00). 1H−1H couplings are assumed to be first order, and peak multiplicities are reported in the usual manner. Silica gel chromatography purifications were performed using prepacked silica gel cartridges (various vendors/various automated chromatography systems). Mass spectral ESI data were determined on a Thermo-Finnigan SSQ7000 spectrometer. Mass spectral APCI data were determined on either a Thermo-Finnigan Navigator or ThermoFinnigan MSQ-Plus mass spectrometer. All final compounds were purified to >95% purity as determined by analytical LCMS. Analytical LCMS was performed on an Agilent series 1100 HPLC system using a Phenomenex Luna Combi-HTS C8(2) 5 μm, 100 Å (2.1 mm × 50 mm) column at 65 °C. The gradient was 3% B for 0.02 min, 3−100% B in 1.28 min with a hold at 100% B for 0.15 min, then 100−3% B in 0.05 min (1.5 mL/min flow rate). The mobile phase A was 0.1% TFA in water, and mobile phase B was HPLC grade MeCN. Detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection under positive APCI ionization conditions or positive ESI ionization conditions. 6653



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residue was partitioned between methyl tert-butyl ether (1 L) and 10% aqueous ammonium chloride (500 mL). The layers were separated, and the organic layer was washed with 10% aqueous ammonium chloride (500 mL) and brine (200 mL). The organic solution was concentrated, taken up in ethanol (750 mL), and filtered. The product was solidified by the slow addition of water (1.125 L) and cooling the resultant slurry to 5 °C. The product was collected by filtration and washed with cold 40% ethanol in water (100 mL) and dried in a vacuum oven at 50 °C to provide (4aR,5S)-5-hydroxy-1methyl-4a-phenyl-4,4a,5,6,7,8-hexahydronaphthalen-2(3H)-one (99.0 g, 70%). 1H NMR (400 MHz, CDCl3) δ ppm 7.54−7.49 (m, 2H), 7.34−7.21 (m, 3H), 3.83 (ddd, J = 10.8, 4.6, 3.6 Hz, 1H), 2.80−2.71 (m, 1H), 2.51−2.42 (m, 1H), 2.36−2.28 (m, 1H), 2.26−2.04 (m, 3H), 2.02−1.97 (m, 1H), 1.93 (d, J = 1.3 Hz, 3H), 1.89−1.75 (m, 2H), 1.74−1.46 (m, 2H). A 2 L Parr stirred pressure reactor was charged with 13.2 g of 5% Pd/C (20 wt % of substrate charged). Under a stream of nitrogen, a solution of (4aR,5S)-5-hydroxy-1methyl-4a-phenyl-4,4a,5,6,7,8-hexahydronaphthalen-2(3H)-one (66 g, 0.257 mol) and tetrahydrofuran (530 mL) and pyridine (130 mL) was added to the reactor. The reactor was purged with nitrogen and hydrogen. The vessel was pressurized to and maintained at 60−100 psig with hydrogen supplied from a high-pressure reservoir of known volume. The mixture was vigorously agitated while keeping the temperature between 22 and 25 °C. The reaction mixture was carefully filtered to remove the palladium catalyst rinsing the reactor and cake with tetrahydrofuran. To the filtrate was added 1,8diazabicyclo[5.4.0]undec-7-ene (6 mL), and the resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was taken up in toluene (500 mL). The resulting solution was washed with 10% HCl (2 × 200 mL) and brine (100 mL), dried over sodium sulfate, and filtered to give the stable epimerized product in toluene solution that was used as is without purification. To this solution was added ethylene glycol (74 mL, 1.3 mol, 5 equiv) and p-toluenesulfonic acid (5 g, 26.5 mL), and the resulting mixture was heated to reflux with Dean−Stark removal of water. After reaction completion, the mixture was cooled to room temperature, and the toluene solution was washed with saturated aqueous sodium bicarbonate (2 × 200 mL) and brine (100 mL), dried over sodium sulfate, filtered, and concentrated to provide (1′S,4a′S,5′S,8a′S)-1′-methyl-4a′-phenyloctahydro-1′H-spiro[1,3-dioxolane-2,2′-naphthalen]-5′-ol which was dissolved in dichloromethane (800 mL), and pyridinium dichromate (199 g, 529 mmol) and magnesium sulfate (6.4 g) were added. The resulting mixture was heated to reflux and stirred overnight. After cooling to room temperature, the mixture was filtered through a plug of silica gel (350 g) rinsing with dichloromethane (2.5 L). The filtrate was concentrated, and the remaining solids were triturated with cyclohexane (300 mL). The solids were collected by filtration, washed with cyclohexane (2 × 50 mL), and dried in a vacuum oven at 50 °C to provide 7 (45.4 g, 27% yield). 1H NMR (400 MHz, CDCl3) δ ppm 7.45−7.14 (m, 5H), 4.00−3.88 (m, 4H), 2.71 (dq, J = 13.0, 6.5 Hz, 1H), 2.29−2.17 (m, 1H), 2.17−2.01 (m, 4H), 2.01−1.86 (m, 3H), 1.71−1.55 (m, 2H), 1.22−1.10 (m, 1H), 1.00 (d, J = 6.6 Hz, 3H). (1′’S,4a′’S,6′’Z,8a′S)-6′-(Hydroxymethylene)-1′-methyl-4a′phenylhexahydro-1′H-spiro[1,3-dioxolane-2,2′-naphthalen]5′(3′H)-one (8). To a solution of 7 (28.93 g, 96 mmol) in ethyl formate (240.0 mL, 2894 mmol) cooled to below 5 °C was added 1 M potassium tert-butoxide solution in tetrahydrofuran dropwise over 30 min. The solution was stirred at reduced temperature for an additional 30 min and then stirred at room temperature for 1 h. The solution was adjusted to pH 7 by addition of 13% potassium phosphate monobasic solution, and then the volatiles were removed under vacuum. The resulting solid was collected by filtration, washed with water, and dried. The solid was reprecipitated: the crude solid was dissolved in ethyl acetate (115 mL), then heptane (800 mL) was added, and the mixture was heated until all solid was dissolved. The solution was cooled and concentrated under vacuum to 50% volume and cooled in a refrigerator below 0 °C for 20 h. The solid was collected by filtration, washed with cold heptane (200 mL), and dried to give 8 (29.5 g, 93%). 1H NMR (400 MHz, CDCl3) δ ppm 0.99 (d,

Figure 5. Dose dependent decrease in reductive glutaminolysis by 13 and 3 (n = 3). A-498 cells were treated with the indicated concentration of (A) compound 13 or (B) compound 3 in the presence of 2 mM 1-13C-Gln for 5 h, and incorporation of the 13C label into citrate was measured. up in dimethyl sulfoxide (500 mL). Pyridinium p-toluenesulfonate (227 g, 903 mmol) and L-phenylalanine (149 g, 903 mmol) were added, and the contents were warmed to 45 °C for 66 h. After cooling, the reaction mixture was poured into saturated aqueous NH4Cl (500 mL), water (500 mL), and methyl tert-butyl ether (500 mL). After mixing for 10 min, the solids were removed by filtration and the layers separated. The aqueous layer was extracted with methyl tert-butyl ether (500 mL). The combined organic layers were washed with water (300 mL) and brine (300 mL), dried over sodium sulfate, filtered, and concentrated. The residue was purified by chromatography over silica gel on a Teledyne Isco CombiFlash Torrent system using a 750 g RediSep silica gel column: ethyl acetate/hexanes 0:1 (2 column volumes) up to 1:1 (gradient over 8 column volumes). Fractions containing product were combined and concentrated under reduced pressure. This crude product (65% ee) was taken up in cyclohexane (500 mL) and stirred overnight. The racemic solids were removed by filtration rinsing with cyclohexane (2 × 100 mL), and the filtrate was concentrated to provide 6 in 95% ee determined using a Chiralpak AS-H column (4.6 mm i.d. × 25 cm, 5 μm) eluting with 10% EtOH in heptane at 1.0 mL/min with the major enantiomer eluting at 10.4 min and the minor enantiomer eluting at 15.1 min. (140 g, 61%). 1H NMR (400 MHz, CDCl3) δ ppm 7.38−7.25 (m, 3H), 7.15−7.10 (m, 2H), 2.82−2.72 (m, 1H), 2.68−2.58 (m, 1H), 2.57−2.48 (m, 1H), 2.46−2.29 (m, 3H), 2.25−2.18 (m, 1H), 2.11− 2.00 (m, 1H), 1.97 (s, 3H), 1.86−1.65 (m, 2H). (1′S,4a′S,8a′S)-1′-Methyl-4a′-phenylhexahydro-1′H-spiro[1,3-dioxolane-2,2′-naphthalen]-5′(3′H)-one (7). A 3 L jacketed round-bottomed flask was charged with 6 (140.5 g, 552 mmol) in ethanol (1.4 L), and the solution was cooled to −5 °C. A solution of sodium borohydride (6.28 g, 166 mmol) in ethanol (1.0 L) was added dropwise while maintaining an internal temperature below 0 °C. The resulting mixture was stirred at −5 °C for 4 h. The reaction mixture was quenched carefully with acetic acid (100 mL) and warmed to 23 °C. After stirring overnight, the mixture was concentrated. The 6654



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Cell Culture. A-498 cells were purchased from ATCC and grown in Eagle’s minimum essential medium containing 10% fetal bovine serum.

J = 6.51 Hz, 3 H), 1.29 (td, J = 13.77, 3.69 Hz, 1 H), 1.63 (dt, J = 13.53, 3.48 Hz, 1 H), 1.80−2.23 (m, 8 H), 2.55 (dt, J = 17.97, 6.47 Hz, 1 H), 3.95 (s, 4 H), 7.20−7.35 (m, 3 H), 7.49 (d, J = 7.70 Hz, 2 H), 13.78 (d, J = 9.87 Hz, 1 H). (6aS,7S,11aS)-7-Methyl-N,11a-diphenyl-5,6,6a,7,11,11ahexahydro[1,2]benzoxazolo[6,5-h]quinazolin-2-amine (12). To a solution of 8 (0.500 g, 1.52 mmol) in dimethylformamide (6 mL) was added phenylguanidine carbonate salt (0.540 g, 2.74 mmol), and the solution was heated at 90 °C for 18 h. The cooled solution was diluted with NaH2PO4 solution and extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulfate, concentrated, and dissolved in THF (5 mL) and then treated with 3 M HCl (2.53 mL, 7.60 mmol) and stirred at ambient temperature for 3 h. The solution was diluted with water, neutralized with sodium bicarbonate, and extracted into ethyl acetate. The organic layer was concentrated and purified by 24 g CombiFlash cartridge, eluting with 0−50% ethyl acetate in heptane to give (6aS,7S,10aS)-2-anilino-7methyl-10a-phenyl-5,6a,7,9,10,10a-hexahydrobenzo[h]quinazolin8(6H)-one (0.25 g 42%). This was treated with ethyl formate (3 mL, 36.9 mmol) and 25% sodium methoxide in methanol (1.0 mL, 4.6 mmol). The reaction mixture was stirred at room temperature for 2 h. The solution was diluted with saturated aqueous NaH2PO4 solution and extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered, and concentrated to provide (6aS,7S,9Z,10aS)-2-anilino-9-(hydroxymethylene)-7-methyl-10a-phenyl5,6a,7,9,10,10a-hexahydrobenzo[h]quinazolin-8(6H)-one (0.237 g, 88%) which was dissolved in ethanol (5 mL) and treated with hydroxylamine hydrochloride (0.080 g, 1.152 mmol). The reaction mixture was heated at 65 °C for 2 h. The cooled solution was diluted with water, neutralized to pH 7 with saturated aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The organic layer was concentrated, and the residue was purified on a Teledyne Isco CombiFlash Rf system using a RediSep 12 g silica gel cartridge, eluting with 0−70% ethyl acetate in heptane to give 12 (0.147 g, 63%). 1H NMR (400 MHz, CDCl3) δ ppm 1.38 (d, J = 6.83 Hz, 3 H), 1.72−1.82 (m, 1 H), 1.93−2.12 (m, 2 H), 2.20−2.32 (m, 1 H), 2.76−2.92 (m, 1 H), 2.95−3.07 (m, 2 H), 3.77 (d, J = 16.81 Hz, 1 H), 6.85 (dd, J = 6.78, 2.98 Hz, 2 H), 6.98 (t, J = 7.37 Hz, 1 H), 7.07 (s, 1 H), 7.11−7.18 (m, 3 H), 7.27 (t, J = 7.92 Hz, 2 H), 7.50 (d, J = 7.70 Hz, 2 H), 8.28 (s, 1 H), 8.34 (s, 1 H); MS (APCI+) m/z 409 (M + H)+. (6aS,7S,10aR)-2-Anilino-7-methyl-8-oxo-10a-phenyl5,6,6a,7,8,10a-hexahydrobenzo[h]quinazoline-9-carbonitrile (13). To a solution of 12 (0.147 g, 0.895 mmol) in methanol (1.5 mL) and tetrahydrofuran (1.5 mL) was added 25% sodium methoxide in methanol (0.391 mL, 1.799 mmol). The reaction solution was stirred at room temperature for 2 h, then diluted with saturated aqueous NaH2PO4 solution, and extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered, and concentrated to provide (6aS,7S,10aS)-2-anilino-7-methyl-8-oxo-10aphenyl-5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazoline-9-carbonitrile. To a solution of (6aS,7S,10aS)-2-anilino-7-methyl-8-oxo-10aphenyl-5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazoline-9-carbonitrile (0.147 g, 0.360 mmol) in dimethylformamide (4 mL) cooled to 0 °C in an ice−water bath was added 1,3-dibromo-5,5-dimethylhydantoin (0.051 g, 0.180 mmol). The solution was stirred at 0 °C for 1 h, pyridine (0.291 mL, 3.60 mmol) was added, and the solution was heated at 50 °C for 2 h. The cooled solution was diluted with water and extracted with ethyl acetate. The organic phase was washed with brine and concentrated. The residue was purified on 12 g RediSep silica gel cartridge using a Teledyne Isco CombiFlash Rf system eluting with 0−25% ethyl acetate in heptane to give 13 (0.142 g, 50% yield). 1H NMR (400 MHz, CDCl3) δ ppm 1.19 (d, J = 6.72 Hz, 3 H), 1.48−1.67 (m, 1 H), 1.92 (dd, J = 13.66, 7.92 Hz, 1 H), 2.28− 2.38 (m, 1 H), 2.45 (td, J = 13.20, 6.67 Hz, 1 H), 2.83−3.06 (m, 2 H), 6.90 (dd, J = 7.92, 1.63 Hz, 2 H), 6.99−7.08 (m, 2 H), 7.28−7.39 (m, 5 H), 7.49 (d, J = 7.70 Hz, 2 H), 8.41 (s, 1 H), 8.78 (s, 1 H); MS (ESI+) m/z 407 (M + H)+, 439 (M + CH3OH + H)+.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00305. Molecular formula strings and some data (CSV) References to chemical synthesis for compounds 10, 11, 14, and 19 and full synthetic details for the preparation of previously unreported compounds 1 and 15−18; Table SI-4 listing X-ray data collection and refinement statistics; protocols for intracelluar concentrations for 11 and 13; orthogonal biophysical confirmation for 11 and 13 by TSA and ITC, IDH1 enzyme assay, and diaphorase counterscreening protocols; MS and covalent linking experimental details; full description of methods for 13C citrate flux measurements (PDF) Accession Codes

PDB codes for ligand bound structures of IDH1: 1 (6BKX), 2 (6BKY), 3 (6BKZ), 11 (6BL0), 13 (6BL1), 15 (6BL2). Authors will release the atomic coordinates and experimental data upon article publication.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (847) 937-5099. E-mail: [email protected]. ORCID

Anup K. Upadhyay: 0000-0002-7536-2183 Anil Vasudevan: 0000-0002-0004-0497 J. Brad Shotwell: 0000-0002-7758-146X Notes

The authors declare the following competing financial interest(s): All authors are current employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

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ACKNOWLEDGMENTS Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DEAC02-06CH11357. We thank Keith Woods for initial chemical triage, coordinating confirmation of initial hits, and resyntheses. We thank Anthony Mastracchio for coordinating syntheses and evaluation of reduced control compounds. We thank Thomas Penning, Michael Michaelides, Scott Ackler, and Stormy Koeniger for helpful discussions.

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ABBREVIATIONS USED IDH, isocitrate dehydrogenase; LPS, lipopolysaccharide; TSA, thermal shift assay; AML, acute myeloid leukemia; NADPH, nicotinamide adenine dinucleotide phosphate; WT, wild type; TCA, tricarboxylic acid cycle/Krebs cycle/citric acid cycle; 6655



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αKG, α-ketoglutarate; GBM, glioblastoma; ROS, reactive oxygen species; ITC, isothermal titration calorimetry

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isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule. J. Biol. Chem. 2015, 290, 762−774. (10) Okoye-Okafor, U. C.; Bartholdy, B.; Cartier, J.; Gao, E. N.; Pietrak, B.; Rendina, A. R.; Rominger, C.; Quinn, C.; Smallwood, A.; Wiggall, K. J.; Reif, A. J.; Schmidt, S. J.; Qi, H.; Zhao, H.; Joberty, G.; Faelth-Savitski, M.; Bantscheff, M.; Drewes, G.; Duraiswami, C.; Brady, P.; Groy, A.; Narayanagari, S. R.; Antony-Debre, I.; Mitchell, K.; Wang, H. R.; Kao, Y. R.; Christopeit, M.; Carvajal, L.; Barreyro, L.; Paietta, E.; Makishima, H.; Will, B.; Concha, N.; Adams, N. D.; Schwartz, B.; McCabe, M. T.; Maciejewski, J.; Verma, A.; Steidl, U. New IDH1 mutant inhibitors for treatment of acute myeloid leukemia. Nat. Chem. Biol. 2015, 11, 878−886. (11) Popovici-Muller, J.; Saunders, J. O.; Salituro, F. G.; Travins, J. M.; Yan, S.; Zhao, F.; Gross, S.; Dang, L.; Yen, K. E.; Yang, H.; Straley, K. S.; Jin, S.; Kunii, K.; Fantin, V. R.; Zhang, S.; Pan, Q.; Shi, D.; Biller, S. A.; Su, S. M. Discovery of the First Potent Inhibitors of Mutant IDH1 That Lower Tumor 2-HG in Vivo. ACS Med. Chem. Lett. 2012, 3, 850−855. (12) Rohle, D.; Popovici-Muller, J.; Palaskas, N.; Turcan, S.; Grommes, C.; Campos, C.; Tsoi, J.; Clark, O.; Oldrini, B.; Komisopoulou, E.; Kunii, K.; Pedraza, A.; Schalm, S.; Silverman, L.; Miller, A.; Wang, F.; Yang, H.; Chen, Y.; Kernytsky, A.; Rosenblum, M. K.; Liu, W.; Biller, S. A.; Su, S. M.; Brennan, C. W.; Chan, T. A.; Graeber, T. G.; Yen, K. E.; Mellinghoff, I. K. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 2013, 340, 626−630. (13) Itsumi, M.; Inoue, S.; Elia, A. J.; Murakami, K.; Sasaki, M.; Lind, E. F.; Brenner, D.; Harris, I. S.; Chio, II; Afzal, S.; Cairns, R. A.; Cescon, D. W.; Elford, A. R.; Ye, J.; Lang, P. A.; Li, W. Y.; Wakeham, A.; Duncan, G. S.; Haight, J.; You-Ten, A.; Snow, B.; Yamamoto, K.; Ohashi, P. S.; Mak, T. W. IDH1 protects murine hepatocytes from endotoxin-induced oxidative stress by regulating the intracellular NADP(+)/NADPH ratio. Cell Death Differ. 2015, 22, 1837−1845. (14) Ye, J.; Gu, Y.; Zhang, F.; Zhao, Y.; Yuan, Y.; Hao, Z.; Sheng, Y.; Li, W. Y.; Wakeham, A.; Cairns, R. A.; Mak, T. W. IDH1 deficiency attenuates gluconeogenesis in mouse liver by impairing amino acid utilization. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 292−297. (15) Metallo, C. M.; Gameiro, P. A.; Bell, E. L.; Mattaini, K. R.; Yang, J.; Hiller, K.; Jewell, C. M.; Johnson, Z. R.; Irvine, D. J.; Guarente, L.; Kelleher, J. K.; Vander Heiden, M. G.; Iliopoulos, O.; Stephanopoulos, G. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 2012, 481, 380−384. (16) Jiang, L.; Shestov, A. A.; Swain, P.; Yang, C.; Parker, S. J.; Wang, Q. A.; Terada, L. S.; Adams, N. D.; McCabe, M. T.; Pietrak, B.; Schmidt, S.; Metallo, C. M.; Dranka, B. P.; Schwartz, B.; DeBerardinis, R. J. Reductive carboxylation supports redox homeostasis during anchorage-independent growth. Nature 2016, 532, 255−258. (17) Mullen, A. R.; Wheaton, W. W.; Jin, E. S.; Chen, P. H.; Sullivan, L. B.; Cheng, T.; Yang, Y.; Linehan, W. M.; Chandel, N. S.; DeBerardinis, R. J. Reductive carboxylation supports growth in tumour cells with defective mitochondria. Nature 2012, 481, 385− 388. (18) Wise, D. R.; Ward, P. S.; Shay, J. E.; Cross, J. R.; Gruber, J. J.; Sachdeva, U. M.; Platt, J. M.; DeMatteo, R. G.; Simon, M. C.; Thompson, C. B. Hypoxia promotes isocitrate dehydrogenasedependent carboxylation of alpha-ketoglutarate to citrate to support cell growth and viability. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 19611−19616. (19) Xu, X.; Zhao, J.; Xu, Z.; Peng, B.; Huang, Q.; Arnold, E.; Ding, J. Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity. J. Biol. Chem. 2004, 279, 33946−33957. (20) Zhao, S.; Guan, K. L. IDH1 mutant structures reveal a mechanism of dominant inhibition. Cell Res. 2010, 20, 1279−1281. (21) Lowther, W. T.; McMillen, D. A.; Orville, A. M.; Matthews, B. W. The anti-angiogenic agent fumagillin covalently modifies a conserved active-site histidine in the Escherichia coli methionine

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Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1

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