Crystallographic investigation and selective inhibition of mutant isocitrate dehydrogenase

Article abstract of DOI:10.1021/ml400036z

Mutations in isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid cycle, have recently been found in ～75% glioma and ～20% acute myeloid leukemia. Different from the wild-type enzyme, mutant IDH1 catalyzes the reduction of α-ketoglutaric

Full text of DOI:10.1021/ml400036z

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Letter
Crystallographic Investigation and Selective
Inhibition of Mutant Isocitrate Dehydrogenase
Baisong Zheng, Yuan Yao, Zhen Liu, Lisheng Deng, Justin L.
Anglin, Hong Jiang, B. V. Venkataram Prasad, and Yongcheng Song
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml400036z • Publication Date (Web): 03 May 2013
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Crystallographic Investigation and Selective Inhibition of Mutant
Isocitrate Dehydrogenase
†,
†,
†,
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Baisong Zheng, § Yuan Yao, § Zhen Liu, § Lisheng Deng, Justin L. Anglin, Hong Jiang, B. V. Venka-
,†
taram Prasad,# Yongcheng Song*
†
Department of Pharmacology, #Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor
College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, United States. Email: ysong@bcm.edu
KEYWORDS. Gene mutation, isocitrate dehydrogenase, Enzyme inhibition, protein crystallography
Supporting Information Placeholder
ABSTRACT: Mutations in isocitrate dehydrogenase (IDH), a key enzyme in the tricarboxylic acid cycle, have recently been
found in ~75% glioma and ~20% acute myeloid leukemia. Different from the wild-type enzyme, mutant IDH1 catalyzes the
reduction of α-ketoglutaric acid to D-2-hydroxyglutaric acid. Strong evidence has shown mutant IDH1 represents a novel
target for this type of cancer. We found two 1-hydroxypyridin-2-one compounds that are potent inhibitors of R132H and
R132C IDH1 mutants with Ki values as low as 120 nM. These compounds exhibit >60-fold selectivity against wild-type IDH1
and can inhibit the production of D-2-hydroxyglutaric acid in IDH1 mutated cells, representing novel chemical probes for
cancer biology studies. We also report the first inhibitor-bound crystal structures of IDH1(R132H), showing these inhibitors
have H-bond, electrostatic and hydrophobic interactions with the mutant enzyme. Comparison with the substrate-bound
IDH1 structures revealed the structural basis for the high enzyme selectivity of these compounds.
Isocitrate dehydrogenases (IDH) are a family of Mg2+
-
HO
O
A
O
O
O
Mg2+, NADP+
O
dependent enzymes catalyzing the oxidative decarboxyla-
tion of isocitric acid (ICT) to α-ketoglutaric acid (α-KG),1
one of the key reactions in the tricarboxylic acid cycle
providing aerobic organisms the majority of energy. Hu-
mans contain three IDH isozymes, i.e., IDH1 in cytoplasm
and IDH2 and IDH3 in mitochondria. IDH1/2 use NADP+ as
their cofactor, as schematically shown in Figure 1a.
HO
OH
HO
OH
wild-type IDH
O
OH
isocitric acid (ICT)
α-ketoglutaric acid (α-KG)
O
O
Mg2+, NADPH
B
O
O
HO
OH
HO
OH
mutant IDH
OH
O
e.g., IDH1(R132H)
α-KG
D-2-hydroxyglutaric acid (D2HG)
Mutations of IDH1/2 have recently been identified in
several types of cancer.2-4 For example, IDH1 mutation is
found in ~75% low-grade gliomas (grade II and III) and
secondary glioblastomas (grade IV glioma with a 5-year
survival of <10%).5,6 ~20% acute myeloid leukemia (AML)
also harbor IDH1/2 mutations.4 These mutations always
occur in one DNA allele with the wild-type (WT) IDH1 gene
in the other, suggesting the WT-IDH1 enzyme is also essen-
tial for the cancer cells. A special feature is that the identi-
fied IDH1 mutations are exclusively located in the Arg132
residue, with R132H being the predominant (~93%).6
These genetic findings suggest IDH1 mutations play im-
portant roles in the initiation and/or progression of the
cancer.
Figure 1. Enzyme reactions catalyzed by (A) wild-type IDH
and (B) mutant IDH.
physiological consequence of IDH mutation is a high cellu-
lar concentration of D2HG, the hallmark of this type of can-
cers. Further studies showed D2HG is an inhibitor of α-KG-
dependent dioxygenases, including important epigenetic
enzymes histone demethylases and 5-methylcytosine hy-
droxylases.3 The inhibition causes genome-wide hyper-
methylation of histone/DNA and blockage of cell differen-
tiation, which could lead to tumorigenesis.9,10 Mutant IDH1
is therefore a novel target for intervention.11-13 It is of in-
terest that during our preparation of this manuscript, a
series of diamide based compounds were disclosed to be
the first inhibitors of mutant IDH1.14 Here, we report the
discovery, activity and synthesis of two novel, selective
inhibitors of mutant IDH1. We also report the first X-ray
crystal structures of inhibitor-bound IDH1(R132H),
providing the exact inhibitor binding mode and the struc-
tural basis for the selectivity.
Biochemical investigation has revealed that all of the
mutant IDH enzymes, including IDH1(R132H) and
IDH1(R132C) (a frequent mutation in AML), are almost
inactive in converting ICT to α-KG. Rather, these mutants
were found to catalyze a new reaction, i.e., the reduction of
α-KG to D-2-hydroxyglutaric acid (D2HG) using NADPH as
the cofactor (Figure 1b).4,7,8 The direct
First, we expressed and obtained the recombinant pro-
teins of WT IDH1, R132H and R132C mutants (Supporting
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Information Experimental Section), which were found to exhibits high cytotoxicity against human WI-38 fibroblast
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have comparable enzyme activity as those reported in pre-
vious studies,4,7,15 respectively. Our previous work on de-
veloping inhibitors of 1-deoxyxylulose-5-phosphate reduc-
toisomerase,16,17 another Mg2+ and NADPH dependent re-
ductase, provided a rich source of compounds that could
target IDH1 mutants. Upon testing this focused library
cells with EC50 values of >50 ꢀM. HT1080 fibrosarcoma
cells, which harbor an IDH1(R132C) mutation,14 was treat-
ed with the most potent inhibitor 2 for 48h. Compound 2
was found to be able to inhibit the production of D2HG
with an EC50 value of 2.4 ꢀM, using a reported HPLC-MS
method.7 This compound is therefore a useful probe to
investigate the cancer biology of IDH1 mutation.
consisting
of
~130
compounds,
5-benzyl-1-
hydroxypyridin-2-one (1) shown in Table 1 was identified
as an inhibitor of IDH1(R132H) and IDH1(R132C) with Ki
values of 8.2 and 6.6 µM, while it has no activity against
WT IDH1 at 100 µM. Compound 1 is a drug-like molecule
with a calculated logP value of 1.6 and is therefore of inter-
est in the context of this study. Medicinal chemistry based
on 1 has yielded 6-substituted 1-hydroxypyridin-2-one
compounds 2 and 3 (Table 1) that are potent inhibitors of
IDH1(R132H) with Ki values of 190 and 280 nM, respec-
tively (Supporting Information Figure S1). It is remarkable
that the 4-methyl group is of importance for the potency,
since compounds 4 and 5 with a 4-H and 4-isopropyl
group, respectively, are ~30× and 50× less active than 2. In
addition, changing the 6-benzyl group in 2 to a phenyl
group in compound 6 completely abrogates the inhibitory
activity. These compounds also exhibited similar inhibitory
activities against IDH1(R132C) mutant, with 2 and 3 being
potent inhibitors of IDH1(R132C) with Ki values of 120 and
270 nM, respectively (Table 1).
8
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Next, we investigated how the novel inhibitors 2 and 3
bind to the mutant IDH1 and what is the structural basis
for the selectivity using X-ray crystallography. Previous
crystallographic studies of R132H mutant as well as WT
IDH1 proteins in complex with isocitric acid (ICT) have
revealed the critical role of Arg132 for the WT enzyme.15
Without the bound ICT, mutant IDH1(R132H) exhibits an
open conformation with an entrance channel of ~20 Å
wide. Upon ICT binding, the protein still adopts the open
conformation and, as shown in Supporting Information
Figure S2a, ICT is located in the ligand binding site I with
H-bond and electrostatic interactions with Arg100, Ser94,
Thr77 as well as NADP. The mutated His132 is not in-
volved in ICT binding as it is ~9.5 Å away. In contrast, WT-
IDH1 behaves differently. While it adopts an open confor-
mation without the bound ICT, upon ligand binding, the
protein undergoes a considerable conformational change
transiting to a closed state with an entrance channel of
~13 Å wide. ICT is located in the ligand binding site II,
which is also the catalytic site ~6.5 Å away from the site I
(Figure S2b). Of interest is that Arg132 is now in the active
site, forming two H-bonds/electrostatic interactions with
the two carboxyl groups of ICT (Figure S2c). The role of
Arg132 is therefore to help pull ICT to the catalytically ac-
tive site II, which occurs concomitantly with the protein
transiting to the closed state. This provides an explanation
as to why IDH1(R132H) loses 95% of its ability to convert
ICT to α-KG. It is likely due to the shorter sidechain of His
as well as the chemical nature of its imidazole group (as
compared to the guanidine in Arg). On the other hand, the
IDH1(R132H):α-KG structure shows a different binding
mode.7 As shown in Figures S2d/e, it mimics the overall
structure of the WT-IDH1:ICT complex. The mutant en-
zyme exhibits a closed conformation with the bound α-KG
located in the ligand binding site II, which is the catalytic
site of the mutant enzyme. His132 has no contacts with α-
KG (5.5 Å away).
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Table 1. Structures and biological activities of 1 – 6.
IDH Enzyme Ki (µM)
R132C
6.6
Cytotoxicity (µM)
R132H
8.2
0.19
0.28
5.9
WT
>50
12.3
16.8
>50
>50
>50
WI-38
>50
>50
>50
>50
>50
>50
1
2
3
4
5
6
0.12
0.27
10.5
14.7
>50
9.5
>50
We determined the X-ray structures of IDH1(R132H) in
complex with NADPH and inhibitors 2 or 3 to a resolution
of 3.3 Å similar to that in the previously reported struc-
tures.15 Statistics for diffraction data and structure refine-
ment are shown in Table S1 and the overall structures of
the two complexes illustrated in Figures 2a and S3a. The
protein crystallizes as a homodimer with both subunits
exhibiting an open conformation with a ~20 Å wide pro-
tein entrance channel. An NADPH molecule was found in
each of the subunits with a similar binding characteristics
as that in the IDH1(R132H):ICT. However, only one inhibi-
tor can be clearly located in the protein based on electron
We tested the activity of compounds 2 and 3 against
WT-IDH1 to determine the enzyme selectivity, since com-
pounds that also strongly block WT-IDH1 are undesirable,
given the important physiological role of this enzyme. As
shown in Table 1, compounds 2 and 3 exhibited very weak
activity against WT-IDH1 with Ki values of 12.3 and 16.8
ꢀM, showing a high selectivity of >60-fold, despite only a
single amino acid difference between the two enzymes.
Moreover, as also can be seen in Table 1, neither 2 nor 3
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Figure 2. (A) The overall structure of IDH1(R132H):2 (ball and stick model); (B) Close-up view of the active site of
IDH1(R132H):2; (C) The active sites of the aligned structures of IDH1(R132H):2 (with carbon and protein chain shown in
green) and IDH1(R132H):ICT (in cyan), showing 2 is located in the ligand binding site I; (D) The aligned structures of
IDH1(R132H):2 (in green), WT-IDH1:ICT (in orange) and IDH1(R132H):α-KG (in blue).
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density and omit maps (Figures S4). Figures 2b and S3b
show the close-up views of the binding sites of 2 and 3,
respectively. Compounds 2 and 3 bind to IDH1(R132H) in
a very similar manner consistent with their chemical struc-
tures. The 4-methyl-1-hydroxypyridin-2-one ring is com-
fortably located in a pocket surrounded by Arg100, Ser94,
Thr77, Asn96, Arg109 and NADPH. The two O atoms of 2
are ~2.7 Å from the two N atoms of the guanidine group of
Arg100, indicating strong H-bonds and electrostatic inter-
actions. The planar -CONH2 of Asn96 is located right un-
derneath the pyridine ring of 2, with the distance of ~4.1
Å. The 4-methyl group of 2/3 fits into a predominantly
hydrophobic cavity, 3.7 - 4.1 Å from -CH3 of Thr77, -CH2-
and -O- of Ser94. Loss of these interactions could account
for the reduced activities of compounds 4 and 5 with a 4-H
and 4-i-Pr, which are either too small or too big to be fa-
vorable at this position. The 6-benzyl group of both inhibi-
tors is involved in hydrophobic interactions with the pyri-
dine ring of NADPH. Although these interactions could also
contribute to the high affinity binding of the inhibitors, lack
of direct binding of the 6-benzyl group to the protein (4.5
Å away from Arg109) renders the electron density of the
moiety less strong than that of the 1-hydroxypyridin-2-one
core. In addition, the 6-phenyl group of compound 6 would
have a severe steric repulsion with NADPH, if its 1-
hydroxypyridin-2-one ring maintains the above mentioned
favorable interactions with IDH1(R132H). This should ac-
count for the loss of activity for compound 6.
Arg100, Ser94, Thr77 and Asn96 (omitted in Fig. 2c for
clarity), 2 (Ki: 0.19 ꢀM) binds to IDH1(R132H) with a much
higher affinity than ICT (Kd >200 ꢀM)15, likely due to in-
creased hydrophobic and electrostatic interactions. Struc-
tural alignment of IDH1(R132H):2 with WT-IDH1:ICT as
well as IDH1(R132H):α-KG further confirmed that 2 is not
located in the catalytically active binding site II (Figure
2d). These studies suggest that the strong binding of 2/3
stabilizes IDH1(R132H) in the catalytically inactive, open
conformation state. It also blocks α-KG from binding to the
site II as well as protein conformational change to its
closed state.
Docking using the program Glide18 was performed to in-
vestigate the observed high selectivity. First, compound 2
was docked into the IDH1(R132H):2 structure and the 10
lowest-energy docking structures shown in Figure S5a are
tightly clustered and mimic the crystal structure of 2, es-
pecially with respect to the conformation of the 4-methyl-
1-hydroxypyridin-2-one ring. Next, docking suggested 2
could not bind favorably to the binding site II in WT IDH1.
As shown in Figure S5b, 4 out of the 10 lowest-energy
docking structures are not located in the site II. In the re-
maining 6 docking structures, the phenyl ring, rather than
the more favorable 1-hydroxypyridin-2-one ring, is located
in the arginine-rich pocket. Moreover, there have been no
WT-IDH1 structures with a ligand bound to the site I, sug-
gesting Arg132 destabilizes this binding site. These studies
provide a rationale for why compounds 2 and 3 exhbit
weak activity against WT IDH1.
We next examined the binding site of 2/3 with respect
to those of ICT and α-KG in mutant and WT IDH1. Thus, the
structure of IDH1(R132H):2 complex was aligned with that
of IDH1(R132H):ICT, showing these two structures are
very similar with a root mean square (rms) deviation of 0.8
Å for the matching Cα atoms. The flexible loop 133-141 for
both structures, including the catalytically important
Tyr139 for the conversion of αKG to D2HG, is largely unor-
dered. As shown in Figure 2c, compound 2 occupies the
ICT binding site I in mutant IDH1. Although there are no
major conformational changes for the key residues
Finally, we describe the synthesis of compounds 1 – 6. 6-
or 5-Bromo-2-methoxypyridine was coupled with benzyl-
magnesium chloride using a palladium-catalyzed coupling
reaction. Using our previous method,16 the benzyl products
were oxidized to a pyridine oxide, followed by removal of
the methyl group to give compounds 1 or 4. An acyl chlo-
ride was reacted with ethyl 3-methyl-2-butenate in the
presence of AlCl3 to afford compound 7 as a mixture of
stereoisomers, which was cyclized to become a single
compound pyron-2-one 8. Since the reaction of 8 with hy-
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droxylamine occurred in a very poor yield (0-20%), 8 was their high selectivity. Moreover, this work suggests target-
converted to more reactive pyron-2-thione 9,19 which was
reacted readily with hydroxylamine to give compounds 2,
3, or 6 in 67-73% yield. 2,6-Dihydroxy-isonicotinic acid
was treated with POBr3 followed by methanolysis to give
dibromo-ester 10, whose 4-methoxycarbonyl group was
converted to an isopropenyl in 11 by treatment with
MeMgBr followed by an elimination reaction. One of the
bromo groups of 11 was substituted by a -OMe and the
other was reacted with n-BuLi and benzaldehyde to give
compound 12. It was hydrogenated and dehydroxylated
using Et3SiH20 to produce 4-isopropyl-2-methoxy-pyridine
13, which was then oxidized and deprotected to afford
compound 5.
ing the binding site I in mutant IDH1 is a viable strategy for
future rational design of potent and selective inhibitors of
mutant IDH1, and our structures could serve as a useful
platform for this purpose.
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AUTHOR INFORMATION
Corresponding Author
9
* Tel: 713-798-7415; E-mail: ysong@bcm.edu.
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Author Contributions
§These authors contributed equally.
ACKNOWLEDGMENT
This work was supported by a grant (R01NS080963) from
National Institute of Neurological Disorders and Stroke
(NINDS/NIH) to Y.S. and a grant (Q1279) from Robert Welch
Foundation to B.V.V.P. We thank the staff of the X-ray Crystal-
lography Facility at Baylor College of Medicine for assistance
in data collection.
Scheme 1. Synthesis for compounds 1 – 6.a
i, ii
iii
Bn
Bn
Br
N
O
N
O
OCH3
N
OCH3
OH
1 or 4
ASSOCIATED CONTENT
v
vii
iv
R
Supporting Information. Supplementary figures S1 – 5, Sup-
plementary table and Experimental Section. This information
is available free of charge via the Internet at
http://pubs.acs.org.
O
X
O
CO2Et
R
vi
O
R
N
CO2Et
OH
8, X = O
9, X = S
7
2, 3 or 6
COOH
COOCH3
Coordinates and structure factors of the IDH1(R132H):2 and
:3 complexes have been deposited in Protein Data Bank as
entries 4I3L and 4I3K, respectively.
viii
ix, x
xi, xii
OH
HO
N
Br
N
Br
N
Br
Br
10
11
ABBREVIATIONS
xiii, ixv
AML, acute myeloid leukemia; IDH, Isocitrate dehydrogenase;
ICT, isocitric acid; α-KG, α-ketoglutaric acid; WT-IDH1, wild-
type IDH1 enzyme; IDH1(R132H), R132H mutant IDH1 en-
zyme; IDH1(R132H), R132H mutant IDH1 enzyme; D2HG, D-
2-hydroxyglutaric acid.
ii, iii
5
N
OCH3
N
OCH3
OH
13
12
aReagents and conditions: (i) BnMgCl, Pd(dppf)Cl2; (ii) 3-
chloroperoxybenzoic acid; (iii) AcCl, reflux, then MeOH;
(iv) RCOCl, AlCl3; (v) H2SO4/AcOH; (vi) P4S10; (vii) NH2OH,
pyridine; (viii) POBr3, 130 oC, then MeOH; (ix) MeMgBr; (x)
methanesulfonyl chloride, NEt3; (xi) NaOMe; (xii) n-BuLi,
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o
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In summary, recent biological studies have shown mu-
tant IDH1 is a drug target for certain cancer. Using rational
compound screening and medicinal chemistry, two 1-
hydroxypyridin-2-one compounds 2 and 3 were found to
be potent inhibitors of IDH1(R132H) and IDH1(R132C),
the frequent mutations in cancer, with Ki values as low as
120 nM. These compounds exhibit >60-fold selectivity over
WT-IDH1 and do not have cytotoxicity against human cells,
representing novel chemical probes for cancer biology
studies of mutant IDH1. In addition, we report the first
crystal structures of IDH1(R132H):inhibitor complexes,
showing 2 and 3 are located in the ligand binding site I,
with several strong H-bond, electrostatic and hydrophobic
interactions with the protein. Inability of these inhibitors
to bind to the catalytically active site II could account for
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