Inhibition of cancer-associated mutant isocitrate dehydrogenases: Synthesis, structure-activity relationship, and selective antitumor activity

Article abstract of DOI:10.1021/jm500660f

Mutations of isocitrate dehydrogenase 1 (IDH1) are frequently found in certain cancers such as glioma. Different from the wild-type (WT) IDH1, the mutant enzymes catalyze the reduction of α-ketoglutaric acid to d-2-hydroxyglutaric acid (D2HG), leading to cancer initiation. Several 1-hydroxypyridin-2-one compounds were identified to be inhibitors of IDH1(R132H). A total of 61 derivatives were synthesized, and their structure-activity relationships were investigated. Potent IDH1(R132H) inhibitors were identified with K_i values as low as 140 nM, while they possess weak or no activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R² of 0.83 and 0.73, respectively. Several inhibitors were found to be permeable through the blood-brain barrier in a cell-based model assay and exhibit potent and selective activity (EC₅₀ = 0.26-1.8 μM) against glioma cells with the IDH1 R132H mutation.

Full text of DOI:10.1021/jm500660f

Article
pubs.acs.org/jmc
Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases:
Synthesis, Structure−Activity Relationship, and Selective Antitumor
Activity
Zhen Liu,†,¶ Yuan Yao,†,¶ Mari Kogiso,⊥ Baisong Zheng,† Lisheng Deng,† Jihui J. Qiu,∥ Shuo Dong,∥
Hua Lv,§ James M. Gallo,§ Xiao-Nan Li,⊥ and Yongcheng Song*,†,‡
†Department of Pharmacology, ⊥Department of Pediatrics-oncology, ∥Department of Medicine, and ‡Dan L. Duncan Cancer Center,
Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, United States
§Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New
York, New York 10029, United States
S
* Supporting Information
ABSTRACT: Mutations of isocitrate dehydrogenase 1
(IDH1) are frequently found in certain cancers such as glioma.
Different from the wild-type (WT) IDH1, the mutant enzymes
catalyze the reduction of α-ketoglutaric acid to D-2-hydrox-
yglutaric acid (D2HG), leading to cancer initiation. Several 1-
hydroxypyridin-2-one compounds were identified to be
inhibitors of IDH1(R132H). A total of 61 derivatives were
synthesized, and their structure−activity relationships were
investigated. Potent IDH1(R132H) inhibitors were identified
with Ki values as low as 140 nM, while they possess weak or no
activity against WT IDH1. Activities of selected compounds against IDH1(R132C) were found to be correlated with their
inhibitory activities against IDH1(R132H), as well as cellular production of D2HG, with R2 of 0.83 and 0.73, respectively. Several
inhibitors were found to be permeable through the blood−brain barrier in a cell-based model assay and exhibit potent and
selective activity (EC50 = 0.26−1.8 μM) against glioma cells with the IDH1 R132H mutation.
particular interest is that all characterized IDH mutant proteins,
such as IDH1(R132H) and IDH1(R132C), almost lose the
catalytic function of wild-type (WT) IDH, but obtain a new
capability: they can catalyze the reduction of α-KG to D-2-
hydroxyglutaric acid (D2HG) using Mg2+ and NADPH as
cofactors,6,12,13 as shown in Figure 1B. These IDH mutant
enzymes therefore cause elevated D2HG concentrations in cell
and plasma. Further studies show a high level of D2HG is very
harmful and could be the culprit for the initiation of the cancer.
Due to its structural similarity to α-KG, D2HG is a broad
inhibitor of α-KG-dependent dioxygenases including histone
demethylases and the TET-family of 5-methylcytosine
hydroxylases,5 which are important enzymes keeping a balanced
histone and DNA methylation status. Overexpression of
IDH1(R132H) can cause hypermethylation of histone and
DNA and block cell differentiation.14,15 These findings suggest
mutant IDH is a novel drug target for intervention,16−18 and its
inhibitors represent useful probes for the investigation of the
biological functions of IDH mutation as well as potential
therapeutics for this type of cancer.
INTRODUCTION
■
Isocitrate dehydrogenase (IDH) is one of the key enzymes in
the tricarboxylic acid cycle, which provides aerobic organisms
the majority of energy by oxidation of the acetyl group derived
from, for example, carbohydrates and fats. IDH catalyzes the
oxidative decarboxylation of isocitric acid (ICT) to α-
ketoglutaric acid (α-KG) using Mg2+ and NADP+ (or NAD+)
as cofactors,1 as shown in Figure 1A. There are three IDH
isozymes in humans, with IDH1 located in cytoplasm and
IDH2 and 3 in mitochondria.2,3 Moreover, α-KG, the product
of the IDH catalyzed reaction, is used as a common cofactor by
∼60 dioxygenases, including important epigenetic enzymes
such as the JmjD family of histone demethylases. Therefore, the
function of IDH enzymes is of importance to normal
physiology.
Recent genetic studies have identified frequent mutations in
IDH genes in several types of cancer.4−6 For example, IDH1
mutations are found in ∼75% of low-grade gliomas (grade II
and III), as well as secondary glioblastoma multiforme, the
grade IV glioma developed from the low-grade tumors.7,8 The
R132H mutation is predominant (>90%) in these gliomas.
Mutations of IDH1 or IDH2 have also been found in ∼20%
acute myeloid leukemia and many sarcomas.6,9−11 The IDH
mutations occur at an early stage of these cancers, suggesting
they could play important roles in cancer initiation. Of
A series of diamide compounds, such as compound 1 shown
in Figure 1C, were reported to be the first inhibitors of mutant
Received: April 28, 2014
© XXXX American Chemical Society
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Figure 1. (A) Reaction catalyzed by WT IDH enzymes in tricarboxylic acid cycle, (B) reaction catalyzed by mutant IDH enzymes, and (C)
structures of current inhibitors of mutant IDH1.
IDH1 with IC50 values as low as 70 nM.19,20 Compound 1 is
Chart 1. Structures and Ki Values of Initial Inhibitors of
able to reduce the cellular D2HG concentration and slow the
proliferation of IDH1 mutated cancer cells. We also reported
several 1-hydroxypyridin-2-one compounds,21 such as com-
pounds 2 and 3 (Figure 1C), are potent inhibitors of mutant
IDH1 with inhibition constant (Ki) values as low as 190 nM,
which exhibit very weak activity against WT IDH1 showing a
high selectivity of >60-fold. In addition, we determined the X-
ray crystal structures of IDH1(R132H) in complex with
inhibitors 2 and 3, which reveal the exact binding mode of
these two compounds as well as the structural basis for the high
selectivity.
IDH1(R132H)
Here, we report the inhibitor discovery, design, synthesis,
and structure−activity relationships (SAR) of several series of
1-hydroxypyridin-2-one compounds targeting cancer-associated
mutant IDH1. Several inhibitors of mutant IDH1 show potent
and selective activity against glioma cells with the IDH1 R132H
mutation.
Medicinal Chemistry To Find Potent IDH1(R132H)
Inhibitors. With the identification of 5- and 6-substituted 1-
hydroxypyridin-2-one compounds, that is, 4, 7, and 8, as the
lead inhibitors of IDH1(R132H), systematic medicinal
chemistry studies were performed in an effort to find
compounds with improved activity. Four series of compounds,
including 5-, 6-, and 4-methyl-6- and 4-methyl-3-substituted 1-
hydroxypyridin-2-one compounds, have been designed, synthe-
sized, and tested against IDH1(R132H).
Table 1 shows the structures and activities of 5- or 6-
substituted 1-hydroxypyridin-2-one compounds. Compounds
10 and 11, having 5-(2-phenylethyl) and 5-(3-phenylpropyl)
substituents, respectively, lose the inhibitory activity. In
addition, compounds 12−14 containing a 5-hydroxamate,
amide, and reversed amide group, respectively, are also inactive.
For 6-substituted 1-hydroxypyridin-2-one compounds, com-
pounds 15 and 16 bearing a 6-phenyl and 6-(1-phenylethyl)
group, respectively, are inactive against IDH1(R132H).
Compound 17 with a 2-phenylethyl group exhibits less activity
(Ki = 10 μM) compared with compound 7 with a 6-benzyl
group. Compound 18, which has an additional -CO2Me, is
considerably less active than 17. These results, together with
those of compounds 4−7, suggest that 6-benzyl is more
favorable. In addition, despite the good activity of compound 8
with a 6-hydroxamic acid (Ki = 4.5 μM), we did not pursue this
RESULTS AND DISCUSSION
■
Identification of the Initial Inhibitors of IDH1(R132H).
Our previous research in developing inhibitors of 1-
deoxyxylulose-5-phosphate reductoisomerase,22−24 another re-
ductase using Mg2+ and NADPH as cofactors, provided an
enriched source of compounds that could inhibit mutant IDH1.
By screening this focused library of ∼130 compounds against
recombinant human IDH1(R132H), the predominant muta-
tion found in the majority of the gliomas, followed by
validation, we found several 5- and 6-substituted 1-hydroxypyr-
idin-2-one compounds to be low micromolar inhibitors. The
structures and activities of these compounds are shown in
Chart 1, together with those of inactive analogs in the library
that are useful for SAR analysis.
5-Benzyl-1-hydroxypyridin-2-one (4) was found to be an
inhibitor of IDH1(R132H) with a Ki value of 8.2 μM, while
compound 5 with a 5-phenyl group has a very weak activity.
Compound 6 with an -O- linkage exhibits a Ki of 46 μM,
suggesting that an electron-rich core is not favored.
Compounds 7 and 8 with a 6-substituent possess an improved
activities (Ki = 5.9 and 4.5 μM) compared with that of 4.
Ciclopirox (9), an antifungal drug, with 4-methyl and 6-
cyclohexyl substituents is inactive against IDH1(R132H).
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Table 1. Structures and Ki Values of 5- or 6-Substituted
Compounds against IDH1(R132H)
Table 2. Structures and Ki Values of 3-, 4-, and 6-Substituted
Compounds against IDH1(R132H)
R5
R6
Ki (μM)
R3
R4
-Me
R6
Ki (μM)
5
-Ph
-Bn
-H
-H
-H
-H
-H
-H
-H
-H
-Ph
-Bn
∼50
8.2
9
2
-H
-H
-cyclohexyl
-Bn
>50
0.19
0.87
>50
9.5
4
-Me
-Me
-Me
-Me
-Me
10
11
6
-(CH2)2Ph
-(CH2)3Ph
-OPh
>50
>50
46
19 -H
20 -H
30 -Bn
-CH2OPh
-Me
-H
12
13
14
15
7
-CONHOH
-CONHBn
-NHCOBn
-H
>50
>50
>50
>50
5.9
31 -CH2(3-OMe-
-H
16.2
Ph)
32 -CH2(3-OH-Ph)
33 -H
-Me
-H
13.6
9.5
-i-Pr
-Bn
-Bn
-Bn
-Bn
-H
34 -H
-OH
0.38
5.5
16
17
18
8
-H
−CH(CH3)Ph
-(CH2)2Ph
>50
10
35 -H
-OMe
-CH2OH
-H
36 -H
5.6
-H
-(CH2)2(4-CO2Me-Ph)
-CONHOH
27
37 -H
-CH2OAc -Bn
4.5
-H
4.5
38 -H
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-CH2(4-OMe-Ph)
-CH2(4-OH-Ph)
-CH2(3-OMe-Ph)
-CH2(3-OH-Ph)
-CH2(2-OMe-Ph)
-CH2(2-OH-Ph)
-CH2(3-F-Ph)
0.56
0.28
0.15
0.14
0.49
0.65
2.2
3
-H
39 -H
40 -H
41 -H
42 -H
43 -H
44 -H
45 -H
46 -H
47 -H
48 -H
49 -H
50 -H
51 -H
52 -H
53 -H
54 -H
55 -H
compound further due to the high polarity as well as poor
pharmacokinetics of the hydroxamate group.
Compound 2, obtained by replacing the 6-cyclohexyl group
of the inactive compound 9 with a benzyl substituent, was
found to be a potent inhibitor of IDH1(R132H) with a Ki value
of 190 nM, as shown in Table 2. It is ∼30× more active than
the analogous compound 7 without a 4-methyl group, showing
the 4-Me is important for the activity. Compound 19 with 4-
methyl-6-phenoxymethyl substituents was found to inhibit
IDH1(R132H) with a Ki of 870 nM, ∼4× less active than 2.
Compound 20 having 4,6-dimethyl substituents was found to
be inactive. These results demonstrate that the combination of
the 4-methyl and 6-benzyl substituents is needed to achieve a
high inhibition.
With the potent inhibitor 2 in hand, efforts were next made
to optimize the 1-hydroxypyridin-2-one core structure. Chart 2
shows the structures and activities of compounds 21−29, each
of which has a different aromatic core structure, while contains
a benzyl substituent for SAR analysis. Compared with
compound 4, lack of activity for compound 21 clearly indicates
the importance of N-OH group. Greatly reduced activities for
compounds 22−24 with a C-substituted -OH group suggest
that the N-substituted -OH in compound 2 increases the
binding affinity to IDH1(R132H). In addition, compared with
compound 7 (Chart 1), lack of activity for compounds 25 and
26 suggests the reversely positioned N-OH and carbonyl
functionalities are disfavored. Furthermore, compared with
compound 2, a 20-fold activity reduction for compound 27 (Ki
= 3.8 μM) with an N-NH2 group again underscores the critical
role of the N-OH group of the 1-hydroxypyridin-2-one core.
Compounds 28 and 29 having a 2-pyrone and 2-thiopyrone
ring, respectively, are also inactive. These SAR studies show
that the 1-hydroxypyridin-2-one ring represents the most
potent core structure for the inhibition of IDH1(R132H).
X-Ray Structure of Inhibitor Bound IDH1(R132H) and
Structure Based Rationalization of SAR. We determined
the X-ray structures of IDH1(R132H) in complex with
NADPH and the potent inhibitors 2 and 3 at 3.3 Å,21 to a
similar resolution as the previously reported structures.25
Detailed structural information has been reported in our
-CH2(penta-F-Ph)
-CH2(4-CN-Ph)
-CH2(3-CN-Ph)
-CH2(3-COOH-Ph)
-CH2(3-CONH2-Ph)
-CH2(4-CF3-Ph)
-CH2(3,5-diMe-Ph)
-CH2(thiophen-3-yl)
-CH2(3-biphenyl)
-CH2(4-OPh-Ph)
-CH2(naphth-1-yl)
2.2
12.5
1.6
>50
8.5
3.2
3.1
0.95
0.60
0.75
0.95
40.5
-CH2(6-OMe-naphth-1-
yl)
56 -H
57 -H
58 -H
-Me
-Me
-Me
-CH2(6-OH-naphth-1-
yl)
27.5
22.5
0.34
-CH2(benzothiophen-2-
yl)
-CH2[4-(4-OMe-Ph)-
Ph]
59 -H
60 -H
-Me
-Me
-CH2[4-(4-OH-Ph)-Ph]
0.25
0.14
-CH2[4-(3-OMe-Ph)-
Ph]
61 -H
62 -H
-Me
-Me
-CH2[4-(3-OH-Ph)-Ph]
-CH2(3,5-diPh-Ph)
0.27
0.30
previous communication,21 while the interactions between the
protein and 2 are briefly summarized to facilitate rationalization
of SARs as well as inhibitor design described below. As shown
in Figure 2a, the 4-methyl-1-hydroxypyridin-2-one ring of
compound 2 is located in a pocket surrounded by Arg100,
Ser94, Thr77, Asn96, Arg109, and NADPH. The planar
-CONH2 of Asn96 is located right underneath the pyridine
ring of 2, with the distance of ∼4.1 Å. The two O atoms of the
1-hydroxypyridin-2-one core form two H-bonds with the two N
atoms of the guanidinium group of Arg100. The calculated pKa
of 6.1 for 2 suggests that the N-OH group may be
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reveals that the 4-Me group has favorable interactions with the
side chains of Thr77 and Ser94. Compared with 2, compound
33 with a 4-isopropyl group was found to exhibit a 50-fold
activity loss (Ki = 9.5 μM), indicating that isopropyl is too
bulky for the pocket. Compound 34 with a 4-hydroxyl group
was found to be also a potent inhibitor of IDH1(R132H) with a
Ki of 380 nM, being comparable to compound 2. Introducing
the -OH was intended to have favorable interactions (e.g., H-
bond) with the side chains of Thr77 (or Ser94), with the
docking results of 34 shown in Figure 2c. However, the 4-OH
in compound 34 is not superior to the 4-Me in 2. In addition,
the 4-methoxy group in compound 35 (Ki = 5.5 μM)
considerably reduces the inhibitory activity. Compound 36
with a 4-hydroxymethyl, as well as its acetyl ester 37, also have
moderate potency with Ki values of 5.6 and 4.5 μM,
respectively. These results show that only small substituents
with a single heavy atom, such as -Me and -OH, are favorable
for the 4-position of 1-hydroxypyridin-2-one ring. Because 1-
hydroxypyridin-2-one compounds with a 4-methyl group is
considerably easier to synthesize and might be more cell
permeable than those with a 4-hydroxyl, we decided to keep the
4-Me for further SAR studies.
Chart 2. Structures of 21−29 and Their Ki Values against
IDH1(R132H)
The SAR studies for the 6-position of compound 2 were
performed to find a substituent that can provide increased
potency. First, we wanted to explore how electron density of
the phenyl ring affects the activity. Compound 38 with a para-
OMe group in its 6-benzyl group exhibits less activity than does
its parent compound 2, while 3 having a para-OH maintains a
comparable activity (Ki = 290 nM). Of interest are the higher
inhibitory potencies of compounds 39 and 40 (Ki = 150 and
140 nM) having a meta-substituted OMe and OH group,
respectively, showing that these two groups are more favorable.
Changing the OMe and OH groups to the ortho-position for
compounds 41 and 42 resulted in ∼2× activity reductions with
their Ki values of 490 and 650 nM, respectively. On the other
hand, an electron-withdrawing fluoro substituent in 43 and 44
(Ki = 2.2 μM) was found to be highly disfavored, leading to
∼10-fold activity decrease. A similar trend can be observed for
compounds 45−49 (Ki 1.6 to >50 μM) bearing electron-
withdrawing -CN, -CO2H, -CONH2, and -CF3 groups, showing
that strongly electron-deficient phenyl ring can reduce the
inhibitory activity of this series of compounds. Compound 50
having two meta-substituted methyl groups possesses a
moderate activity of 3.1 μM. Compound 51 bearing a
thiophenyl side chain was found to be a good inhibitor (Ki =
950 nM). The increased activity for compounds 39 and 40
might be due to increased interactions with Arg109, as show in
Figure 2d for the docking results of 39. Introducing strong
electron-withdrawing groups (e.g., -F, -CF3, and -CN) for
compounds 43−49 is disfavored because this could reduce the
electrostatic interactions between the phenyl rings and the
electron-deficient nicotinamide ring of NADPH.
deprotonated, which, together with the (partially negative) O
atom of the 2-oxo group, also provides strong electrostatic
interactions with the positively charged side chain of Arg100.
This should explain why the core structures in compounds 21−
24 and 27−29 with either a single O atom or a higher pKa value
are considerably less potent. The 4-methyl group is nicely fitted
into a mainly hydrophobic cavity, ∼3.7−4.2 Å from -CH3 of
Thr77 and -CH2- and -O- of Ser94. Loss of these favorable
interactions should account for the reduced activity of
compound 7. The phenyl ring of the 6-benzyl has favorable
hydrophobic (as well as electrostatic) interactions with the
nicotinamide ring of NADPH, while the other side of the
phenyl ring is ∼5 Å away from the side chain of Arg109. In
addition, the C4 atom of the phenyl ring is ∼11 Å away from
Leu250′ (omitted in Figure 2a for clarity) from the other
monomeric protein. Therefore, Arg109, Leu250′, and NADPH
form a relatively large pocket that could be explored for
inhibitor design (described below). The crystal structures show
compounds bearing a branched 6-substituent, such as 9, 15, and
16, would cause severe steric conflicts with NADPH if their 4-
methyl-1-hydroxypyridin-2-one ring maintains the favorable
interactions with the protein. This should account for the loss
of activity for 9, 15, and 16.
Structure Guided Inhibitor Development and SAR. In
the structure of the IDH1(R132H)/2 complex, there is a
pocket near the 3-position of the 1-hydroxypyridin-2-one core,
surrounded by Gly97, Lys93, Arg100, and Asn101. Compound
30, having a 3-benzyl as well as the favorable 4-methyl group,
was designed and synthesized. Although it can be docked into
the structure of IDH1(R132H)/2 with the binding pose of 1-
hydroxypyridin-2-one core similar to that of 2 (Figure 2b),
compound 30 exhibits a moderate inhibitory activity with a Ki
of 9.5 μM (Table 2), being 50× less active than compound 2.
Compounds 31 and 32 with additional meta-substituted -OMe
and -OH are less active than 30. These results indicate that a
benzyl group at the 3-position of the 1-hydroxypyridin-2-one
ring is considerably less favorable than that at the 6-position.
We next sought to optimize the 4-position of the 1-
hydroxypyridin-2-one ring. The IDH1(R132H)/2 structure
Analogs with two or more aromatic rings at the 6-position of
1-hydroxypyridin-2-one were synthesized because of the
relatively large pocket surrounded by NADPH, Arg109, and
Leu250′ described above. Compounds 52, 53, and 54 having a
meta-biphenyl, para-phenoxyphenyl, and 1-naphthyl group,
respectively, were found to be strong inhibitors with Ki values
of 600, 750, and 950 nM. However, compounds 55 and 56,
which are derivatives of 54 with a 6-OMe or -OH in the
naphthyl ring, respectively, exhibit very weak activity (Ki > 25
μM). Similarly weak activity was observed for the benzothio-
phenyl derivative 57 (Ki = 22.5 μM). These three compounds
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Figure 2. X-ray crystal and docking structures of inhibitors of IDH1(R132H). (A) The close-up view of the active site of the crystal structure of
IDH1(R132H)/2, with the backbone of one monomeric protein shown in blue lines and that of the other monomer in yellow. Compound 2 is
shown as a ball and stick model. Only selected residues with the interactions with 2 are shown for clarity. (B) Ten docking structures of compound
30 (with C atoms in green), superimposed with the crystal structure of 2 (in yellow), showing that the 3-benzyl group of 30 is predicted to occupy
an empty pocket. (C) Ten docking structures of compound 34 (with C atoms in green), superimposed with the crystal structure of 2 (in yellow),
showing the 4-OH group of 34 is predicted to have favorable interactions with Thr77. (D) Ten docking structures of compound 39 (with C atoms
in green), superimposed with the crystal structure of 2 (in yellow), showing the 6-(3-OMe-Ph) group of 39 is predicted to have favorable
interactions with Arg109.
are >23× less active than their parent compounds 54 and 51.
Compounds 58 and 59 with a para-OMe and para-OH
substituted para-biphenyl group were, however, found to be
potent inhibitors of IDH1(R132H) with Ki values of 340 and
250 nM, respectively. Moving the OMe and OH groups to the
meta-position for compounds 60 and 61 resulted in more
potent inhibition, with the Ki values of these two compounds
being 140 and 270 nM, respectively. Finally, compound 62
bearing a meta-terphenyl group was found to be still a potent
inhibitor (Ki = 300 nM). Activity data of these compounds
show a large variety of substituents with different sizes can
replace the phenyl moiety of compound 2 while maintaining
comparable or having even improved activity. More mod-
ifications at this position could further improve the inhibitory
activity.
kinetic parameters.6 The R132C mutation also causes elevated
levels of D2HG in these cancer patients.
We selected 10 representative IDH1(R132H) inhibitors,
with Ki values ranging from 0.14 to 9.5 μM, and tested the
activity of these compounds against recombinant IDH1-
(R132C), using our previous method.21 The results, together
with their Ki values against IDH1(R132H), are summarized in
Table 3. These compounds were found to also be inhibitors of
IDH1(R132C) with Ki values ranging from 0.12 to 14.7 μM,
which shows a good correlation with those for the R132H
mutant enzyme, with R2 of 0.83 as well as slope of 1.04 (Figure
3A). These results suggest that the residue Cys132 seems to
have a similar function as His132 does for catalyzing the
reduction of α-KG to D2HG.
Next, given that WT IDH1 plays an important role in normal
physiology, an ideal inhibitor should have a good selectivity for
the mutant IDH1 enzymes. The inhibitory activities of the
above 10 compounds were tested against recombinant WT
IDH1 and the results are also shown in Table 3. Compounds 2,
3, and 58−60 were found to have only weak inhibitory
Inhibition of IDH1(R132C) and WT IDH1. Although rare
in glioma, Arg132 mutation to cysteine in IDH1 is frequently
found in acute myeloid leukemia and sarcomas.6,9−11 The
mutant protein IDH1(R132C) possesses the same enzymatic
function as IDH1(R132H) (Figure 1C) with comparable
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3 in the binding site I, while R132 in WT IDH1 can induce
protein conformational changes and stabilize the binding of
ICT/α-KG in the site II. Our modeling studies show that the
distinct environment for binding site I represents the structural
basis for the high selectivity of inhibitors 2 and 3 for mutant
IDH1.21
Table 3. Activity of Selected Inhibitors of Mutant IDH1
IDH enzyme Ki (μM)
IC50 (μM) for
WT
R132H
R132C
inhibition of D2HG
2
12.3 3.1
16.8 3.0
0.19 0.04
0.28 0.07
8.2 2.1
0.12 0.03
0.27 0.07
6.6 2.6
2.4 0.5
8.5 2.5
a
3
Inhibition of Cellular Production of D2HG. The
hallmark of IDH mutated cancer is the significantly elevated
D2HG concentrations in cancer cells as well as in plasma. We
next measured the ability of the 10 inhibitors in Table 3 to
reduce the cellular production of D2HG. Human fibrosarcoma
HT1080 cells, which harbor the IDH1 R132C mutation,26 were
treated with increasing concentrations of these compounds for
48 h. No significant cytotoxicity to HT1080 cells was observed
for up to 30 μM. D2HG concentrations of the cells were
quantitatively determined by using HPLC-MS. Are shown in
Table 3, except for the weak inhibitors 7 and 33 (used as
negative controls), inhibitors of IDH1(R132C) were able to
inhibit the production of D2HG in HT1080 with the IC50
values of 1.1−9.7 μM. In addition, the cell activities of these
compounds also exhibit a good correlation with their enzyme
activity, showing a R2 value of 0.73 (Figure 3B).
In vitro Blood−Brain Barrier (BBB) Permeability. Since
inhibitors of mutant IDH1 are required to penetrate the
blood−brain barrier (BBB) for glioma treatment, a cell-based
model system using MDCK-MDR1 cells27,28 was used to
determine the BBB permeability potential for selected
compounds 2 and 39, as well as for the known inhibitor 1 as
a comparison. The cells are grown on polycarbonate supports
separating two chambers to allow compound flux in either
direction, apical [A] to basolateral [B] and vice versa, to be
measured. The cells are highly confluent to approximate the
tight junctions characteristic of endothelial cells that comprise
the BBB. In addition, the multidrug resistance 1 (MDR1) gene
is overexpressed in the cell, producing a high level of P-
glycoprotein (the product of MDR1), a known BBB efflux
pump, on the apical side that limits brain distribution for many
low molecular weight drugs.
4
>50
7
>50
>50
>50
>30
>30
>30
5.9 1.3
10.5 3.2
2.4 0.4
>30
8
4.5 1.7
a
33
34
39
40
58
59
60
9.5 2.9
14.7 6.3
1.8 0.5
>30
0.38 0.08
0.15 0.05
0.14 0.07
0.34 0.08
0.25 0.1
0.14 0.04
9.7 1.8
1.1 0.2
3.8 0.8
1.1 0.3
3.2 0.3
6.3 0.8
0.26 0.07
0.42 0.11
0.80 0.31
0.56 0.24
0.62 0.21
14.0 2.9
15.2 5.1
13.0 3.5
a
Not tested.
activities (Ki 12.3−16.8 μM) against WT IDH1, while
compounds 7, 33, 34, 39, and 40 do not inhibit the enzyme.
Selectivity of >41-fold was observed for these compounds,
compared with their Ki values against IDH1(R132H),.
The activities of these compounds against the three IDH1
enzymes indicate that Arg132 exhibits a drastically different role
from His132 or Cys132. Previous structural studies of WT and
R132H mutant IDH1 show IDH1(R132H) has two ligand
binding sites, I and II (Supporting Information Figure S1A),25
with the site II being the catalytic site ∼6.5 Å away from site I,
while WT IDH1 only has the binding site II. ICT binds to site I
in IDH1(R132H), while α-KG occupies the catalytically active,
binding site II.12,25 His132 is not involved in the binding of
either ICT or α-KG. ICT is located in site II in WT IDH1. The
function of Arg132 is to have two H-bonds as well as an
electrostatic interaction with the two carboxyl groups of ICT,
which can enhance the binding of ICT in site II as well as
stabilize the overall structure of the IDH1/ICT complex. Due
to the shorter length as well as the chemical nature of the side
chain, His132 (as well as Cys132) cannot play the same role as
Arg132. This explains why R132H and R132C mutant proteins
have the similar function, while WT IDH1 having Arg132 is a
different enzyme. It is noted that the two binding sites I and II
do not coexist in IDH1(R132H). With ligand induced protein
conformational changes, IDH1(R132H) exhibits either its
binding site I or II.
As can be seen in Table 4, 1-hydroxy-pyridin-2-one
compounds 2 and 39 were both found to have analogous cell
Table 4. Cell Permeability Rates (10−6 cm/s) for Mutant
IDH1 Inhibitors
a
rate (apical to basolateral) rate (basolateral to apical) efflux ratio
1
0.15
5.45
5.39
10.4
6.34
8.88
72.6
1.3
Our X-ray crystallographic studies showed that compounds 2
and 3 are located in the ligand binding site I of IDH1(R132H)
(Supporting Information Figure S1B,C).21 The R132H (as well
as R132C) mutation causes the mutant protein to bind ICT/2/
2
39
1.7
a
Measured as rate(B→A)/rate(A→B)
.
Figure 3. Correlations between inhibition of IDH1(R132C) and that of (A) IDH1(R132H) and (B) cellular production of D2HG.
F
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permeability values of 5.45 and 5.39, respectively, in the A → B
direction, and possessed comparable rates of 6.34 and 8.88 for
the reverse direction. The low efflux ratios (1.3 and 1.7) due to
the comparable directional permeability rates suggest that these
two compounds are not likely substrates for P-glycoprotein. On
the other hand, compound 1 exhibited a low rate of cell
permeability (0.15) in the A → B direction and much higher
rate of 10.4 in the B → A direction leading to a high efflux ratio
of 72.6, suggesting that compound 1 is a substrate of P-
glycoprotein and likely BBB impermeable.
Selective Activity against Glioma Stem-Like Cells with
IDH1 R132H Mutation. Next, we examined the activity of the
IDH1(R132H) inhibitors against BT-142 glioma cells having
the R132H IDH1 mutation,29 as well as two glioma cells, BXD-
4687 and -3752, without an IDH1 mutation as controls.30,31
Unlike normal cell culture conditions under which cells are
grown as a monolayer attached to a plate, these glioma cells
were cultured in serum-free media and grown as neurospheres,
colonies of glioma cells with a diameter of 30−300 μm. The so-
called cancer stem cells (CSC) are enriched in these
neurospheres.32−34 CSCs represent a small fraction of cancer
cells with a distinct phenotype that can form new tumors when
transplanted into immunocompromised mice.30,35−37 While
proliferating rapidly, the bulk non-stem cancer cells fail to do
so. CSCs possess certain key traits as normal stem cells,
including the ability to unlimitedly self-renew and differentiate.
It has now well documented that CSCs play important roles in
antitumor-drug resistance, cancer relapse, and metastasis. It is
therefore important to find compounds having selective activity
against CSCs.
against IDH1(R132H)-containing BT-142 cells with an EC50 of
370 nM, while it had significantly weaker activity against BXD-
4687 and -3752 without an IDH1 mutation (EC50 6.8 and 5.9
μM, respectively), showing a high selectivity of ∼16-fold. In
addition, compound 2 possesses negligible cytotoxicity against
the proliferation of normal fibroblast WI-38 cells (EC50 > 50
μM, Table 4). These results show that potent IDH1(R132H)
inhibitor 2 possesses selective activity against the proliferation
of CSCs of BT-142, which is dependent upon the cellular
environment caused by a high concentration of D2HG. The
moderate activity of 2 against BXD-4687 and -3752 suggests
the compound could have nonspecific activity against the
proliferation of these two glioma cells. Indeed, while inactive
against IDH1(R132H), compound 9 (ciclopirox) was reported
to have similar (low micromolar) antiproliferative activity
against, for example, breast cancer and leukemia cells.38,39 This
suggests compound 2 exerts dual activities on BT-142 cells, that
is, by inhibiting IDH1(R132H) and blocking rapid proliferation
simultaneously, which could provide a synergistic (or additive)
effect. This could explain the high potency of the dual-role
compound. Similarly, compounds 39, 40, 58, and 59 exhibited
selective activity against BT-142 glioma cells, with compound
58 being the most potent. It inhibited neurosphere formation
of BT-142 with an EC50 of 260 nM, while it showed >10-fold
less activity against the two glioma cells without an IDH1
mutation. Moreover, none of these compounds possess
significant cytotoxicity against the proliferation of normal
fibroblast WI-38 cells, showing potentially low toxicity. Given
temozolomide resistance in a significant portion of glioma/
glioblastoma as well as poor BBB permeability and weak
antiglioma activity of compound 1, these results demonstrate
the importance for further development of these 1-hydrox-
ypyridin-2-one inhibitors as potential therapeutics for IDH1
mutated glioma.
The most potent IDH1(R132H) inhibitors 2, 39, 40, 58, and
59 were tested to inhibit the formation of neurospheres of the
three glioma cells. Also included in the assay are compound 1
and temozolomide, the first-line chemotherapy for glioblasto-
ma. As shown in Table 5, all of the three clinical glioblastoma
Chemistry. Scheme 1 shows the general methods for the
synthesis of the 4-methyl-6-substituted 1-hydroxypyridin-2-one
and related compounds, which include the most active
inhibitors (e.g., 2 and 39) of mutant IDH1. An acyl chloride
was reacted with ethyl 3-methyl-2-butenate in the presence of
AlCl3 to produce a mixture of stereoisomeric esters, which
without purification were cyclized under acidic conditions to
generate a single compound, pyron-2-one 63, with a yield of
50−85% from the acyl chloride. Because the reaction of 63 with
hydroxylamine occurred in a very poor yield (0−20%),
compound 63 was converted to more reactive pyron-2-thione
64,40 which reacted readily with hydroxylamine or hydrazine to
give 4-methyl-6-substituted 1-hydroxy (or amino)-pyridin-2-
one compounds in 67−73% yield. We found that compound
63a with a 6-chloromethyl group is a useful common
intermediate, which can undergo a mild Suzuki coupling
reaction to give 63b. With this route, compounds 3 and 38−62
were synthesized without each starting from a different acyl
chloride. In addition, strong acid sensitive R-groups, for
example, thiophenyl in 51, can be introduced.
The majority of the mono-5- or 6-substituted 1-hydroxypyr-
idin-2-one compounds were synthesized using a palladium
catalyzed coupling reaction starting from a bromo-substituted
2-methoxypyridine (Scheme 2). By our previous methods,21
the 2-methoxypyridine moiety of the coupling products was
converted to the 1-hydroxypyridin-2-one core by oxidation with
3-chloroperoxybenzoic acid followed by treatment with acetyl
chloride. To synthesize compounds 36 and 37, 2,6-dihydrox-
yisonicotinic acid was heated with POBr3 followed by
Table 5. EC50 Values (μM) of Selected Inhibitors against
Glioma and Normal Fibroblast WI-38 Cells
BT-142
>50
BXD-4687
BXD-3752
WI-38
temozolomide
>50
>50
b
>50
>50
>50
>50
>50
>50
a
1
>20
>20
>20
2
0.37 0.2
0.63 0.2
1.8 0.6
6.8 1.2
1.2 1.3
7.5 3.1
7.6 1.4
16 2.0
5.9 1.1
2.5 1.3
6.2 1.2
2.8 1.3
5.1 1.1
39
40
58
59
0.26 0.2
0.69 0.4
a
EC50 of 1 cannot be accurately determined; 1 (2 μM) exhibited
∼40% of inhibition, but increasing to 20 μM still showed ∼40%
b
inhibition. Not tested.
cells are resistant to temozolomide (EC50 > 50 μM), suggesting
that these cells overexpress MDR1, which is the main cause for
temozolomide resistance. Compound 1 exhibits no activity
against BXD-4687 and -3752 (without an IDH1 mutation) and
normal fibroblast WI-38 cells. For BT-142 glioma cells with the
IDH1 R132H mutation, 1 was found to inhibit neurosphere
formation by ∼40% at 2 μM, but increase of the concentration
of 1 to 20 μM did not result in higher inhibition, presumably
due to the high efflux or poor permeability of 1 (Table 4).
Consistent with the results in Table 4, activity of compound 2
(as well as other 1-hydroxypyridin-2-one analogs) is not
affected by these factors. Compound 2 exhibited potent activity
G
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a
Scheme 1. Synthesis for 4-Methyl-6-substituted 1-Hydroxypyridin-2-one and Related Compounds
a
Reagents and conditions: (i) AlCl3, CH2Cl2, reflux; (ii) H2SO4/HOAc; (iii) P4S10, benzene, reflux; (iv) NH2OH or NH2NH2, pyridine, reflux; (v)
R-B(OH)2, Pd(dppf)Cl2, dioxane, reflux.
a
the nitration at the 4-position of 2,6-dibromopyridine to give
compound 68. Upon reduction of the pyridine-oxide, treatment
with NaOMe resulted in double substitutions of the 2-Br and 4-
NO2 groups to give 6-bromo-2,4-dimethoxypyridine (70). A
palladium catalyzed reaction to introduce the 6-benzyl group
followed by conversion to 1-hydroxypyridin-2-one gave rise to
compound 35, whose -Me was removed by treatment with BBr3
to produce compound 34.
Scheme 2. Synthesis for Other Compounds
CONCLUSION
■
IDH1 mutations at the residue Arg132 are frequently found in
low grade gliomas/secondary glioblastomas (∼75%), AML
(∼20%), and certain sarcomas. Studies have shown that these
mutant proteins acquire a new enzyme function, that is, the
reduction of α-KG to D2HG, causing D2HG accumulation in
the patients. High concentrations (in millimolar levels) of
D2HG can inhibit a broad range of α-KG-dependent
dioxygenases, including histone demethylases and DNA
hydroxylases, leading to abnormal levels of histone/DNA
methylation, which block cell differentiation and eventually
cause cancer initiation. Mutant IDH1 proteins are therefore
drug targets for these types of cancer. Upon screening of
compounds targeting another Mg2+/NADPH-dependent en-
zyme, 1-hydroxypyridin-2-one compounds 4 and 7 were
identified to be low micromolar inhibitors of IDH1(R132H).
Guided by SAR as well as the X-ray structure of the
IDH1(R132H)/2 complex, a total of 61 derivatives were
designed and synthesized, among which several potent
inhibitors were identified with Ki values of 140−270 nM.
SAR analysis shows that (1) the 1-hydroxypyridin-2-one core
structure is required to be active, (2) a very small group such as
-Me or -OH at the 4-position of the core is needed to achieve a
submicromolar potency, (3) a (substituted) benzyl group at the
6-position of 1-hydroxypyridin-2-one is more favored compared
with a phenyl or other groups at the 3- and 5-positions, and (4)
strongly electron-withdrawing groups are disfavored as a
substituent for the 6-benzyl, while -OMe or -OH, for example,
that in compounds 39, 40, and 60, provides an improved
activity. Ten selected compounds were tested for their activity
in inhibiting IDH1(R132C), as well as the production of
D2HG in HT1080 fibrosarcoma cells, and the results indicate
that there are good correlations between these inhibitory
activities. In addition, these compounds exhibit potent and
selective activity in inhibiting the proliferation of BT-142
glioma cells with the R132H IDH1 mutation, with the most
potent compound 58 having EC50 of 0.26 μM, while it is
considerably less active against two glioma cells without an
a
Reagents and conditions: (i) a Pd-catalyzed coupling reaction, for
example, Suzuki or Heck reaction; (ii) 3-chloroperoxybenzoic acid;
(iii) AcCl, reflux, then MeOH; (iv) POBr3, 130 °C, then MeOH; (v)
NaBH4, reflux; (vi) MOMCl, Et3N; (vii) NaOMe, reflux; (viii) BuLi,
−78 °C, then PhCHO, 25 °C; (ix) Et3SiH, trifluoroacetic acid, 50 °C;
(x) 3 N HCl; (xi) NaOH; (xii) H2O2, TFA, reflux; (xiii) HNO3,
H2SO4; 60 °C; (xiv) PBr3; (xv) NaOMe, MeOH/THF, 40 °C; (xvi)
BnZnBr, Pd(PPh3)4, THF, reflux; (xvii) BBr3, CH2Cl2.
methanolysis to give 2,6-dibromo ester 65, which was reduced,
protected with a methoxymethyl (MOM) group, and treated
with NaOMe to produce compound 66 with the desired 2-
OMe and 6-Br. Treatment with BuLi followed by benzaldehyde
afforded a secondary alcohol, whose -OH was reduced by the
reaction with Et3SiH and trifluoroacetic acid41 to give
compound 67. Deprotection of MOM as well as conversion
to 1-hydroxypyridin-2-one led to the formation of compound
37. Hydrolysis of 37 with NaOH produced compound 36.
Compounds 34 and 35 were prepared from 2,6-dibromopyr-
idine (Scheme 2). The oxidation of the pyridine ring facilitated
H
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concentration, Ki values were calculated using the Cheng−Prusoff
equation Ki = IC50/(1 + [S]/Km), where [S] is the concentration of α-
KG (1 mM) and Km is the literature value of 0.965 mM for R132H12
and 0.295 mM for R132C.6 For compounds with IC50 < 1 μM, Ki
values were calculated using the Morrison tight inhibition modeling in
Prism.
IDH1 mutation (EC50 2.8 and 7.6 μM), showing a selectivity of
>10-fold. Low cytotoxicity (EC50 > 50 μM) of these
compounds against normal fibroblast WI-38 cells was observed.
Moreover, using a cell based BBB model assay, 1-hydroxypyr-
idin-2-one inhibitors were found to be BBB permeable, while
inhibitor 1 with another chemo-type has a high efflux ratio,
which might be responsible for the observed weak activity of 1
(EC50 > 20 μM) against BT-142 glioma cells. All these results
suggest that this work could lead to a new treatment for IDH1
mutated glioma and further development of 1-hydroxypyridin-
2-one inhibitors is warranted.
Determination of the activity/inhibition of WT-IDH1 is based on
the initial linear production of NADPH. In brief, the enzyme activity
assay was performed in a 96-well microplate using the purified IDH1
(15 nM), 4 mM MgCl2, 200 μM sodium D-isocitrate (Km = 65 μM),12
and 1 mM NADP+ (≫Km for NADP) in 50 mM HEPES buffer (pH =
7.5). The reaction can be readily monitored by an increase in optical
absorbance at 340 nm. Activity and compound inhibition can be
determined similarly as described for mutant IDH1 using Prism 5.0.
Docking. Docking studies were performed with our previous
methods21−23 using the Schrodinger suite (version 2013, Schrodinger,
EXPERIMENTAL SECTION
■
̈
̈
All reagents were purchased from Alfa Aesar (Ward Hill, MA) or
Aldrich (Milwaukee, WI). Compounds were characterized by 1H
NMR on a Varian (Palo Alto, CA) 400-MR spectrometer, and the
purities monitored by a Shimadzu Prominence HPLC with a
Phenomenex C18 column (4.6 mm × 250 mm, methanol/H2O
60:40, monitored at 254 and 280 nm). The purities of all compounds
were found to be >95%.
LLC, New York, NY, 2013), which includes all of the programs
described below. The crystal structure of IDH1(R132H)/2 (PDB
4I3L) was prepared using the module “protein preparation wizard” in
Maestro (version 9.5) using default protein parameters, with all water
molecules removed, hydrogens added, inhibitor extracted, and
NADPH retained in the protein structure for docking. H-bonds
were then optimized, and the protein was energy-minimized using
OPLS-2005 force field. A receptor grid, which is large enough to
contain the whole active site, was generated using Glide (version 5.5)
without constraints. Compounds were built, minimized using OPLS-
2005 force field in Maestro, and then docked into the prepared protein
structure using Glide (docking parameters: standard-precision and
dock flexibly).
Synthesis and Characterization of Compounds 2−62. Details
of compound synthesis and characterization can be found in
Supporting Information, Experimental Section.
Expression and Purification of Human WT and R132H
Mutant IDH1. The wild-type IDH1 gene was cloned using 5′-
GATCCGAATTCGATGTCCAAAAAAATCAGTG-3′ and 5′-TGG-
TGCTCGAGTAAGTTTGGCCTGAGCTAG-3′ as forward and
reverse primers, respectively, and inserted into pET-24b vector.
Correctness of the inset was verified by sequencing. Escherichia coli
BL21-CodonPlus strain (Agilent) was transformed with the plasmid
and cultured at 37 °C in LB medium containing kanamycin (50 μg/
mL) and chloramphenicol (34 μg/mL). Upon reaching an optical
density of ∼0.6 at 600 nm, IDH1 expression was induced by addition
of 0.1 mM isopropylthiogalactoside at 18 °C for 20 h. Cells were
harvested, lysed, and centrifuged at 20000 rpm for 20 min, and the
supernatant was collected and purified using Ni-affinity (HisTrap HP,
GE Healthcare) followed by Superdex 75 (GE Healthcare) column
chromatography. WT-IDH1 was obtained with ∼90% purity (SDS-
PAGE).
Cell Growth Inhibition. The cytotoxicity assay was done using
our previous method.21,22 In brief, 105 WI-38 fibroblast cells were
inoculated into each well of a 96-well plate and cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum at 37 °C in a 5% CO2 atmosphere with 100% humidity
overnight for cell attachment. After addition of compounds (from 1 to
50 μM), plates were incubated for 48 h, after which cell viability was
assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay, using a commercially available kit (Sigma).
IC50 value of each compound was calculated from dose−response
curves by Prism 5.0.
D2HG Production Inhibition in HT1080 Cells. The HT1080
fibrosarcoma cell line, which harbors an IDH1(R132C) mutation, was
obtained from ATCC (Manassas, VA). The D2HG production
inhibition assay followed our previous protocol.21 In brief, 105 cells/
well were seeded into wells of a six-well plate and cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% dialyzed fetal bovine serum at 37 °C in a 5% CO2 atmosphere
with 100% humidity overnight for cell attachment. Cells were treated
with an increasing concentration of compounds in 2 mL of culture
medium for 48 h. Medium was collected and diluted with 8 mL of
MeOH. After shaking, the mixture was centrifuged to remove any
precipitate, and the supernatant was subjected to HPLC-MS to
separate and quantitate the amount of D2HG. HPLC was run using a
Phenomenex C18 column (250 mm × 4.6 mm, 5 μm) with 50%
tributylamine buffer/50% MeOH as an eluent at a flow rate of 0.5 mL/
min (tributylamine buffer: 10 mM tributylamine, 15 mM acetic acid,
3% MeOH in water). A single ion monitoring (SIM) for 147 Da was
used to detect and quantitate the amount of D2HG (parameters:
interface voltage, −4.2 kV; detector voltage, 1.3 kV; nebulizing gas, 1.5
L/min; drying gas, 15 L/min; desolvation line temperature, 250 °C;
heat block temperature, 200 °C; Pirani gauge vacuum, 102 Pa; ion
gauge vacuum: 5 × 10−4 Pa). Authentic D2HG purchased from Sigma-
Aldrich (St Louis, MO) was used to validate and calibrate the HPLC-
MS assay conditions before measuring the D2HG concentrations
secreted by the HT1080 cells untreated or treated with 2. EC50 was
calculated from the dose−response curve by Prism 5.0.
R132H and R132C mutant IDH1 genes were generated from the
wild-type IDH1 plasmid, using QuikChange site-directed mutagenesis
kit (Agilent) following the manufacturer’s protocol. Correctness of the
gene sequences was verified. The mutant genes were then transferred
to pGEX-KG vector for better expression. Expression of mutant IDH1
enzymes were performed similarly to that for the wild-type protein.
Cells were harvested, lysed, and centrifuged at 20000 rpm for 20 min,
the supernatant was collected, and the recombinant protein was
trapped in glutathione sepharose resin (GE Healthcare). The GST-
IDH1 fusion protein was eluted with 10 mM glutathione solution, and
the GST tag was removed by thrombin digestion overnight at 4 °C.
IDH1(R132H) and IDH1(R132C) were obtained in ∼90% purity
(SDS-PAGE) using a glutathione sepharose column followed by
Superdex 75 gel filtration column chromatography.
Enzyme Inhibition Assays. Determination of the activity and
inhibition of IDH1(R132H) and IDH1(R132C) is based on the initial
linear consumption of NADPH in the reaction. The enzyme activity
assay was performed in a 96-well microplate using the purified IDH1
mutant (100 nM), 4 mM MgCl2, 1 mM α-KG, and 100 μM NADPH
(≫Km for NADPH) in 50 mM 4-(2-hydroxyethyl)-1-piperazineetha-
nesulfonic acid (HEPES) buffer (pH = 7.5) containing 0.1 mg/mL
bovine serum albumin. For inhibition assays, triplicate samples of
compounds were incubated with the protein for 5 min before addition
of α-KG to initiate the reaction. The optical absorbance of each well
was monitored every 30 s at 340 nm, where NADPH has maximum
absorption, using a Beckman DTX-880 microplate reader. The data
were imported into Prism (version 5.0, GraphPad), and the IC50 values
were calculated with a standard dose−response curve fitting. For
compounds with IC50 values much greater than the enzyme
In Vitro Blood−Brain Barrier (BBB) Permeability Assay.
MDCK-MDR1 cells were obtained from NIH (Bethesda, MD) and
maintained in DMEM with 10% standard fetal bovine serum, 100 U/
mL penicillin, 100 μg/mL streptomycin, and 80 ng/mL colchicine. For
I
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transport experiments, cells with passage numbers of 24−33 were
seeded at a density of 60000 cells/cm2 on Transwell plates (pore size
0.4 μm; diameter 6.5 mm; insert growth area 0.33 cm2; Costar,
Pittston, PA) and maintained in culture as previously described.28 In
brief, confluent MDCK-MDR1 monolayers expressing P-glycoprotein
were obtained 3−4 days postseeding, and their integrity was assessed
by measurement of the transepithelial electrical resistance (TEER, Ω·
cm2) with a volt−ohm meter (Millicell-ERS, Millipore Corpration,
Billerica, MA). After subtraction of the background TEER (i.e., the
resistance exhibited by the filter alone), only MDCK-MDR1 cell
monolayers that exhibited a TEER > 1000 Ω·cm2 throughout
[measured before and after the study] the experiments were used.
Drug transport across the cell monolayers was measured in both
apical to basolateral (A-B) and basolateral to apical (B-A) directions.
Experiments were performed in HBSS (Hank’s balanced salt solution
containing 50 mM HEPES buffer, pH ≈ 7.4) at 37 °C using
monolayers that were preincubated for 30 min with prewarmed HBSS.
At the start of the experiment, fresh HBSS was added to the receiver
compartments, and the compounds were independently added to the
donor compartments at an initial concentration of 10 μM (diluted
from 10 mM DMSO stock to a final DMSO concentration of 0.1%)
and then incubated at 37 °C for 90 min, after which samples were
collected from both the receiver and donor compartments.
Detection and quantification of the compounds were performed
with an LC/MS/MS system (HPLC, Shimadzu, Kyoto, Japan; MS,
QTrap 5500, Applied Biosystems, Foster City, California) using an
electrospray ionization (ESI) interface and operated in positive ion
mode. Instrument control, data acquisition, and processing for both
chromatography and MS were performed using the Analyst 1.5.1
software (Applied Biosystems MDS Sciex, Ontario, Canada). The
chromatographic separation system consisted of a guard cartridge
(C18, 4.0 × 2.0 mm2; Phenomenex, Torrance, California), an analytic
column (Luna C18, 3 μm particle size, 50 × 2.0 mm2; Phenomenex),
and a mobile phase of acetonitrile/10 mM ammonium formate (65:35,
v/v), delivered isocratically at a flow rate of 0.2 mL/min. To 10 μL
samples from both the apical or basolateral sides, 40 μL of cold
acetonitrile was added; samples were mixed and centrifuged at 15000
rpm for 5 min; 10 μL of the resultant supernatant was injected into the
LC/MS-MS system. Compound quantification was performed by ESI-
selected reaction monitoring. The column effluent was monitored at
the following precursor−product ion transitions: m/z 214.9 → 197.1
for compound 2, m/z 244.9 → 212.3 for 39, and m/z 463.2 → 123.0
for 1 with a dwell time of 100 ms for each ion transition.
ASSOCIATED CONTENT
* Supporting Information
Alignments of crystal structures of IDH1 forms bound to
substrates and inhibitors and experimental procedures provid-
ing details of compound synthesis and characterization. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: 713-798-7415. E-mail: ysong@bcm.edu.
■
Author Contributions
¶Z.L. and Y.Y. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant (R01NS080963) from
National Institute of Neurological Disorders and Stroke
(NINDS/NIH) and a grant (RP140469) from Cancer
Prevention and Research Institute of Texas (CPRIT) to Y.S.,
as well as a grant (R01CA127963) to J.M.G.
ABBREVATIONS
■
α-KG, α-ketoglutaric acid; BBB, blood−brain barrier; D2HG,
D-2-hydroxyglutaric acid; ICT, isocitric acid; IDH, isocitrate
hydrogenase; MDR1, multidrug resistance 1; R132H, Arg132
mutation to His; R132C, Arg132 mutation to Cys; SAR,
structure−activity relationship; WT, wild-type
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