Biochemical, cellular, and biophysical characterization of a potent inhibitor of mutant isocitrate dehydrogenase IDH1

Article abstract of DOI:10.1074/jbc.M113.511030

Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC₅₀ = 68 nM), whereas the (-) isomer is over 400-fold less active (IC₅₀ = 29 μM) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)- ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC ₅₀ >36 μM). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α- ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC₅₀=250 nM) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.

Full text of DOI:10.1074/jbc.M113.511030

Molecular Bases of Disease:
Biochemical, Cellular, and Biophysical
Characterization of a Potent Inhibitor of
Mutant Isocitrate Dehydrogenase IDH1
Mindy I. Davis, Stefan Gross, Min Shen,
Kimberly S. Straley, Rajan Pragani, Wendy A.
Lea, Janeta Popovici-Muller, Byron
DeLaBarre, Erin Artin, Natasha Thorne,
Douglas S. Auld, Zhuyin Li, Lenny Dang,
Matthew B. Boxer and Anton Simeonov
J. Biol. Chem. 2014, 289:13717-13725.
doi: 10.1074/jbc.M113.511030 originally published online March 25, 2014
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 20, pp. 13717–13725, May 16, 2014
Published in the U.S.A.
Biochemical, Cellular, and Biophysical Characterization of a
Potent Inhibitor of Mutant Isocitrate Dehydrogenase IDH1*
Received for publication, September 5, 2013, and in revised form, March 5, 2014 Published, JBC Papers in Press, March 25, 2014, DOI 10.1074/jbc.M113.511030
Mindy I. Davis‡1, Stefan Gross§1, Min Shen‡, Kimberly S. Straley§, Rajan Pragani‡, Wendy A. Lea‡,
Janeta Popovici-Muller§, Byron DeLaBarre§, Erin Artin§, Natasha Thorne‡, Douglas S. Auld‡¶, Zhuyin Li‡,
Lenny Dang§, Matthew B. Boxer‡, and Anton Simeonov‡2
From the ‡NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health,
Rockville, Maryland 20892, §Agios Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, and the ¶Center for Proteomic
Chemistry, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139
Background: IDH1 R132H, implicated in glioblastoma and AML, produces the oncometabolite 2-HG.
Results: A detailed binding mechanism of a small molecule inhibitor (ML309) is proposed.
Conclusion: ML309 competes with ␣-KG but is uncompetitive with NADPH and rapidly and reversibly affects cellular 2-HG
levels.
Significance: Understanding IDH1 R132H inhibition sets the stage for targeting IDH1 R132H for the treatment of cancer.
Two mutant forms (R132H and R132C) of isocitrate dehydro-
genase 1 (IDH1) have been associated with a number of cancers
Recent advances in tumor genome analysis have suggested
novel roles for previously unappreciated genes in establish-
including glioblastoma and acute myeloid leukemia. These ment and maintenance of the oncogenic state (1–3). Muta-
tions in isocitrate dehydrogenase 1 (IDH1),3 a metabolic
enzyme responsible for the conversion of isocitrate to ␣-keto-
glutarate (␣-KG), were annotated at a high frequency in an
analysis of 22 human glioblastoma multiforme tumor samples.
Interestingly, all these mutations were G395A, resulting in the
conversion of an arginine at position 132 to a histidine (4).
Because proteins with the IDH1 R132H missense mutation dis-
play decreased efficiency in the conversion of isocitrate to
␣-KG in vitro because Arg-132 is one of the substrate-binding
arginine triads in the enzyme active site, these were at first
believed to be loss-of-function mutations (5). However, the dis-
covery of gain of function where IDH1 R132H results in a neo-
morphic enzymatic activity (Fig. 1), namely the conversion of
␣-KG to 2-hydroxyglutarate (2-HG), has profound implica-
tions for the role of IDH1 and its close homologue IDH2 in the
metabolic activities of the cancer cell (6). As a dead-end metab-
olite, 2-HG accumulates to millimolar levels in cells with neo-
active IDH1 (i.e. R132H or R132C) and IDH2 mutations (i.e.
R172K) (7), and acts as an inhibitor of the ␣-KG-dependent
epigenetic machinery (8, 9), blocking differentiation and pro-
moting the proliferation of undifferentiated tumorous cells. It
has recently been shown that 2-HG alone can promote leuke-
mogenesis (10). Additionally, 2-HG suppresses the tricarboxy-
lic acid (TCA) cycle and results in enhanced lipid metabolism
(11). Inhibitors of 2-HG production by mutant IDH1 and IDH2
could have important clinical applications in the treatment of
IDH mutated glioblastoma and acute myeloid leukemia (4, 5,
12, 13). Moreover, such inhibitors could help elucidate mecha-
nism by which these mutations function in the context of the
cancer cell metabolome. Therefore, there is a need for the
development of inhibitors for mutant IDH1 and to gain an
understanding of their mechanisms of action.
mutations confer a neomorphic activity of 2-hydroxyglutarate
(2-HG) production, and 2-HG has previously been implicated as
an oncometabolite. Inhibitors of mutant IDH1 can potentially
be used to treat these diseases. In this study, we investigated the
mechanism of action of a newly discovered inhibitor, ML309,
using biochemical, cellular, and biophysical approaches. Sub-
strate binding and product inhibition studies helped to further
elucidate the IDH1 R132H catalytic cycle. This rapidly equili-
brating inhibitor is active in both biochemical and cellular
assays. The (؉) isomer is active (IC50 ؍ 68 nM), whereas the (؊)
isomer is over 400-fold less active (IC50 ؍ 29 ␮M) for IDH1
R132H inhibition. IDH1 R132C was similarly inhibited by (؉)-
ML309. WT IDH1 was largely unaffected by (؉)-ML309 (IC50
>36 ␮M). Kinetic analyses combined with microscale thermo-
phoresis and surface plasmon resonance indicate that this
reversible inhibitor binds to IDH1 R132H competitively with
respect to ␣-ketoglutarate and uncompetitively with respect to
NADPH. A reaction scheme for IDH1 R132H inhibition by
ML309 is proposed in which ML309 binds to IDH1 R132H after
formation of the IDH1 R132H NADPH complex. ML309 was
also able to inhibit 2-HG production in a glioblastoma cell line
(IC50 ؍ 250 nM) and had minimal cytotoxicity. In the presence of
racemic ML309, 2-HG levels drop rapidly. This drop was sus-
tained until 48 h, at which point the compound was washed out
and 2-HG levels recovered.
* This research was supported by the Molecular Libraries Initiative of the
National Institutes of Health Roadmap for Medical Research, National Insti-
tutes of Health (U54 MH084681).
1 Both authors contributed equally to this work.
2 To whom correspondence should be addressed: NIH Chemical Genomic
Center, National Center for Advancing Translational Sciences, National 3 The abbreviations used are: IDH1, isocitrate dehydrogenase 1; ␣-KG, ␣-
Institutes of Health, 9800 Medical Center Dr., Rockville, MD 20892. Tel.:
301-217-5721; Fax: 301-217-5736; E-mail: asimeono@mail.nih.gov.
ketoglutarate; PK, pharmacokinetics; 2-HG, 2-hydroxyglutarate; rac-
ML309, racemic ML309; DMSO, dimethyl sulfoxide.
MAY 16, 2014•VOLUME 289•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 13717

Mutant IDH1 Inhibitor Mechanism of Action
molecular formulae was accomplished using electrospray ioni-
zation in the positive mode with the Agilent Masshunter soft-
ware (version B.02).
Preparation of (Ϫ)- and (ϩ)-2-(2-(1H-Benzo[d]imidazol-1-
yl)-N-(3-fluorophenyl)acetamido)-N-cyclopentyl-2-o-tolylacet-
amide ((Ϫ)-ML309 and (ϩ)-ML309)—A solution of isocyano-
cyclopentane (196 mg, 2.064 mmol), 3-fluoroaniline (229 mg,
2.064 mmol), and 2-(1H-benzo[d]imidazol-1-yl)acetic acid
(364 mg, 2.064 mmol) in MeOH (6.9 ml) was treated with
2-methylbenzaldehyde (248 mg, 2.064 mmol). The reaction was
warmed to 40 °C and stirred for 4 h and then concentrated
under reduced pressure. The crude product was purified by
silica chromatography (2:98 to 5:95 MeOH/dichloromethane),
which afforded racemic ML309 (rac-ML309) (216 mg, 0.446
mmol) in 22% yield.
FIGURE 1. Enzyme reactions catalyzed by WT IDH1 and IDH1 R132H.
A previously reported high-throughput screen identified the
first potent series of inhibitors of IDH1 R132H that were fur-
ther optimized (14). The series consists of a phenyl-glycine
scaffold with one stereocenter. One enantiomer was shown to
be predominantly responsible for the activity of the racemic mix-
ture. The inhibitor series was selective for mutant IDH1 over wild-
type (WT) IDH1 and had excellent cell activity (IC50 ϭ 70 nM),
including the ability to lower 2-HG levels by ϳ90% in an in vivo
U87MG IDH1 R132H mouse tumor xenograft model (14).
Recently, a member of this series was shown to delay growth and
promote differentiation of glioma cells (15).
rac-ML309 (216 mg, 0.446 mmol) was separated by chiral
chromatography to afford (Ϫ)-ML309 (73 mg, 0.151 mmol,
Ͼ99% enantiomeric excess) and (ϩ)-ML309 (66 mg, 0.136
mmol, Ͼ99% enantiomeric excess). (Ϫ)-ML309: LC-MS reten-
tion time: t1 (Chiralcel OD column; 60:40 EtOH/hexane iso-
cratic) ϭ 2.91 min; [␣]D22 ϭ Ϫ72.1° (c ϭ 0.86, MeOH); 1H NMR
(400 MHz, MeOH-d4) ␦ ppm 8.01 (s, 1 H), 7.89 (br s, 1 H), 7.65
(d, J ϭ 7.8 Hz, 1 H), 7.41 (d, J ϭ 7.8 Hz, 1 H), 7.32 (t, J ϭ 6.4 Hz,
1 H), 7.27 (t, J ϭ 6.8 Hz, 1 H), 7.14 (d, J ϭ 6.8 Hz, 1 H), 7.07 (t, J ϭ
7.2 Hz, 1 H), 6.99 (dt, J ϭ 2.4, 8.0 Hz, 1 H), 6.88 (t, J ϭ 7.2 Hz, 1
H), 6.81 (d, J ϭ 7.2 Hz, 1 H), 6.56 (br s, 1H), 6.36 (s, 1H), 5.02 (d,
J ϭ 17.2 Hz, 1 H), 4.81 (d, J ϭ 17.6 Hz, 1 H), 4.18 (tt, J ϭ 6.8, 6.8
Hz, 1 H), 2.45 (s, 3 H), 1.94–1.83 (m, 2 H), 1.67–1.27 (m, 8 H);
19F NMR (282 MHz, MeOH-d4) ␦, Ϫ113.03, Ϫ114.12 ppm; high
resolution mass spectrometry (electrospray mass ionization)
ML309, described herein, is a newly identified and character-
ized member of the phenyl-glycine series. ML309 is active in
both biochemical and cell assays. The time dependence of the
effect on 2-HG levels in cells was explored. To gain a deeper
understanding of how the substrates, and phenyl-glycine scaf-
fold inhibitors, such as ML309, interact with IDH1 R132H
enzyme, a detailed characterization using kinetic and biophys-
ical approaches was undertaken. Based on these results, a com-
pound binding model was proposed that provides a plausible
explanation of the inhibitory mechanism and that can be used
for future structure and activity relationship studies.
ϩ
EXPERIMENTAL PROCEDURES
m/z (MϩH) calculated for C29H29FN4O2, 485.2385; found
General Methods for Chemistry—All air- or moisture-sen- 485.2335. (ϩ)-ML309: LC-MS retention time: t1 (Chiralcel OD
sitive reactions were performed under positive pressure of column; 60:40 EtOH/hexane isocratic) ϭ 5.56 min; [␣]D22 ϭ ϩ
nitrogen with oven-dried glassware. Anhydrous solvents, such 75.3° (c ϭ 0.76, MeOH); high resolution mass spectrometry
ϩ
as dichloromethane, N,N-dimethylformamide, acetonitrile, (electrospray mass ionization) m/z (MϩH) calculated for
methanol, and triethylamine, were purchased from Sigma-Al- C29H29FN4O2, 485.2385; found 485.2367.
drich. Analytical analysis was performed on an Agilent 1200
Absorption, Distribution, Metabolism, and Excretion, PK, Solu-
series LC/MS (Agilent Technologies, Santa Clara, CA). Sample bility, and Chemical Stability—rac-ML309 stability in aqueous
dissolution in MeOH and analysis were performed at room solution, 5 mM glutathione, assay buffer, mouse and human
temperature. The analytical column used was a Chiralcel OD microsomes, and human plasma and aqueous solubility were
column (4.6 ϫ 150 mm, 5 ␮m). The mobile phase was 60:40 tested following methods described previously (16, 17). A 20
ethanol (absolute, 200 proof)/hexane (0.1% diethyl amine) at mM DMSO stock of rac-ML309 was dispensed in assay buffer
1.0 ml/min. The sample was detected with a diode array detec- (final concentrations of 2% DMSO, 15% MeCN, 83% assay
tor at 220 and 254 nm. Optical rotation was determined with an buffer), 5 mM glutathione (final concentrations of 2% DMSO,
in-line polarimeter (PDR-Chiral). Purity and enantiomeric 49% MeCN, 49% PBS), or Dulbecco’s PBS buffer (2% DMSO,
excess were determined by this analytical method. All of the 25% MeCN, 73% Dulbecco’s PBS), and the remaining com-
analogues tested in the biological assays have a purity greater pound was monitored by the area under the curve of the peak at
than 95%. Preparative purification of racemic material was per- 220 nm by LC/MS at 0, 0.5, 1, 2, 6, 24, and 48 h. The half-life of
formed on an Agilent 1200 series Prep/LC (Agilent Technolo- rac-ML309 was tested by in vivo mouse pharmacokinetics (PK)
gies, Santa Clara, CA). The column used was a Chiralcel OD using wild-type BALB/c mice (18).
column (5 ϫ 50 cm, 20 ␮m). The mobile phase was 60:40 eth-
General Methods for Biology—Full-length IDH1 (EC 1.1.1.42
anol (absolute, 200 proof)/hexane (0.1% diethyl amine) at 35 and UniProt accession number O75874), IDH1 R132H, and
ml/min. Fraction collection was triggered by UV absorbance IDH1 R132C were expressed and purified as described pre-
1
(220 nm). H NMR spectra were recorded on a Varian 400- viously (6, 14). Both IDH1 R132H and IDH1 R132C protein
MHz spectrometer. Chemical shifts are reported in ppm were expressed with N-terminal His6 tags and purified by
(CHD2OD at 3.31 ppm as internal standard) for MeOH-d4 standard metal-chelate affinity chromatography techniques;
solutions. High resolution mass spectrometry was recorded on IDH1 R132H was expressed in Escherichia coli, and IDH1
Agilent 6210 time-of-flight LC/MS system. Confirmation of R132C was expressed in insect cells using the BaculoDirect
13718 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 20•MAY 16, 2014

Mutant IDH1 Inhibitor Mechanism of Action
(Invitrogen) baculovirus expression system. Proteins were buf- ML309 was titrated, i.e. ␣-KG was fixed at 150 mM and rac-
fer-exchanged by G25 gel-filtration chromatography into 500
mM NaCl, 50 mM Tris, pH 8.0, 10% glycerol and stored at
Ϫ80 °C. NADPH, ␣-KG, and buffer components were pur-
chased from Sigma. Experiments were run at room tempera-
ture unless otherwise specified.
ML309 was titrated down from 500 ␮M or NADPH was fixed at
50 ␮M and rac-ML309 was titrated down from 500 ␮M. The
final DMSO concentration for each protein-compound sample
was 0.5% DMSO. Samples were loaded into Monolith NT
hydrophilic treated capillaries (NanoTemper Technologies). A
capillary scan was performed followed by the successive mea-
surement of thermophoresis in each capillary on a NanoTem-
per Monolith NT.115 instrument at room temperature. The
IR laser power and the LED power conditions are described
in the figure legends. A laser on-time of 30 s and a laser-off
time of 5 s were used. The experiment was performed in
duplicate, and the fits are reported as mean Ϯ S.D. Data
normalization and curve fitting were performed using
GraphPad Prism 5.
IDH1 R132H, IDH1 R132C, and IDH1 WT Biochemical
Assays—The activity of IDH1 R132H and IDH1 R132C was
measured in 384-well plates by coupling NADPH consumption
to a diaphorase/resazurin-based detection system and mea-
suring resorufin production (Ex544/Em590) as described previ-
ously (6, 14). For testing the potency of small molecule inhibi-
tors, an end point-coupled assay system was used, with 2–4 nM
enzyme. In this assay, the reactions were run in the ␣-KG to
2-HG direction with the concomitant oxidation of NADPH to
NADP. For IDH1 R132H, a final concentration of 1 mM ␣-KG
and 4 ␮M NADPH was used; for IDH1 R132C, a final concen-
tration of 200 ␮M ␣-KG and 4 ␮M NADPH was used. The
NADPH remaining at the end of a 1-h incubation reaction was
measured by the addition of 20 ␮M resazurin and 10 ␮g/ml
diaphorase, which facilitated the stoichiometric conversion of
resazurin to the highly fluorescent resorufin. To test the revers-
ibility of the binding of compound to enzyme, 10ϫ IC50 of the
compound and 100ϫ enzyme were incubated for 1 h, at which
point the sample was diluted 100-fold and the enzyme reaction
was run in kinetic mode monitoring the consumption of
NADPH at 340 nm. Data were normalized to control columns
representing maximum signal (no enzyme) and minimum sig-
nal (all components). WT IDH1 activity was also measured with
this coupled diaphorase/resazurin system, but in this case prod-
uct (NADPH) could be measured directly. Readout interfer-
ence was tested with a counterscreen assay that contained all
components except the proteins. The data were fit in GraphPad
Prism 5 (La Jolla, CA) using nonlinear least-squares curve
fitting.
Surface Plasmon Resonance Binding Measurements—Bind-
ing experiments between IDH1 R132H and either NADPH or
NADPH/rac-ML309 were performed using SPR measurements
on a ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.) at
25 °C. IDH1 R132H (“ligand”) was immobilized at high surface
densities (6000–7000 resonance units) on an activated
ProteOn HTE nickel-nitrilotriacetic acid sensor chip (Bio-Rad
Laboratories, Inc.) through the His tag on the IDH1 R132H
protein according to the manufacturer’s instructions. The
ligand was injected five times at a concentration of 20 ␮g/ml at
a flow rate of 30 ␮l/min for 1000 s across two of the six available
channels. To perform the binding assays, the various com-
pounds (“analytes”) were injected at different concentrations in
running buffer at a flow rate of 100 ␮l/min for 120 s (NADPH or
rac-ML309 alone) or 180 s (rac-ML309 with NADPH), and sen-
sorgrams were recorded. Blank surfaces and interspots (a fea-
ture of the ProteOn interaction array system) were used for
background corrections using the ProteOn software. The sen-
sorgrams were fit using the ProteOn analysis software. The pro-
tein rapidly lost activity on the chip, and therefore the chip was
regenerated and the ligand was reloaded for each enzyme-com-
pound test. The analytes tested included NADPH (a five-point
1:3 dilution series starting at 1 ␮M) and NADPH/rac-ML309 (at
1 ␮M NADPH constant and a five-point 1:3 dilution of rac-
ML309 starting at 3 ␮M). The running buffer used for ligand
loading and testing of NADPH alone or rac-ML309 alone was
TBS. For the tests with rac-ML309 and NADPH co-injections,
the running buffer was TBS with 1 ␮M NADPH and 0.25%
DMSO.
Stopped-Flow Kinetics and Assessment of Km(app), Ki, and
V
max(app)—For determination of the Km(app) of substrates,
IDH1 R132H was assayed in 150 mM NaCl, 20 mM Tris-Cl, pH
7.5, 10 mM MgCl2, 0.03% BSA. Reactions where studied in an
SFM-400 stop-flow spectrophotometer by monitoring the oxi-
dation state of the co-factor at Ex340/Em450 and used 5–100 nM
recombinant protein. Typical reactions were monitored for ϳ5
s, and steady-state rates were extracted by linear regression
with fluorescence converted to NADPH concentration by a
standard curve of NADPH in assay buffer. The equations used
to fit the substrate inhibition and product inhibition are
described in detail in Ref. 19.
Cell Line Culture (U87)—U87MG (human glioblastoma)
cells were obtained from American Type Culture Collection
(ATCC) (Manassas, VA). U87MG is cultured in RPMI 1640, 2
mM L-Glutamine, 10 units/ml penicillin/streptomycin, and 10%
FBS (Life Technologies). Cells were maintained in 5% CO2 at
37 °C.
Microscale Thermophoresis Measurements of Binding Kinetics—
IDH1 R132H was labeled with a fluorescent dye NT-647 using
the manufacturer’s protocol (NanoTemper Technologies,
München, Germany). A 16-point titration series of rac-ML309
in DMSO, NADPH in water, ␣-KG in water, or combinations
thereof was transferred to the protein (20 nM) in a buffer con-
taining 0.05% BSA, 0.01% Tween 20, 2 mM ␤-mercaptoethanol,
20 mM Tris, pH 7.5, and 150 mM NaCl and equilibrated for 15
min. The final top concentrations were 25 ␮M for NADPH, 25
mM ␣-KG, or 500 ␮M rac-ML309 for the single agent tests. For
Generation of IDH1 R132H-expressing Cell Lines—U87MG
cells were infected with lentivirus containing full-length IDH1
R132H (U87MG pLVX-IDH1 R132H-neo or U87MG IDH1
R132H for short). For infection, cells were plated with the
viral supernatant supplemented with 8 ␮g/ml Polybrene
(5.0 ϫ 104 cells/ml viral supernatant) and incubated in 5%
the competition tests, the substrate was held constant and rac- CO2 at 32 °C for 24 h. After infection, transduced cells were
MAY 16, 2014•VOLUME 289•NUMBER 20 JOURNAL OF BIOLOGICAL CHEMISTRY 13719

Mutant IDH1 Inhibitor Mechanism of Action
FIGURE 2. Biochemical and cellular IC50 values for ML309. A, chemical structure of ML309. B, IDH1 R132H biochemical assay for (ϩ)-ML309 (triangles) and
(Ϫ)-ML309 (open squares). C, IDH1 R132C biochemical assay for (ϩ)-ML309 (triangles) and (Ϫ)-ML309 (open squares). D, IDH1 WT biochemical assay for
rac-ML309. E, cytotoxicity of U87MG IDH1 R132H cells for (ϩ)-ML309 (triangles) and (Ϫ)-ML309 (open squares). Error bars are mean Ϯ S.D. from two duplicate
experiments. F, inhibition of 2-HG in U87MG IDH1 R132H cells for (ϩ)-ML309 (triangles) and (Ϫ)-ML309 (open squares). Error bars are mean Ϯ S.D. from two
replicates.
TABLE 1
IC50 values (in nM) of rac-ML309, (؉)-ML309, and (؊)-ML309
Average values with S.E. are shown for two or three experimental replicates.
Compound
IDH1 R132H
IDH1 R132C
IDH1 WT
Cell 2-HG levelsa
150 Ϯ 1.7
250 Ϯ 1.7
Noneb
Cell cytotoxicitya
rac-ML309
(ϩ)-ML309
(Ϫ)-ML309
96 Ϯ 1.2
62 Ϯ 1.2
36,000 Ϯ 1.1
Noneb
Noneb
Noneb
68 Ϯ 1.2
30 Ϯ 1.4
Noneb
29,000 Ϯ 1.3
15,000 Ϯ 1.5
Noneb
a U87MG IDH1 R132H cells were used. Cell 2-HG refers to an assay that measures 2-HG levels, and Cell pertains to results from cytotoxicity testing.
b None indicates that there was little to no effect over the concentration range tested, and no IC50 fit was obtained.
selected using G418 (1 mg/ml) for 2 weeks to generate stable tions. Cells were plated into 6-well dishes. Upon each time
expression cells.
point, cells were washed three times with PBS and harvested,
U87MG pLVX-IDH1 R132H-neo Cell-based Assays—U87MG and 2-HG was extracted as described previously (6).
pLVX-IDH1 R132H-neo cells were maintained in DMEM con-
RESULTS
taining 10% FBS, 1ϫ penicillin/streptomycin, and 500 ␮g/ml
G418. Cells were seeded at a density of 5000 cells/well into
ML309 Is a Selective Inhibitor of the IDH1 Mutants R132H
96-well white solid-bottom microtiter plates and incubated and R132C in a Biochemical Assay in Comparison with Wild-
overnight at 37 °C and 5% CO2. The next day compounds were type Enzyme—Containing a chiral center, rac-ML309 was sep-
prepared in 100% DMSO and then diluted in medium for a final arated into its (ϩ) and (Ϫ) enantiomers (Fig. 2A). A coupled
concentration of 0.2% DMSO. Medium was removed from the biochemical assay was used to determine the IC50 values for
cell plates, and 200 ␮l of the compound dilutions were added to IDH1 R132H for the (ϩ)-ML309 and (Ϫ)-ML309 enantiomers,
each well. After 48 h of incubation with compound at 37 °C, 100 which were 68 Ϯ 1.2 and 29,000 Ϯ 1.3 nM, respectively (Fig. 2B
␮l of medium was removed from each well and analyzed by and Table 1), affirming the former as the eutomer and the latter
LC-MS for 2-HG concentrations as described in Ref. 6. The cell as the distomer. Very similar activity was observed for the IDH1
plates were then allowed to incubate another 24 h. At 72 h after R132C enzyme, giving IC50 values of 30 Ϯ 1.4 and 15,000 Ϯ 1.5
compound addition, Promega CellTiter-Glo reagent (Madison, nM for (ϩ)-ML309 and (Ϫ)-ML309, respectively (Fig. 2C and
WI) was added to each well, and the plates were read for lumi- Table 1). rac-ML309 was weakly active in the IDH1 WT assay
nescence to determine any compound effects on growth inhi- (IC50 Ͼ36 ␮M; Fig. 2D and Table 1). rac-ML309 showed no
bition (GI50) as described in Refs. 6 and 14).
readout interference with the coupled diaphorase/resazurin
For compound time course and washout studies, HT1080 detection system (data not shown). The binding of rac-ML309
cells (cultured as described in Refs. 6 and 14) that have endog- to IDH1 R132H was found to be reversible by the method of
enous expression of IDH1 R132C were incubated with com- compound/enzyme preincubation followed by rapid dilution
pound similar to the cell-based assays with the following excep- and measurement of remaining enzyme activity (20) (Fig. 3).
13720 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 20•MAY 16, 2014

Mutant IDH1 Inhibitor Mechanism of Action
Characterization of the Mechanism of Inhibition of rac-
ML309—To better understand the effects of inhibitor binding on
IDH1 R132H, we first determined the order of substrate binding.
Steady-state enzymatic activity was measured while ␣-KG was
held constant at 2 mM and NADPH was titrated from 64 to 5000
nM. Kinetic analysis of ␣-KG binding to IDH1 R132H was also
undertaken, and our experiments revealed substrate inhibition
at high concentrations of ␣-KG. This substrate inhibition was
relieved at increasing concentrations of NADPH. Investiga-
tions of this phenomenon were performed at NADPH concen-
trations of 1, 5, 10, and 20 ␮M while ␣-KG was varied from 78
␮M to 40 mM. Data for Km(app), Vmax(app), and h (Hill coefficient)
were fit globally, whereas Ki was fit for each concentration of
NADPH. These experiments are presented in Fig. 4, and the
kinetic parameters obtained from these experiments are sum-
marized in Table 2; results are suggestive of an obligate order of
binding with NADPH binding preceding ␣-KG and confirm
previously reported results (6, 21).
FIGURE 3. Reversibility test of ML309. Activity values represent normalized
(E ϭ 100%, vehicle alone (Veh alone) ϭ 0%) values. The error bars represent
the error from the fit to the slope of enzyme activity versus time. E ϭ IDH1
R132H, veh ϭ DMSO, and I ϭ ML309. pre-inc indicates that the compound
(concentration ϭ 10ϫ IC50) was preincubated with 100ϫE for 30 min and
then diluted 100ϫ (achieving inhibitor concentration ϭ IC10 and enzyme
concentration ϭ 1ϫ) rather than the standard protocol of adding the inhibi-
tor at the same final concentration and then directly measuring activity.
Next, we determined the mechanism of inhibition of rac-
ML309 with regard to NADPH and ␣-KG binding. Fig. 5
describes the effect on Vmax(app) and Km(app) as a function of
FIGURE 4. Characterization of binding affinity of the substrate ␣-KG and co-factor NADPH. A, Km(app) determination for NADPH. [IDH1 R132H] ϭ 5 nM;
[␣-KG] ϭ 2 mM. [NADPH] as shown on x-axis of the graph. B, relief of ␣-KG substrate inhibition by increasing concentrations of the co-factor NADPH at 1 ␮M
(circles), 5 ␮M (squares), 10 ␮M (triangles), and 20 ␮M (inverted triangles). [IDH1 R132H] ϭ 5 nM; [␣-KG] as shown on the x-axis of the graph. Data were fit to an
allosteric sigmoidal binding model with substrate inhibition model. Error bars for A and B are mean Ϯ S.D. from triplicate experiments. C, typical stopped-flow
reaction trace. [IDH1 R123H] ϭ 5 nM; [␣-KG] ϭ 2 mM and [NADPH] ϭ 100 ␮M. Note that in this experiment, the NADPH and the enzyme were premixed for 60
min in one syringe and the ␣-KG was premixed in a second syringe. This pre-equilibration was done for all experiments as appropriate for the assay setup.
TABLE 2
Kinetic parameters of substrate binding and inhibition of IDH1 R132H
a
a
Fixed
Varied
Vmax(app)
Km(app)
ha,b
Kia
␮M/min/mg
2.2 Ϯ 0.028
1.8 Ϯ 0.025
␮M
mM
␣-KG
NADPH
␣-KG
␣-KG
␣-KG
␣-KG
␣-KG
0.044 Ϯ 0.011
1.6 Ϯ 0.12
1.1 Ϯ 0.20
(Ϫ)
NADPH (global fit)
NADPH 1 ␮M
820 Ϯ 11
6.9 Ϯ 0.43
58 Ϯ 5.7
101 Ϯ 11
140 Ϯ 17
NADPH 5 ␮M
NADPH 10 ␮M
NADPH 20 ␮M
a Average values with S.D. are shown for three experimental replicates.
b
h ϭ Hill coefficient.
MAY 16, 2014•VOLUME 289•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 13721

Mutant IDH1 Inhibitor Mechanism of Action
FIGURE 5. Mechanism of action of ML309. A, stopped-flow kinetic investigation of the effect of rac-ML309 on the Vmax(app) and Km(app) of IDH1 R132H when
NADPH is held constant at 100 ␮M and ␣-KG is varied as shown on the x-axis of the graph ([ML309]: 0 nM, circles; 50 nM, squares; 100 nM, triangles; 150 nM, inverse
triangles; and 300 nM, diamonds). [IDH1 R132H] ϭ 5 nM. Error bars represent the mean and S.D. of three replicates at each point. B, simultaneous titration of ␣-KG
and rac-ML309 increases the Km(app) (squares) of the reaction, but not the Vmax(app) (circles), suggesting a competitive mode of action with regard to ␣-KG. Error
bars represent the mean and S.D. of three replicates at each point. C, stopped-flow kinetic investigation of the effect of rac-ML309 on the Vmax(app) and Km(app)
of IDH1 R132H when ␣-KG is held constant at 2 mM and NADPH is varied as shown on the x-axis of the graph ([ML309]: 0 nM, circles; 50 nM, squares; 100 nM,
triangles; 150 nM, inverse triangles; and 300 nM, diamonds). [IDH1 R132H] ϭ 5 nM. Error bars represent the mean and S.D. of three replicates at each point.
D, simultaneous titration of NADPH and rac-ML309 at constant NADPH decreases both the Vmax(app) (squares) and the Km(app) (circles) indicative of an
uncompetitive inhibitor mechanism of action with regard to NADPH. Error bars represent the mean and S.D. of three replicates at each point. E,
microscale thermophoresis of ␣-KG shows the effect on the IC50 of rac-ML309 upon the addition of ␣-KG. Triangles, rac-ML309 with 150 mM ␣-KG; open
squares, rac-ML309 without 150 mM ␣-KG. Laser power was 80%, and LED power was 50%. F, microscale thermophoresis of NADPH shows the effect on
the rac-ML309 IC50 upon the addition of NADPH. Triangles, rac-ML309 with 50 ␮M NADPH; open squares, rac-ML309 without 50 ␮M NADPH. Laser power
was 80%, and LED power was 50%.
inhibitor concentration. Increasing concentrations of rac-
Microscale thermophoresis was also used to measure the
ML309 increased the apparent Km(app) of ␣-KG of the reaction effect on the Kd of rac-ML309 binding in the presence and
while not affecting the apparent Vmax(app), indicating that the absence of saturating amounts of NADPH. As shown in Fig. 5F,
inhibitor is competitive with respect to substrate. Increasing con- the Kd decreases dramatically in the presence of NADPH (from
centrations of rac-ML309 decreased both the apparent Km(app) 20 Ϯ 0.88 ␮M to 410 Ϯ 29 nM, consistent with the uncompeti-
and the apparent Vmax(app) of the reaction with regard to NADPH, tive binding with respect to NADPH).
indicating that the mechanism of action is uncompetitive.
On/off Rate Analysis and Kd Determination from SPR—
To further characterize this effect, microscale thermophore- NADPH binding to IDH1 R132H was measured using SPR. The
sis methods were used to determine the apparent Kd of rac- data were fit in both kinetic (Fig. 6A) and equilibrium modes
ML309 binding in the absence of ␣-KG and in the presence of a (Fig. 6B). From a kinetic analysis, the Kd was determined to be
saturating concentration of ␣-KG (Fig. 5E). There is a right shift 80 Ϯ 0.94 nM with an on rate of ϳ 420,000 Ϯ 8000 1/ms and an
in the Kd in the presence of 150 mM ␣-KG (from 20 Ϯ 0.88 ␮M off rate of ϳ0.034 Ϯ 0.033 1/s. This agreed well with the equi-
to Ͼ500 ␮M), indicating that ␣-KG and rac-ML309 compete librium analysis, which produced a Kd of 88 Ϯ 0.71 nM. These
for binding to IDH1 R132H.
Kd values compare well with the recently reported Kd of 43 Ϯ 9
13722 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 20•MAY 16, 2014

Mutant IDH1 Inhibitor Mechanism of Action
FIGURE 6. Surface plasmon resonance characterization of binding of NADPH and/or rac-ML309. A, NADPH alone in kinetic mode. Top concentration is 1
␮M, and dilution is 1:3. Residuals to the fit are shown below the plot. RU, resonance units. B, NADPH alone in equilibrium mode. Top concentration is 1 ␮M, and
dilution is 1:3. C, rac-ML309 alone in kinetic mode. Top concentration is 3 ␮M, and dilution is 1:3. D, NADPH and rac-ML309 together in kinetic mode. Top
concentration of rac-ML309 is 3 ␮M, and dilution is 1:3. NADPH is held constant at 1 ␮M. Residuals to the fit are shown below the plot. For A, C, and D, one of the
two replicates is shown with the fit. For B, the plot is of the averaged data and the corresponding fit, and the error bars were smaller than the symbol size.
Injection start and stop times are indicated by vertical dashed lines.
nM (22). Additionally, the SPR data correlated well with the glioblastoma cell line transfected with IDH1 R132H, and it was
stopped-flow kinetic data described above, which yielded a found that (ϩ)-ML309 inhibited the production of 2-HG with
Km(app) of 0.044 Ϯ 0.011 ␮M. rac-ML309 alone has very little an IC50 of 250 Ϯ 1.7 nM, whereas (Ϫ)-ML309 showed no inhi-
affinity for IDH1 R132H, and no binding was observed up to 3 bition up to 5 ␮M (Fig. 2F). Notably, the shift between inhibitory
␮M (Fig. 6C). In the presence of saturating NADPH, the binding potencies of the compound in the biochemical and cell assays is
of rac-ML309 to IDH1 R132H increased. Fig. 6D shows the minimal, roughly 4-fold. As part of the above assay, the cyto-
binding isotherms for rac-ML309 in the presence of NADPH. toxicity was measured, and both the (ϩ)-ML309 and the (Ϫ)-
Fitting of the isotherms indicates that the on rate is ϳ 32,000 Ϯ ML309 were nontoxic at the concentrations tested (Fig. 2E).
3100 1/ms and the off rate is ϳ 0.0015 Ϯ 0.000071 1/s. A
Time Dependence of 2-HG Reduction upon rac-ML309 Treat-
binding constant of 48 Ϯ 0.24 nM was determined, in good ment and Recovery after Compound Washout—HT1080 cells,
agreement with the IC50 observed in the enzymatic assay (see which harbor the IDH1 R132C mutation, were treated with 5
above).
␮M rac-ML309, and the 2-HG levels were monitored as
ML309 Has Reasonable PK Properties—rac-ML309 has a described in Refs. 6 and 14. 2-HG levels rapidly decreased as
kinetic aqueous solubility greater than 50 ␮M, making it useful shown in Fig. 7 (t ⁄ ϳ 5 h) and remained low. The effect was
1
2
for biochemical, biophysical, and cell-based studies. rac-ML309 reversed upon compound washout after 48 h.
was found to be highly stable in aqueous solution, 5 mM gluta-
DISCUSSION
thione, assay buffer, and human plasma (98% remaining after
30 min in all instances). Despite the low stability in human
IDH1 R132H and IDH1 R132C have been identified as
and mouse microsomes, 6.2 and 7.7% remaining after 30 important targets for the development of therapeutics for glio-
min, respectively, in mouse PK studies, the half-life of the blastoma and acute myeloid leukemia. Cells that harbor these
compound was greater than 3 h. However, no brain exposure mutations have significantly higher levels of 2-HG relative to
was observed.
WT cells (6). Decreasing the levels of 2-HG through inhibition
ML309 Is Active in Cell Assays—In cells that contain the of the 2-HG-producing IDH1 mutant enzymes is a potential
IDH1 R132H mutation, ␣-KG (the product of the WT IDH1 avenue for therapeutic treatment.
reaction) is converted into the oncometabolite 2-HG. The
Recent work at Agios Pharmaceuticals, Inc., resulted in the
impact on 2-HG formation was determined in U87MG human identification of a phenyl-glycine chemical series that showed
MAY 16, 2014•VOLUME 289•NUMBER 20 JOURNAL OF BIOLOGICAL CHEMISTRY 13723

Mutant IDH1 Inhibitor Mechanism of Action
guished here, but hopefully a crystal structure with a member of
the phenyl-glycine series will be forthcoming. Currently, a crys-
tal structure is available for IDH1 with NADPH (6), and a struc-
ture with a small molecule (1-hydroxypyridine 2-one scaffold)
inhibitor was published (24).
Understanding the inhibition modality is important when
comparing different compound series, which may have differ-
ent mechanisms of action. It is also important for properly set-
ting up SPR and other biophysical measurements. For an
uncompetitive inhibitor (or a mixed inhibitor in which the
affinity is much greater for the ES form), a build-up of substrate
leads to enhanced inhibitor affinity in contrast to competitive
inhibitors, which can be overcome by high concentrations of
substrate The cell activity was determined in both a cytotoxicity
assay and an assay measuring product 2-HG formation. (ϩ)-
FIGURE 7. Time dependence of 2-HG levels following 5 ␮M rac-ML309
treatmentandwashoutafter48hinHT1080cells, whichharbortheIDH1
R132C mutation. Error bars are mean Ϯ S.D. from three replicates.
potent inhibition in both biochemical and cell assays (14). A rep- ML309 is not very toxic during the course of the in vitro cyto-
resentative inhibitor from the phenyl-glycine series (ML309) was toxicity assay, consistent with previous data on a member of the
studied in detail here to provide a deeper understanding of the phenyl-glycine series that indicated that the compound did not
mechanism of inhibition. rac-ML309 was very stable in human induce apoptotic cell death (15) but that it does have a profound
plasma and had a half-life of over 3 h in a mouse PK study. The effect on 2-HG formation. 2-HG is thought to behave as an
integrity of the compound is expected to be stable during the oncometabolite by inhibiting multiple ␣-KG-dependent dioxy-
cell assays described herein. Although there was no observed genases, which are important in a variety of biological pro-
penetration of the blood brain barrier in these normal healthy cesses, such as histone and DNA demethylation (8, 13). Indeed,
mice, there could still be penetration in a glioma setting in it may be that epigenetic changes due to the presence of 2-HG
which the blood brain barrier may be compromised (23), and contribute to tumorigenesis. A recent study showed that the
this compound could provide a good starting point for further effect of 2-HG on promoting leukemogenesis (i.e. growth factor
optimization to improve drug-like properties and potency.
independence and impaired differentiation) was reversible (10).
(ϩ)-ML309 was potent against both IDH1 R132H and IDH1 The authors tested AGI-5198, a previously published member
R132C in a biochemical assay, indicating that it could be useful of the phenyl glycine series (14), and showed that this inhibitor
for both glioblastoma and acute myeloid leukemia treatment. It reversed the impaired differentiation and growth factor inde-
is exquisitely selective for the mutant forms of IDH1 over the pendence of IDH1 R132H-expressing TF-1 cells. Inhibition of
WT form. Although a crystal structure is not currently available the formation of 2-HG should help mitigate the deleterious
for ML309 bound to IDH1 R132H to definitively show the bind- effect of the R132C and R132H mutations of IDH1.
ing mode, one possibility for the preference of binding to IDH1
To further understand the effect of ML309 on 2-HG levels, a
R132H over WT IDH1 is that IDH1 R132H has a more open time course was performed. Upon treatment of HT1080 cells,
conformation than WT IDH1 (22). The Kd measured for rac- which harbor the IDH1 R132C mutation, with 5 ␮M rac-
ML309 binding to IDH1 R132H in the presence of NADPH ML309, 2-HG levels rapidly dropped with a t ⁄ ϳ 5 h and
1
2
(48 Ϯ 0.24 nM) by SPR was very similar to the IC50 measured in remained low until 48 h, at which time the compound was
the IDH1 R132H biochemical assay (68 Ϯ 1.2 nM). washed out and 2-HG levels recovered. The rapid recovery is
The substrate titration experiments allow us to propose a consistent with reversible binding of rac-ML309. This rapid
kinetic mechanism for the inhibitor. Our work suggests that recovery of 2-HG levels also has implications for dosing regimens
NADPH is the first to bind the enzyme followed by ␣-KG. A and indicates that continual coverage of the target may be neces-
previous study on IDH1 R132H using a combination of dead- sary to keep 2-HG levels below deleterious levels, although this
end inhibition studies and primary kinetic isotope effects also remains to be tested.
concluded that NADPH is likely to bind first followed by ␣-KG
The two enantiomers of ML309 had vastly differing activities
(22). Given the uncompetitive nature of rac-ML309 with regard to in the enzymatic and cellular assays, with (ϩ)-ML309 emerging
NADPH and competitive nature with regard to ␣-KG, we propose as the active enantiomer. Enantiopure (ϩ)-ML309 was pre-
that rac-ML309 binds after NADPH and prevents the binding of pared by synthesizing rac-ML309 and isolating the enantiopure
␣-KG, stopping the catalytic cycle, which produces the oncome- isomer by chiral HPLC. The absolute stereochemistry of (ϩ)-
tabolite 2-HG.
ML309 could not be verified by an enantioselective synthesis
An understanding of the mechanism of action can provide because racemization occurs in the Ugi reaction used to pre-
assistance in series selection and lead optimization. NADPH pare rac-ML309. However, it is likely that the (ϩ)-ML309 enan-
binding may orient IDH1 R132H for productive inhibitor bind- tiomer is (S)-ML309 and the (Ϫ)-ML309 enantiomer is (R)-
ing. It is possible that the (ϩ)-ML309-binding site overlaps with ML309 by comparison with the previously characterized chiral
that of ␣-KG and that the competitive inhibition is due to direct phenyl-glycine series in which the activity was predominantly
competition for the same binding site. Alternatively, (ϩ)- contained within the (S)-configured analogues (14).
ML309 could bind remotely and thus allosterically alter the
In summary, the detailed mechanism of action of an inhibitor
␣-KG-binding pocket. These two possibilities cannot be distin- of IDH1 R132H was determined and validated using a battery of
13724 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 20•MAY 16, 2014

Mutant IDH1 Inhibitor Mechanism of Action
linked to EGLN activation. Nature 483, 484–488
biochemical, cellular, and biophysical methods. The inhibitor
binds to IDH1 R132H predominantly in the presence of
NADPH and is competitive with respect to ␣-KG binding. The
compound binds reversibly to the enzyme with modest on and
off rates. rac-ML309 rapidly affected 2-HG levels in cells, and
this effect could be reversed by compound washout. The
detailed mechanistic evidence and proposed reaction diagram
presented here will hopefully aid in the development of future
mutant IDH1 inhibitors and in their evaluation as treatments
for diseases, such as glioblastoma and acute myeloid leukemia.
10. Losman, J. A., Looper, R. E., Koivunen, P., Lee, S., Schneider, R. K., Mc-
Mahon, C., Cowley, G. S., Root, D. E., Ebert, B. L., and Kaelin, W. G. (2013)
(R)-2-Hydroxyglutarate is sufficient to promote leukemogenesis and its
effects are reversible. Science 339, 1621–1625
11. Miyata, S., Urabe, M., Gomi, A., Nagai, M., Yamaguchi, T., Tsukahara, T.,
Mizukami, H., Kume, A., Ozawa, K., and Watanabe, E. (2013) An R132H
mutation in isocitrate dehydrogenase 1 enhances p21 expression and in-
hibits phosphorylation of retinoblastoma protein in glioma cells. Neurol.
Med. Chir. (Tokyo) 53, 645–654
12. Paschka, P., Schlenk, R. F., Gaidzik, V. I., Habdank, M., Krönke, J.,
Bullinger, L., Späth, D., Kayser, S., Zucknick, M., Götze, K., Horst, H. A.,
Germing, U., Döhner, H., and Döhner, K. (2010) IDH1 and IDH2 muta-
tions are frequent genetic alterations in acute myeloid leukemia and con-
fer adverse prognosis in cytogenetically normal acute myeloid leukemia
with NPM1 mutation without FLT3 internal tandem duplication. J. Clin.
Oncol. 28, 3636–3643
Acknowledgments—We are grateful for the technical support teams
from Bio-Rad-ProteOn and NanoTemper-MST (microscale thermo-
phoresis) for helpful discussions. We acknowledge Ed Kerns, Amy
Wang, and Kimloan Nguyen for the absorption, distribution, metab-
olism, and excretion and in vivo PK data and helpful discussions as
well as William Leister and Jim Bougie for the help with chiral
chromatography.
13. Xu, W., Yang, H., Liu, Y., Yang, Y., Wang, P., Kim, S. H., Ito, S., Yang, C.,
Wang, P., Xiao, M. T., Liu, L. X., Jiang, W. Q., Liu, J., Zhang, J. Y., Wang, B.,
Frye, S., Zhang, Y., Xu, Y. H., Lei, Q. Y., Guan, K. L., Zhao, S. M., and Xiong,
Y. (2011) Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of
␣-ketoglutarate-dependent dioxygenases. Cancer Cell 19, 17–30
14. 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., and Su, S. M. (2012)
Discovery of the first potent inhibitors of mutant IDH1 that lower tumor
2-HG in vivo. ACS Med. Chem. Lett. 3, 850–855
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