Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases by 2-Thiohydantoin Compounds

Article abstract of DOI:10.1021/acs.jmedchem.5b00684

Somatic mutations of isocitrate dehydrogenase 1 (IDH1) at R132 are frequently found in certain cancers such as glioma. With losing the activity of wild-type IDH1, the R132H and R132C mutant proteins can reduce α-ketoglutaric acid (α-KG) to d-2-hydroxyglutaric acid (D2HG). The resulting high concentration of D2HG inhibits many α-KG-dependent dioxygenases, including histone demethylases, to cause broad histone hypermethylation. These aberrant epigenetic changes are responsible for the initiation of these cancers. We report the synthesis, structure-activity relationships, enzyme kinetics, and binding thermodynamics of a novel series of 2-thiohydantoin and related compounds, among which several compounds are potent inhibitors of mutant IDH1 with K_i as low as 420 nM. X-ray crystal structures of IDH1(R132H) in complex with two inhibitors are reported, showing their inhibitor-protein interactions. These compounds can decrease the cellular concentration of D2HG, reduce the levels of histone methylation, and suppress the proliferation of stem-like cancer cells in BT142 glioma with IDH1 R132H mutation.

Full text of DOI:10.1021/acs.jmedchem.5b00684

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Article
Inhibition of Cancer-Associated Mutant Isocitrate
Dehydrogenases by 2-thiohydantoin compounds
Fangrui Wu, Hong Jiang, Baisong Zheng, Mari Kogiso,
Yuan Yao, Chao Zhou, Xiao-Nan Li, and Yongcheng Song
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00684 • Publication Date (Web): 17 Aug 2015
Downloaded from http://pubs.acs.org on August 23, 2015
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Inhibition of Cancer-Associated Mutant Isocitrate
Dehydrogenases by 2-thiohydantoin compounds
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Fangrui Wu,†,§ Hong Jiang,†,§ Baisong Zheng,†,§ Mari Kogiso,¶ Yuan Yao,† Chao Zhou,† Xiao-Nan Li,¶
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‡
Yongcheng Song*,†,
† Department of Pharmacology, ¶ Department of Pediatrics-oncology, ‡ Dan L. Duncan Cancer Center,
Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.
CORRESPONDING AUTHOR FOOTNOTE:
*To whom correspondence should be addressed. Address: Department of Pharmacology, Baylor College
of Medicine, 1 Baylor Plaza, Houston, TX 77030. Tel: 713-798-7415. E-mail: ysong@bcm.edu.
§ These authors contributed equally.
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ABSTRACT
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Somatic mutations of isocitrate dehydrogenase 1 (IDH1) at R132 are frequently found in certain cancers
such as glioma. With losing the activity of wild-type IDH1, the R132H and R132C mutant proteins can
reduce α-ketoglutaric acid (α-KG) to D-2-hydroxyglutaric acid (D2HG). The resulting high
concentration of D2HG inhibits many α-KG-dependent dioxygenases, including histone demethylases,
to cause broad histone hypermethylation. These aberrant epigenetic changes are responsible for
initiation of these cancers. We report the synthesis, structure activity relationships, enzyme kinetics and
binding thermodynamics of a novel series of 2-thiohydantoin and related compounds, among which
several compounds are potent inhibitors of mutant IDH1 with Ki as low as 420 nM. X-ray crystal
structures of IDH1(R132H) in complex with two inhibitors are reported, showing their inhibitor-protein
interactions. These compounds can decrease the cellular concentration of D2HG, reduce the levels of
histone methylation, and suppress proliferation of stem-like cancer cells in BT142 glioma with IDH1
R132H mutation.
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INTRODUCTION
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Isocitrate dehydrogenase (IDH) catalyzes oxidative decarboxylation of isocitric acid (ICT) to α-
ketoglutaric acid (α-KG) (Figure 1A), which is one of the key reactions in the tricarboxylic acid cycle.1
Among the three isoforms of IDH in humans, frequent somatic mutations of IDH1 have recently been
found in certain cancers.2-4 Particularly noted is that ~75% of lower grade (grade II and III) gliomas, the
most common form of brain tumors, contain IDH1 mutations.5,6 Although growing relatively slowly,
these gliomas are invasive and eventually develop to become secondary glioblastoma multiforme
(GBM, grade IV glioma), a highly malignant tumor with 5-year survival rates of <10%. Distinct from
many other mutations, the mutation site in IDH1 is always located in the R132 residue, with R132H
being predominant (~93%). IDH mutations (including IDH1 and IDH2) have also been found in ~20%
acute myeloid leukemia (AML) and ~50% of certain sarcomas.4,7 Moreover, clinical data suggest the
IDH mutation is an early event in cancer initiation and development.5,6
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Figure 1. Reactions catalyzed by (A) Wild-type IDH1, (B) Mutant IDH1, and (C) α-KG-dependent
histone demethylases. D2HG, the product of mutant IDH1, inhibits α-KG-dependent histone
demethylases, resulting in histones with more methylated lysine residues; (D) The structures of two
known inhibitors of mutant IDH1.
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Biochemically, all of the characterized mutant IDH1 proteins in cancer almost lose the wild-type
(WT) enzyme activity. However, these mutant enzymes can reduce α-KG to D-2-hydroxyglutaric acid
(D2HG), using NADPH as the cofactor (Figure 1B).4,8,9 This new function causes a high concentration
of D2HG to be accumulated in cancer cells (as well as in patient plasma). D2HG was found to be an
inhibitor of many α-KG-dependent dioxygenases, including the JmjC family of histone demethylases
(Figure 1C).3 This inhibitory activity leads to genome-wide hypermethylation of histones in IDH
mutated cancers.10,11 Given the crucial roles of histone lysine methylation in gene regulation as well as
cancer initiation, the observed aberrant epigenetic changes were hypothesized to be responsible for the
initiation and/or maintenance of the IDH mutated cancers.12,13
As representatively shown in Figure 1D, three chemo-types of inhibitors (1 – 3) of mutant IDH1
have been reported.14-18 These compounds can reduce the cellular levels of D2HG, showing on-target
activity in cells. Compounds 1 and 2 can also inhibit proliferation of IDH1 mutated cancer cells,15,17
demonstrating that mutant IDH1 is a drug target for these cancers. Here, we report the synthesis and
structure activity relationships (SAR) of a series of 2-thiohydantoin-containing compounds as a new
chemo-type of inhibitors of mutant IDH1. X-ray crystallographic, enzyme kinetics as well as binding
thermodynamics studies of selected compounds showed the binding structures and the mode of action.
Biological activity testing demonstrated these compounds can reduce cellular concentration of D2HG,
inhibit histone lysine methylation, and impair self-renewal ability of IDH1 mutated glioma cells.
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RESULTS AND DISCUSSION
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Synthesis and structure activity relationship studies. Recombinant human IDH1 R132H mutant
protein was expressed in E. coli and purified with a series of column chromatography, using our
previously reported methods.16,17 The activity and inhibition of IDH1(R132H) were determined based on
initial linear consumption of NADPH (Figure 1B) monitored at 340 nm, where NADPH has the
maximal optical absorption. Through compound screening using our published biochemical method,16,17
3-benzyl-5-(3,4-dihydroxybenzylidene)-2-thiohydantoin (4)
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was identified to be an inhibitor of IDH1(R132H) with a Ki value of 4.7 µM against the substrate α-KG.
This compound is of interest since it possesses good inhibitory activity and is amenable for medicinal
chemistry studies.
Scheme 1. General synthetic method.a
aReagents and conditions: (i) R1NCS, KOH, H2O-EtOH; (ii) H2SO4, Acetone; (iii) an aldehyde, e.g., 6-
methoxynicotinaldehyde, NaOAc, HOAc, reflux; (iv) HCl, reflux.
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Medicinal chemistry was performed with modifications on the 3-substituent (-R1, Table 1), 5-
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substituent (-R2, Table 1) and the 2-thiohydantoin core (Chart 1) of compound 4. These compounds
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were efficiently synthesized using a general method as shown in Scheme 1. Glycine was reacted with R1
isothiocyanate and the resulting thiourea product was cyclized in the presence of sulfuric acid to give 3-
R1-substituted 2-thiohydantoin. The condensation reaction of a 2-thiohydantoin (or related heterocyclic)
compound with an aldehyde in the presence of sodium acetate in refluxing acetic acid to give a R2-
substituted 5-methylene-2-thiohydantoin compound in 60-90% yield. The synthesis and characterization
of compounds 4-41 are described in the Supporting Information Experimental Section.
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Compound 5 (Table 1) having a 3,4-dimethoxyphenyl group was found to be inactive (Ki > 50
µM) against IDH1(R132H). Similarly, compounds 6 and 7, which contain the same R2 group of 3,4-
dimethoxyphenyl, are also inactive. This observation suggests that at least one of the two –OH groups in
compound 4 is important for the activity. Indeed, after removal of the methyl groups of 6 and 7, the
corresponding hydroxyl compounds 8 and 9 can inhibit the mutant enzyme with Ki values of 16.7 and
13.4 µM, respectively, although they are ~3-fold less active than compound 4 (R1 = benzyl), which
should be due to a different R1 group (i.e., phenyl and 4-OH-benzyl for 8 and 9 versus benzyl in 4).
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Table 1. Structures and Ki values of 5- or 6-substituted compounds against IDH1(R132H).
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R1
Bn
R2
Ki (µM)
>50
3,4-OMe-Ph
3,4-OMe-Ph
3,4-OMe-Ph
3,4-OH-Ph
5
6
Ph
>50
4-OMe-Bn
Ph
>50
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16.7
13.4
>50
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4-OH-Bn
Bn
3,4-OH-Ph
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Pyridin-3-yl
2-OMe-pyridin-5-yl
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Bn
>50
Bn
2-Pyridinone-5-yl
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12
Et
2-OMe-pyridin-5-yl
2-Pyridinone-5-yl
2-OMe-pyridin-5-yl
2-Pyridinone-5-yl
2-OMe-pyridin-5-yl
2-Pyridinone-5-yl
2-OMe-pyridin-5-yl
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Et
Me
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Me
H
H
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>50
-CH2COOH
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-CH2COOH
2-Pyridinone-5-yl
1-Me-2-pyridinone-5-yl
1-Bn-2-Pyridinone-5-yl
1-Oxo-2-OMe-pyridin-5-yl
>50
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Me
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Me
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Me
Me
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Me
Me
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>50
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H
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Because undesirable properties associated with the 3,4-dihydroxyphenyl (catechol-containing)
group, which is a strong metal chelator and is easily oxidized, compounds 10 – 12 were synthesized in
an effort to find another R2 group that has comparable or improved activity. Compound 10 with a 3-
pyridinyl and compound 11 with a 2-methoxy-5-pyridinyl group have one and two H-bond acceptors.
Both compounds are inactive. However, compound 12 with a 2-pyridinone-5-yl group possesses a good
inhibitory activity with a Ki value of 4.8 µM against IDH1(R132H), showing a comparable activity to
compound 4.
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Next, compounds 13 – 20 (Table 1) were synthesized to optimize the R1 group. Although
compounds 13, 15 and 17 containing a 2-methoxy-5-pyridinyl R2 group are still moderate or weak
inhibitors of IDH1(R132H) with Ki values of 9.2, 25 and 7.8 µM, their activities are superior to that of
compound 11 (Ki > 50 µM), suggesting a smaller R1 group (i.e., -Et in 13, -Me in 15 and -H in 17) is
more favorable. The demethylated compounds 14, 16 and 18 (with a 2-pyridinone-5-yl R2 group) were
found to possess strong activities. While compound 14 with an -Et R1 group has a Ki of 3.6 µM,
compounds 16 and 18 with a -Me and -H R1 group, respectively, showed potent inhibitory activities with
Ki values of 750 and 420 nM. The increasing activities of compounds 12, 14, 16 and 18 suggest a SAR
trend for the R1 group of Bn < Et < Me < H, with small substituents Me and H being most favorable.
Compounds 19 and 20 were synthesized to find if a carboxyl group or its ester is a good R1. However,
none of these compounds are active.
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Compounds 21 – 34 (Table 1) were synthesized to investigate the SAR for the R2 group.
Compound 21 with an N-methyl substituent on the 2-pyridinone ring was found to have a slight
improved activity with a Ki of 470 nM, as compared with 16. However, an N-benzyl substituent
considerably reduces the inhibitory activity of compound 22 (Ki = 4.6 µM). Compound 23 and its
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demethylated compound 24, which have an O atom (or –OH) attached to N1 of the pyridine-2-one ring
also show decreased activities (Ki = 4.3 and 5.9 µM, respectively). Compounds with other pyridinyl R2
substituents were synthesized for SAR studies. Compounds 25 and 26 containing 2-methoxy-pyridin-3-
yl and pyridine-2-one-3-yl group, respectively, were found to have very weak or no inhibitory activity
(Ki ≥ 25 µM) against IDH1(R132H). Compound 27 with 6-methoxypyridin-2-yl R2 group is inactive,
while the demethylated compound 28 has a modest activity (Ki = 11.3 µM). Compounds bearing a 4-
pyridinyl R2 group are of interest. Although compound 29 with a 4-pyridinyl group is inactive (Ki > 50
µM), compounds 30 and 31 having 2-methoxy-4-pyridinyl and pyridin-2-one-4-yl groups are good
inhibitors with Ki values of 2.6 and 9.2 µM, respectively. In addition, compound 30 is the only -OMe
containing compound that is considerably more active than its -OH analog 31, suggesting a more
hydrophobic group might be favorable at this position and several compounds were therefore
synthesized. Loss of activity for compound 32 indicates the 3-bromo-4-pyridinyl R2 group is disfavored,
while compound 33 bearing 3-phenyl-4-pyridinyl group showed good inhibitory activity (Ki = 5.6 µM).
Similarly, compound 34 having a 2,5-substituted furan R2 group also exhibited a good Ki of 3.2 µM.
The SAR studies described above show that a good R2 group requires a strong H-bond acceptor
(e.g., the 4-O atom in 4 and 2-O in 12, 14, 16, 18, 21, 22 and 24) at the para-position. The potent
activity of compound 21 (as well as that of 22) indicates that an H-bond donor (e.g., 3-OH in 4 and 1-
NH in pyridin-2-one containing compounds) is not essential.
SARs for the 2-thiohydantoin ring were investigated and the results are shown in Chart 1. We
synthesized compounds 35 and 36 with a 2-thioxothiazolidin-4-one core structure, 37 and 38 with a
hydantoin, and compound 39 with a 2-amino-imidazol-4-one core. The activities of compounds 35 and
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36 (Ki = >50 and 3.1 µM, respectively) are significantly lower than those of the corresponding 2-
thiohydantoin compounds 17 and 18 (Ki = 7.8 and 0.42 µM), showing -NH- at the 1-position of the ring
is more favorable than -S-. Complete loss of activity for compounds 37 and 38 was unexpected,
indicating the 2-carbonyl O atom of the hydantoin ring is highly disfavored, as compared to the
corresponding S atom of 2-thiohydantoin ring in 17 and 18. Similarly, compound 39 having an –NH2 at
this position is also inactive. In compound 40, the methyl group attached to the S atom causes the
compound to completely lose the inhibitory activity against IDH1(R132H). Moreover, adding a methyl
group to the N3 atom of the 2-thiohydantoin ring in compound 41 (Ki = 16.5 µM) resulted in a 22-fold
activity reduction, as compared to compound 16 (Ki = 0.75 µM).
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Chart 1. Structures and activities of compounds 35-41.
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Activity against R132C and WT IDH1. Compounds 16, 18 and 22 were selected to be tested for their
abilities to inhibit R132C (a frequent mutation in leukemia and sarcoma) mutant as well as WT IDH1.
These two recombinant IDH1 proteins were also expressed in E. coli and purified with our previously
reported methods.16,17 The activity and inhibition of IDH1(R132C) were similarly determined as those of
the R132H mutant protein. For WT IDH1, the reaction rate was calculated based on the production of
NADPH (Figure 1B), resulting in an increase of optical density at 340 nm.
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As can be seen in Table 2, these three compounds were found to be also inhibitors of
IDH1(R132C). While compound 18 is the most potent inhibitor of IDH1(R132H) (KiR132H = 0.42 µM), it
is less inhibitory against the R132C mutant protein with a Ki of 2.3 µM. Compound 16 exhibited a Ki
value of 1.2 µM against IDH1(R132C), which is, however, comparable to that against IDH1(R132H)
(KiR132H = 0.75 µM). In addition, compound 22 showed a less inhibitory active against IDH1(R132C)
with a Ki of 12.5 µM. These three compounds have moderate to good selectivity for the mutant IDH1
enzymes. Compounds 16, 18 and 22 were found to be relatively weak inhibitors of WT IDH1, with Ki
values of 8.8, 10.3 and 32.9 µM, respectively. Thus, compound 16 shows selectivity indices of 11.7 and
7.3 (Table 2) for the R132H and R132C mutant protein, respectively. Compound 18 was found to be
24.5- and 4.5-fold more active for the R132H and R132C mutants, as compared to the WT enzyme.
Similarly, compound 22 possesses selectivity indices of 7.2 and 2.6-fold for inhibition of R132H and
R132C, respectively.
Table 2. Activity (Ki, µM) and selectivity of compounds 16, 18 and 22.
Selectivity Index
R132H
R132C
1.2 ± 0.3
2.3 ± 0.7
12.5 ± 0.7
WT
KiWT/KiR132H KiWT/KiR132C
0.75 ± 0.39
0.42 ± 0.21
4.6 ± 0.7
8.8 ± 0.4
10.3 ± 0.5
32.9 ± 1.2
11.7
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2.6
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22
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X-ray crystallography. X-ray crystallography was used to find how these novel 2-thiohydantoin
inhibitors bind to IDH1(R132H). We determined the tertiary structures of IDH1(R132H) in complex
with NADPH and compounds 16 and 22 to a resolution of 3.2 Å, similar to those reported
previously.16,19 Statistics for diffraction data and structural refinement are in Supporting Information
Table S1. As shown in Figure 2A and Supporting Information Figure S1, the protein IDH1(R132H) was
found to crystallize as a homodimer with one NADPH molecule co-crystallized in each protein subunit.
Only one molecule of 2-thiohydantoin inhibitor 16 (or 22) can be found in the protein homodimer, based
on the electron density and omit maps (Figures 2B, C and Figure S2). These observations were also
found in our previous structures of IDH1(R132H) in complex with 1-hydroxypyridinone inhibitors (e.g.,
compound 2).16
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Figure 2. X-ray structures of IDH1(R132H):inhibitor complexes. (A) The overall structure of
IDH1(R132H) in complex with compound 16 (ball & stick model) and NADPH (tube model); (B) The
2Fo-Fc electron density map of IDH1(R132H):16 at the inhibitor-binding site, contoured at 1σ; (C) The
Fo-Fc omit map of IDH1(R132H):16, contoured at 3σ; (D) The superimposed structures of
IDH1(R132H) (electrostatic surface model) in complex with 16 (with C atoms in green) and 22 (in
pink); and (E) The close-up views of compound 16 and (F) compound 22 in IDH1(R132H). The protein
backbones are shown as blue lines and H-bonds as purple dotted lines.
Compounds 16 and 22 are located deeply inside a cleft between the two protein homodimers
(Figure 2D), sitting in a pocket surrounded by the residues Thr77, Ser94, Asn96, Gly97, Arg100,
Asn101, Arg109 and NADPH. Figures 2E and S3A show the close-up view of the binding site and the
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ligand-protein interactions of compound 16. The almost flat inhibitor molecule lies on a “bed surface”
composed of Gly97, Asn96 and Ser94, with the distance between the two almost parallel planes being
~3.3-3.6 Å. The two partially negatively charged O atoms of 16 form three H-bonds as well as
electrostatic interactions with the positively charged side chains of Arg100 and Asn101. The S atom of
compound 16 was found to have favorable, mostly hydrophobic interactions with Thr77 and NADPH.
Figures 2F and S3B show the binding site and the ligand-protein interactions of compound 22. The
conjugated rings of 2-thiohydantoin and pyridinone adopt a similar binding pose as observed for
compound 16, having favorable interactions with Gly97, Asn96 and Ser94. In addition, the two O atoms
of 22 also have three H-bond and electrostatic interactions with Arg100 and Asn101. Therefore, it
appears that these two types of common interactions between the protein and compounds 16 and 22
provide the majority of binding affinity for these inhibitors. The 2-thiohydantoin ring of compound 22
was found to orientate slightly differently from that of compound 16 (Figure 2D), with, for example, the
distance between the two S atoms being ~2.2 Å. While such a movement for 22 causes loss of the
interactions between the S atom and NADPH and Thr77, it renders the 3-methyl group of 22 to have van
der Waals interactions with the side chain of Arg109. The benzyl group of compound 22 bends and
inserts into a deep side-pocket, having mostly hydrophobic interactions with the side chains of Lys93,
Gly97 and Thr98 (Figure 2D and F).
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Previous crystallographic studies showed that unlike the WT protein, the R132H IDH1 mutant
has two ligand binding sites.16,19 Site I is where ICT as well as 1-hydroxypyridinone inhibitors (e.g.,
compound 2) bind to IDH1(R132H). The binding site II is the catalytically active site where the
substrate α-KG is located.8 It is also noted that the two binding sites in IDH1(R132H) do not co-exist. A
large protein conformational change occurs to switch the two ligand binding sites in IDH1(R132H) that
recognize a different ligand (e.g., ICT versus α-KG). However, in the WT IDH1, there is only one ligand
binding site that corresponds to site II in IDH1(R132H).
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Superposition of the crystal structures of IDH1(R132H) in complex with ICT, α-KG and
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compounds 16 and 22, shown in Figure 3A, demonstrated that the two inhibitors occupy the ligand
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binding site I, the same location where ICT and 1-hydroxypyridinone inhibitors (e.g., 2) are located in
IDH1(R132H). α-KG is ~6 Å away in the binding site II. Figure 3B shows the large protein
conformational differences between the two binding states of IDH1(R132H). In addition, the binding
poses of NADPH in the two structures also differ significantly (Figure 3B).
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Figure 3. (A) Superimposed structures of IDH1(R132H):16 (protein as blue lines) and IDH1(R132H):α-
KG (protein as brown lines), showing the two ligand binding sites. Also aligned are the crystal structures
of IDH1(R132H):ICT (with C atoms in gray) and :22 (in purple), showing both ligands are also located
in the binding site I; (B) Superposition of the structures of IDH1(R132H):16 (protein as blue ribbons)
and IDH1(R132H):α-KG (as yellow ribbons), showing a large protein conformational difference.
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Compound 16 (C atoms in green) and α-KG (in brown) are shown as ball and stick models. In addition,
the conformation as well as the binding site of NADPH of the two structures (tube models in green and
brown, respectively) are also significantly different.
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Enzyme kinetic studies. We also performed a steady-state enzyme kinetic study for the most potent
compound 18, with which an X-ray crystal structure of IDH1(R132H) cannot be obtained. Initial
velocities of IDH1(R132H) were determined in the presence of increasing concentrations of compound
18, the substrate α-KG and cofactor NADPH. These data were imported into the program SigmaPlot and
fitted to competitive, uncompetitive and noncompetitive kinetic models, using a Lineweaver-Burk or
Michaelis-Menten plot. The best model, as judged by the R2 and AICc values, showed the mode of
action for the inhibitor. Thus, compound 18 was found to exert a competitive mode of action against the
substrate α-KG, as shown in Figures 4A (competitive fitting) and S4A (uncompetitive and
noncompetitive fitting). In addition, compound 18 was determined to be a noncompetitive inhibitor
against the cofactor NADPH (Figures 4B and S4B). Moreover, enzyme kinetic results for compound 18
are in line with the crystal structure of compound 16 described above, which also suggests that it is
competitive to α-KG and noncompetitive to NADPH.
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Figure 4. The best fitting models for enzyme kinetic studies of compound 18. (A) Competitive
inhibition model with variable concentrations of α-KG using a Lineweaver-Burk plot; and (B)
Noncompetitive inhibition model with variable concentrations of NADPH using a Michaelis-Menten
plot.
Binding thermodynamics studies. We performed isothermal titration calorimetry (ITC) experiments to
further characterize the binding as well as mode of action for compound 18. First, binding
thermodynamics of compound 18 to IDH1(R132H) (100 µM) without NADPH was investigated in 30
mM Tris buffer (pH 7.4) with 4 mM MgCl2 and 150 mM NaCl at 298K. Use of a relatively high
concentration of the protein was because of the small amount of heat generated for the binding. As
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shown representatively in Figure 5A, the binding of 18 to IDH1(R132H) alone was slightly exothermic
(ΔH = -1.5 kJ/mol) with a Ka (affinity constant) value of 1.68 × 105 M-1, or a Kd (dissociate constant)
value of 5.9 µM. This equals a ΔG value of -29.8 kJ/mol, showing the binding is mostly entropy driven
(with a calculated ΔS of 95.0 J K-1 mol-1). Next, binding of compound 18 to IDH1(R132H) (133 µM) in
the presence of NADPH (300 µM) was found to be slightly more exothermic (ΔH = -2.5 kJ/mol) with a
Kd value of 8.9 µM (Figure 5B). Comparable Kd values as well as similar thermodynamics for these two
experiments suggest that the binding of compound 18 to IDH1(R132H) is not significantly affected by
NADPH. These two molecules could bind to different binding sites in the protein. Third, compound 18
was titrated into IDH1(R132H) (100 µM) with NADPH (300 µM) and α-KG (300 µM). As shown
representatively in Figure 5C, the observed binding affinity of 18 to the protein (Kd = 46.0 µM) was
found to be reduced by ~5-folds in the presence of α-KG, suggesting inhibitor 18 and α-KG are
competitive to each other. These results are consistent with the enzyme kinetics data as well as the X-ray
crystallographic studies of compounds 16 and 22.
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Figure 5. Representative ITC results and fitting curves for (A) compound 18 binding to IDH1(R132H)
(100 µM); (B) 18 binding to IDH1(R132H) (100 µM) in the presence of NADPH (300 µM); and (C) 18
binding to IDH1(R132H) (100 µM) in the presence of NADPH (300 µM) and α-KG (300 µM).
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Biological activity evaluation. First we tested if the most potent inhibitors 16 and 18 inhibit mutant
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IDH1 enzymes in vivo (cells), using human fibrosarcoma cell line HT1080, which harbors R132C
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mutation of IDH1, as a cell model. Thus, HT1080 cells in an exponential growth phase were treated with
increasing concentrations of compounds 16 and 18 for 2 days, after which the concentrations of D2HG
were determined by HPLC-MS using our previous method.17 As can be seen in Figure 6A, treatment
with compounds 16 and 18 significantly reduced the production of D2HG, showing these inhibitors are
cell membrane permeable and on-target in cells.
Figure 6. Cellular activity of mutant IDH1 inhibitors. (A) Treatment of compounds 16 and 18 caused
significantly reduced amount of D2HG in HT1080 cells; (B) Treatment of 18 significantly reduced
methylation levels on histone lysine residues in HT1080 cells, while inactive compound 20 did not
significantly affect histone methylation levels; (C) Treatment of compounds 18, 16 and 1 inhibited
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neurosphere formation of BT142 glioma cells with IDH1 R132H mutation, but (D) did not affect
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neurosphere formation of BXD-3752 glioma cells without an IDH1 mutation.
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Upon confirming their ability to inhibit cellular mutant IDH1, we next investigated whether the
activity can alter levels of histone methylation. Since D2HG broadly inhibits α-KG-dependent histone
lysine demethylases,3 it is expected that treatment with inhibitors of mutant IDH1 can restore the activity
of these histone demethylases and therefore reduce the levels of histone lysine methylation. HT1080
cells were treated with compound 18 for 4 days. Histone proteins from both control and treated cells
were extracted and subjected to Western blot analysis, probed by specific antibodies of human histone
H3, trimethylated and dimethylated histone H3 lysine 4 (H3K4me3 and H3K4me2), dimethylated
histone H3 lysine 27 (H3K27me2) and dimethylated histone H4 lysine 20 (H4K20me2). As shown in
Figure 6B, compound 18 was found to significantly reduce the methylation levels of these histone lysine
residues in a dose dependent manner. To further confirm the effects were due to inhibition of mutant
IDH1, inactive compound 20 (Table 1) was used in the experiments and it did not significantly reduce
D2HG (data not shown) as well as histone lysine methylation levels in HT1080 (Figure 6B). These
results show that by reducing the cellular concentration of D2HG, the inhibitors of mutant IDH1 can
maintain the functions of histone demethylases and reverse the histone hypermethylation phenotype in
HT1080 with IDH1 R132C mutation.
Compounds 16 and 18 were next tested for their in vitro antitumor activity, together with potent
known inhibitor 1 as a comparison. These three compounds were found to have no or negligible activity
(EC50 > 50 µM) against proliferation of MCF-7 (breast), A549 (lung) cancer cells as well as WI-38
normal fibroblast cells. In addition, compounds 1, 16 and 18 did not significantly inhibit the growth of
HT1080 cells (EC50 > 50 µM). These results showed these potent inhibitors of mutant IDH1 do not have
general cytotoxicity. Lack of growth inhibition against these tumor and normal cells also suggests that
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compounds 16 and 18 do not appear to affect critical cellular metabolism and signaling pathways,
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showing possibly good selectivity, although an enzyme selectivity profiling needs to be done for future
studies.
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We further tested whether these compounds inhibit cancer stem cells (CSC) containing an IDH1
mutation. CSCs represent a small fraction of cancer cells that possess certain stem cell traits including
ability to self-renew and differentiate. Studies have found that only CSCs can generate new tumors when
transplanted into a new host.20 In the perspective of cancer treatment, CSCs are more resistant to
chemotherapies and also believed to be responsible for tumor relapse and metastasis. It is therefore of
importance to find compounds have selective activity against CSCs. To this end, patient sample derived
BT142 glioma cells21 bearing IDH1 R132H mutation were cultured in serum-free media and grew as
“neurospheres”, which are colonies of cells with a diameter of 30 - 300 µm. Stem-like glioma cells are
found to be enriched in these neurospheres.22-24 BXD-3752 glioma cells25 without an IDH1 mutation
were included as a control cell model. As shown in Figure 6C, compounds 16 and 18 were able to
significantly inhibit proliferation of BT142 glioma stem-like cells, although these compounds are non-
cytotoxic to normal and malignant cancer cells. However, compounds 16 and 18 did not have activity
against proliferation of BXD-3752 glioma stem-like cells (Figure 6D), showing their anti-CSC activity
is selective for IDH1 mutated cancer cells. Moreover, 2-thiohydantoin inhibitors 16 and 18 exerted
comparable anti-CSC activity to that of known inhibitor 1 (Figures 6C and D). Compound 1 can inhibit
growth of IDH1 mutated BT142 glioma stem-like cells, but did not affect BXD-3752 cells without IDH1
mutation. These results from the two chemo-types of inhibitors (i.e., compounds 1, 16 and 18) show that
the function of mutant IDH1 seems of importance for maintenance of glioma stem cells bearing an IDH1
mutation. However, presumably due to the presence of other gene mutations, inhibition of mutant IDH1
alone does not significantly affect proliferation of the bulk of non-stem like cancer cells, such as those in
HT1080. This probably explains the reason why these mutant IDH1 inhibitors did not affect the overall
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proliferation rate of HT1080, but impaired self-renewal ability of the HT1080 stem-like cells.
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Although 2-thiohydantoin and related compounds have been reported to be associated with a
number of other biological activities and implicated as promiscuous inhibitors,30 our SAR studies
showed this is unlikely to be the case, because our compounds showed a wide range of activity (Ki: 0.4 -
>50 µM) as well as several SARs for inhibition of IDH1(R132H). Enzyme kinetics and ITC studies also
suggested that compound 18 is a competitive inhibitor of IDH1(R132H) against the substrate α-KG. Our
X-ray crystallographic investigation further revealed the exact binding structures of compounds 16 and
22 in IDH1(R132H), which could serve as a useful platform for more rational inhibitor design and
development.
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Conclusion
Genetic studies have shown frequent mutations of IDH are found in certain cancers, especially in
gliomas. Biochemical studies have revealed that the mutant IDH enzymes can reduce α-KG, an
important cellular metabolite, to D2HG, which inhibits histone lysine demethylation. Further biological
and pharmacological studies demonstrated that mutant IDH1 is a viable drug target for these cancers.
However, there have been a limited chemotypes of inhibitors of mutant IDH1.
Several series of 5-substituted 2-thiohydantoin and related compounds were synthesized, among
which several compounds were found to be potent and selective inhibitors of mutant IDH1 with Ki
values as low as 420 nM. SAR studies of these compounds showed that 1) 2-thiohydantoin ring is the
most favorable core, 2) a small substituent, such as –H or –Me, is more favored at the 3-position, and 3)
2-pyridinone-5-yl group at the 5-postion is the most potent for inhibition of mutant IDH1. X-ray
crystallographic studies revealed the binding structures of compounds 16 and 22 in IDH1(R132H),
providing a platform for further structure-based inhibitor design. Enzyme kinetics as well as binding
thermodynamics studies indicated that the most potent inhibitor 18 is a competitive inhibitor with
respect to the substrate α-KG and exerts a noncompetitive mode of action to the cofactor NADPH. Cell
biology studies showed that mutant IDH1 inhibitors 16 and 18 can lower down the concentrations of
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D2HG in HT1080 cells. These compounds can also reverse the hypermethyled phenotype of histones,
and selectively suppress self-renewal ability of glioma stem-like cells with IDH1 R132H mutation.
These compounds are therefore novel chemical probes for studies of mutant IDH1 in cancer and
represent a new scaffold for drug discovery targeting IDH1 mutated cancers.
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Experimental Section
All reagents were purchased from Alfa Aesar (Ward Hill, MA) or Aldrich (Milwaukee, WI).
1
Compounds were characterized by H NMR on a Varian (Palo Alto, CA) 400-MR spectrometer and
identities of selected compounds 16, 18 and 22 were confirmed with high resolution mass spectra (HRMS)
using a ThermoFisher LTQ-Orbitrap mass spectrometer. Compound purity was monitored by a Shimadzu
Prominence HPLC with a Phenomenex C18 column (4.6 x 250 mm, Methonal:H2O 60:40, monitored at
254 and 280 nm). The purities of all compounds were found to be >95%.
Synthesis and characterization of compounds 4 – 41 can be found in Supporting Information
Experimental Section.
Expression, purification and inhibition of human WT and mutant IDH1. Expression, purification
and inhibition of the WT, R132H and R132C IDH1 enzymes followed our previous published
methods.16,17 Enzyme inhibition assay for IDH1(R132H) and IDH1(R132C) was performed in a 96-well
microplate in 50 mM HEPES buffer (pH = 7.5) containing the enzyme (100 nM), 4 mM MgCl2, 100 μM
NADPH and increasing concentrations an inhibitor. Upon incubation for 5 min, α-KG (1 mM) was
added to initiate the reaction. The optical absorbance of each well was monitored every 30s at 340 nm
with a Beckman DTX-880 microplate reader. The inhibition assay of WT IDH1 was carried out in 50
mM HEPES buffer containing the enzyme (30 nM), 4 mM MgCl2, 1 mM NADP+, an inhibitor as well as
200 μM sodium (D)-isocitrate. The reaction was monitored by an increase in optical absorbance at 340
nm. The data were imported into Prism (version 5.0, GraphPad) and IC50s were calculated by using a
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standard dose response curve fitting. For compounds with IC50s >> [enzyme], Ki was 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 R132H and 0.295 mM for R132C. For compounds with IC50s
< 1 µM, Ki values were determined with the Morrison tight inhibition modeling in Prism. The reported
results are the average from at least three independent experiments.
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Crystallization and structure determination. IDH1(R132H) (10 mg/mL) was crystallized together
with 2 mM of compounds 2 or 3, 10 mM NADPH and 10 mM CaCl2 in a solution of 1.75 M (NH4)2SO4
and 0.1 M NaOAc (pH 5.6). Prism-like single crystals appeared in ~5 days. Diffraction data were
collected using a Rigaku FR-E+ SuperBright X-ray source at Baylor College of Medicine and processed
with the program the program HKL2000.26 The initial structure was obtained by molecular replacement
using the program Phaser,27 with the coordinates of 3MAP as a target. Structural refinement was
performed using the program Refmac28 or PHENIX29. The coordinates of IDH1(R132H) in complex
with 16 and 22 were deposited into Protein Data Bank as entries 4XRX and 4XS3, respectively.
Steady-state kinetic study. Steady-state kinetic inhibition experiment was conducted by measuring the
initial velocities of reactions catalyzed by IDH1(R132H) while varying the concentrations of compound
18, α-KG and NADPH. Data were imported into SigmaPlot (version 13, Systat Software, Inc.) and fitted
to competitive, noncompetitive and uncompetitive inhibition models. The best kinetic models were
determined by the highest R2 and lowest AICc values. Lineweaver-Burk or Michaelis-Menten plots were
generated by Sigmaplot.
Isothermal titration calorimetry study. ITC experiments were performed using a Nano ITC LV (190
μL) instrument from TA Instruments (New Castle, DE) at 298 K. A 190 μL solution containing
specified concentrations of IDH1(R132H), NADPH and α-KG in 30 mM Tris-HCl buffer (pH = 7.4)
with 4 mM MgCl2 and 150 mM NaCl was added into the sample cell. Compound 18 (1 mM) in the same
buffer solution (50 μL) was transferred into the syringe of the instrument. Upon reaching temperature
equilibrium, 25 injections (2 μL each) of the inhibitor were used for the titration. ITC data were then
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imported into NanoAnalyze software (TA Instruments, New Castle, DE). The titration baseline was
corrected and the ΔH and Ka values for the binding of the inhibitor were obtained by fitting into the
independent model in the software. All experiments were repeated for at least one time to ensure the
reported data are reliable.
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Cell culture and growth inhibition for cells that attach to a plate. 5,000 - 105 cells/well 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 (FBS) at 37 °C in a 5% CO2 atmosphere with
100% humidity. Upon culturing overnight for cell attachment, increasing concentrations of a compound
were added and incubated for 48-96 h. Cell viability was then assessed with an MTT assay.
D2HG production inhibition in HT1080 cells. Inhibition of cellular mutant IDH1 was determined
following our previous protocol.17 105 HT1080 cells/well were cultured in DMEM containing10%
dialyzed FBS and treated with increasing concentrations of an inhibitor for 48h. Medium was collected,
diluted with MeOH (8 mL) and centrifuged (20,000 rpm for 3 min). D2HG in the supernatant was
determined by HPLC-MS using our previous published methods.17
Inhibition of the proliferation of glioma stem-like cells. Two glioma cells BT142 and BXD-3752
were cultured as neurospheres in serum-free cell growth media at 37 °C in a 5% CO2 atmosphere with
100% humidity as described previously.17 2000 cells/well were added into 96-well plates and treated
with increasing concentrations of a compound for 14 days. Cell viability was determined by Cell
Counting Kit-8 (Dojindo Molecular Technologies, Rockville, MD) according to the manufacturer’s
instructions.
Acknowledgment. This work was supported by a grant (RP140469) from Cancer Prevention and
Research Institute of Texas (CPRIT) and a grant (R01NS080963) from National Institute of Neurological
Disorders and Stroke (NINDS/NIH) to Y.S.
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Supporting Information Available. Table S1, Figure S1-S4 and Experimental Section showing
compound synthesis and characterization. This material is available free of charge via the Internet at
http://pubs.acs.org.
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ABBREVATIONS: α-KG, α-ketoglutaric acid; D2HG, D-2-hydroxyglutaric acid; ICT, isocitric acid;
IDH, isocitrate hydrogenase; ITC, isothermal titration calorimetry; R132H, Arg132 mutation to His;
R132C, Arg132 mutation to Cys; SAR, structure activity relationship; WT, wild-type.
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