Identification of fumarate hydratase inhibitors with nutrient-dependent cytotoxicity

Article abstract of DOI:10.1021/ja5101257

Development of cell-permeable small molecules that target enzymes involved in energy metabolism remains important yet challenging. We describe here the discovery of a new class of compounds with a nutrient-dependent cytotoxicity profile that arises from p

Full text of DOI:10.1021/ja5101257

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Communication
Identification of Fumarate Hydratase Inhibitors
with Nutrient-Dependent Cytotoxicity
Toshifumi Takeuchi, Paul T Schumacker, and Sergey A Kozmin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja5101257 • Publication Date (Web): 03 Dec 2014
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Identification of Fumarate Hydratase Inhibitors with Nutrient-
Dependent Cytotoxicity
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Toshifumi Takeuchi, † Paul T. Schumacker § and Sergey A. Kozmin*,†
†Department of Chemistry, University of Chicago, Chicago, Illinois 60637, United States
§Department of Pediatrics, Division of Neonatology, Feinberg School of Medicine, Chicago, Illinois 60611, United States
Supporting Information
We screened a collection of nearly 6000 small molecules in sev-
eral human cancer cell lines under different growth conditions.
ABSTRACT: Development of cell-permeable small molecules
that target enzymes involved in energy metabolism remains im-
The chemical library was assembled over a number of years by a
series of parallel synthetic strategies and incorporated over 40
different drug-like heterocycles, including imidazopyridines, tet-
rahydroindolones, tetrahydroquinolinones and hexahydroacri-
dinones.6,7,8 In addition to high level of structural diversity, this
compound collection also featured favorable physicochemical
properties, including molecular weight, lipophilicity, polar surface
area, as well as numbers of rotatable bonds, hydrogen-bond do-
nors and acceptors. In the course of this study, we found that
pyrrolidinone 1 (Figure 1A) elicited a nutrient-dependent cytotox-
icity in a number of cancer cell lines. For example, compound 1
displayed increased growth-inhibitory activity towards SW620
cell line grown in the absence of glucose while a substantially
lower activity was observed using a standard glucose-containing
Dulbecco's Modified Eagle's (DME) medium (Figure 1B). In
search for more selective and potent pharmacological agents, we
next constructed a focused library of structural analogs of pyrroli-
dinone 1 (Figure S1 in Supporting Information). The design of a
secondary library was guided by the structure-activity data from
the primary screen of the original library, which incorporated a
number of derivatives of 1 with both peripheral and core altera-
tions. We found that truncation of propargyl moiety to a methyl
group was beneficial. The resulting pyrrolidinone 2 (Figure 1C)
demonstrated not only an increase in cell-growth inhibitory activi-
ty against SW620 cell line but also an enhanced selectivity (Fig-
ure 1D). Replacing propargyl group with larger substituents abol-
ished both the activity and selectivity of the resulting compounds
(Figure S1). Conversion of the ethyl ester 2 to the corresponding
carboxylic acid 3 (Figure 1E) was tolerated albeit with a slightly
diminished activity but a similar nutrient dependence on its anti-
proliferative activity (Figure 1F). We also established that this
class of compounds was configurationally stable at pH 6-8 (Figure
S11).
portant yet challenging. We describe here the discovery of a new
class of compounds with a nutrient-dependent cytotoxicity profile
that arises from pharmacological inhibition of fumarate hydratase
(also known as fumarase). This finding was enabled by a high-
throughput screen of a diverse chemical library in a panel of hu-
man cancer cell lines cultured under different growth conditions,
followed by subsequent structure-activity optimization and target
identification. While the highest cytotoxicity was observed under
low glucose concentrations, the antiproliferative activities and
inhibition of oxygen consumption rates in cells were distinctly
different from those displayed by typical inhibitors of mitochon-
drial oxidative phosphorylation. The use of a photoaffinity label-
ing strategy identified fumarate hydratase as the principal phar-
macological target. Final biochemical studies confirmed dose-
dependent, competitive inhibition of this enzyme in vitro, which
was fully consistent with the initially observed growth inhibitory
activity. Our work demonstrates how the phenotypic observations
combined with a successful target identification strategy can yield
a useful class of pharmacological inhibitors of an enzyme in-
volved in the operation of tricarboxylic acid cycle.
Glycolysis, tricarboxylic acid (TCA) cycle and oxidative phos-
phorylation (OXPHOS) are the three main components of eukary-
otic energy metabolism. The ability to pharmacologically modu-
late individual biochemical steps of such pathways using cell-
permeable small molecules provides new avenues for research in
human biology and drug discovery. Many highly effective phar-
macological modulators of OXPHOS have been identified over
the years.1,2,3 Such compounds have been particularly valuable in
elucidating and studying the electron transport chain (ETC) of
mitochondria.4 In contrast, there is only a limited arsenal of po-
tent and selective small-molecule modulators of enzymes in-
volved in glycolysis and TCA cycle. We have previously report-
ed a simple and effective strategy for identification of inhibitors
of glycolysis, which was enabled by the ability of such com-
pounds to block ATP production in human cells with chemically
impaired mitochondria.5 We describe here the discovery of a new
We next examined the generality of the observed nutrient depend-
ence on the activity of this class of compounds in a number of
histologically different cancer cell lines, which were grown in
standard DME medium or in L-15 medium.9 Representative
dose-dependent activity data for the most potent compound 2 in
five representative cell lines cultured in the absence of glucose are
shown in Figure 2A. All cell lines were found to be highly sensi-
tive to this agent with a mean IC50 of 2.2 µM. When the same cell
lines were grown in the presence of glucose, the growth inhibitory
activity of 2 was diminished more than tenfold (Figure 2B). Fur-
thermore, replacing glucose with galactose or pyruvate resulted in
substantially lower survival of SW620 cells that were grown
class of compounds with
a highly characteristic nutrient-
dependent cytotoxicity that arises from pharmacological inhibi-
tion of fumarate hydratase, an enzyme of the TCA cycle. This
finding was enabled by a high-throughput screen of a diverse
chemical library in a panel of human cancer cell lines under dif-
ferent growth conditions, followed by structure-activity optimiza-
tion and subsequent protein target identification using a pho-
toaffinity labeling strategy.
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Figure 2. Effects of compound 2 on growth of human cancer cell
lines under different conditions. (A) Antiproliferative activities of
compound 2 on SW620, ACHN, HCT-116, PC3 and SK-MEL-28
cells cultured in DME medium. (B) Antiproliferative activities of
compound 2 on SW620, ACHN, HCT-116, PC3 and SK-MEL-28
cells cultured in L-15 medium. (C) Effect of compound 2 on pro-
liferation of SW620 cells after 48 h under several different growth
conditions using glucose-free DME medium supplemented with
glucose (2 mM), galactose (2 mM) or sodium pyruvate (1 mM).
(D) Cellar ATP levels in SW620 cells after rapid (30 min) expo-
sure to compound 2 and glycolysis inhibitors, including 2-deoxy-
glucose (10 mM) or cytochalasin B (10 µM). Each value is a
mean ± SEM of triplicate values from three independent experi-
ments.
Figure 1. Structure and effects of compounds 1, 2 and 3 on
growth of SW620 cell line cultured in DME medium with or
without glucose. Each value is a mean ± SEM of triplicate values
from three independent experiments.
under such alternative carbon sources in the presence of com-
pound 2 (Figure 2C and S2). Such observations strongly suggest-
ed that compounds 1-3 might exert their effects on a mitochondri-
al target since the activity of this organelle would be required for
survival if glycolysis was suppressed.10 We next examined the
effect of compound 2 on ATP production in SW620 cells in the
presence of known glycolytic inhibitors.5 We found that ATP
concentration was rapidly (within 30 min) decreased by 2 in dose-
dependent manner in the presence of 10 mM concentration of 2-
deoxyglucose (Figure 2D). However, compound 2 did not block
ATP production in SW620 cells in the presence of cytochalasin B
(Figure 2D) or other glucose transport inhibitors (Figure S3). The
latter effect was distinctly different from that displayed by
OXPHOS inhibitors, which blocked ATP production in cells
treated with glucose transport inhibitors.5 Furthermore, while
NADH levels typically rapidly rise in cells treated with OXPHOS
inhibitors,12 such effects were not observed for any of the com-
pounds 2 and 3 (Figure S4), suggesting that they did not block
mitochondrial electron transport chain (ETC) or ATP synthase.
gradually reduced cellular respiration of SW620 cells (Figure 3B
and S5). Further experiments demonstrated that while cellular
respiration was inhibited by either 2 or rotenone, the respiration
was reinitiated by succinate, indicating that 2 did not inhibit com-
plex II-IV activities (Figure S5 and S6).14 We further established
that 2 did not have any effects on activity of complex I of ETC
using submitochondrial particles generated from SW620 cells
(Figure S7). These studies conclusively demonstrated that com-
pound 2 did not directly block any of the complexes of mitochon-
drial ETC and its effect may therefor arise from blocking the TCA
cycle. Such inhibitory activity would be expected to impair cellu-
lar respiration. 15
We also used Clark-type oxygen electrode to examine the effects
of carboxylic acid 3 on cellular respiration in the same cell line.
Interestingly, the oxygen consumption was reduced immediately
upon treatment of cells with 3 (Figure 3C and S8) with the same
overall profile displayed by 2. The difference in the kinetics of
cellular respiration inhibition suggested that ester 2 might be hy-
drolyzed in the cell to acid 3, serving as a pro-drug with an in-
creased cellular permeability.16 Indeed, a detailed LC-MS analy-
sis confirmed the presence of carboxylic acid 3 in the extracts of
SW620 cells treated with ester 2 (Figure S9 and S10). We further
demonstrated that 3 did not have any effects on activity of any of
the complexes of mitochondrial ETC (Figure S8 and S12).
We next examined the effects of newly identified compounds on
cellular respiration. While inhibition of both OXPHOS and TCA
cycle may impair oxygen consumption rate (OCR), such studies
can be used to clearly distinguish between such effects on cells.
We first analyzed OCR of SW620 cells treated with the most
potent compound 2 at several concentrations in the range of 0.5-
5.0 µM. As shown in Figure 3A, the respiration rate was reduced
in a dose dependent manner. At the highest concentration tested,
compound 2 blocked cellular respiration in SW620 cells com-
pletely. This effect could not be rescued by a proton uncoupling
agent, such as carbonyl cyanide-4-(trifluoro-methoxy)-
phenylhydrazone (FCCP), which clearly established that 2 did not
inhibit ATP synthase. Since it appeared that OCR was reduced by
2 gradually within the first 30 min of treatment of cells, we next
used Clark-type oxygen electrode to analyze the kinetics of this
effect.13 This study confirmed that pyrrolidinone 2 slowly and
To identify the cellular target of this compound class, we generat-
ed a chemical probe 4 armed with a benzophenone group and an
alkyne tag (Figure 4A). The benzophenone group, which replaced
a biphenyl moiety of the initial compounds, was designed for
covalent photoaffinity labeling of the cellular target.17,18 The
alkyne moiety would facilitate identification of the photocross-
linked proteins.19 Compound 4 displayed similar activity profile to
that observed for the initial set of compounds 1-3, indicating
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Figure 4. Target identification by photoaffinity labeling. (AB)
Structures of probes 4 and 5, which were used for photoaffinity
labeling of binding proteins in SW620 cells. The cells were treat-
ed with either of the above probe 4 (5 µM) or 5 (5 µM) in the
absence or presence of 3 (10 µM), irradiated at 365 nm and lysed.
Photocrossslinked proteins were next labeled with azide-
conjugated Alexa Fluor 488 and analyzed by SDS-PAGE. (C)
Fluorescence imaging and coomassie brilliant blue (CBB) staining
of SDS-PAGE separated proteins from whole SW620 cell lysate
following treatment with photoaffinity probes, UV irradiation and
fluorescent dye labeling. (D) Fluorescence imaging and CBB
staining of SDS-PAGE separated proteins from mitochondrial
fraction of SW620 cells following treatment with photoaffinity
probes, UV irradiation and fluorescent dye labeling.
Figure 3. Effects of compounds 2 and 3 on cellular respiration.
(A) Real-time measurement of OCR in SW620 cells treated with
several concentrations of compound 2 (0, 0.5, 0.75, 1.5, 5 µM).
OCR was measured using XFe96 analyzer. Positive controls
included oligomycin (1 µM), FCCP (0.5 µM), and rotenone (1
µM)/antimycin A (1 µM) mixture. Error bars indicate standard
errors. (B) Real-time measurement of oxygen consumption using
Clark-type oxygen electrode of SW620 cells treated with com-
pound 2 (C) Real-time measurement of oxygen consumption us-
ing Clark-type oxygen electrode of SW620 cells treated with
compound 3.
that the benzophenone group and the terminal alkyne were well
tolerated (Figure S13). We also prepared a closely related nega-
tive control 5 (Figure 4B), which showed no biological activity
(Figure S13). The photoaffinity labeling experiments were car-
ried out with SW620 cells. Cells were treated with photoaffinity
probe 4, followed by irradiation at 365 nm, lysis of cells and la-
beling of any alkyne-containing proteins with azide-conjugated
Alexa Fluor 488 dye using Cu-catalyzed azyde-alkyne cycloaddi-
tion.20 The proteins were analyzed by SDS-PAGE and visualized
by in-gel fluorescence scanning.
Figure 5. Inhibition of fumarate hydratese with compound 3 in
vitro. (A) Dose-dependent inhibition of fumarate hydratase, which
was isolated from SW620 cells, by compound 3. (B) Lineweaver-
Burk plot of the inhibition of fumarate hydratase by 3. Kinetic
parameters: Ki = 4.5 µM (Competitive inhibition), Km = 1.3 mM,
Vmax = 1.1 µM/min.
cells (Figure S15). We also performed similar photoaffinity label-
ing experiments with the ester-containing derivative of 4 (Figure
S14), which revealed Hsp70 and fumarate hydratase as the two
main protein bands (Table S3). Identification to Hsp70 is likely
not functionally significant and may simply represent binding of a
more lipophilic small molecule to this protein. Appearance of the
fumarate hydratase band, however, is consistent with hydrolysis
of the ester prior to protein cross-linking.
The results of such experiments using whole cell extract and the
mitochondrial fraction are shown in Figures 4C and 4D, respec-
tively. In both cases, the use of a photoaffinity probe 4 resulted in
the appearance of a major protein band at 50 kDa. Similar studies
using negative control 5 did not produce the same result, indicat-
ing that the 50 kDa protein may be the cellular target of this com-
pound class. Photoaffinity labeling experiments using probe 4
following treatment of cells with compound 3 reduced the intensi-
ty of the 50 kDa-band, further supporting the evidence that the 50
kDa protein was a high affinity binding protein (Figure S15).
Excision of the 50 kDa band, followed by in-gel digestion and
LC-MS analysis, unambigiuously established this protein as
fumarate hydratase (Table S1 and S2), which is known to reside
in mitochondria21 and serve as a component of the TCA cycle.
Indeed, confocal fluorescence microscopy studies confirmed lo-
calization of probes 1 and 4 in mitochondria of SW620
We next carried out in vitro fumarate hydratase activity assays.
The activity of this enzyme was measured using a well-
established assay that monitored the conversion of fumarate into
L-malate and subsequent oxidation of L-malate to oxaloacetate by
malate dehydrogenase (Scheme S1). Initial controls established
that neither the carboxylic acid 3 nor ester 2 inhibited malate de-
hydrogenase (Figure S16B). Using this two-enzyme protocol we
found that carboxylic acid 3 inhibited fumarate hydratase in a
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dose-dependent fashion in vitro (Figure 5A). However, ester 2 did
not exert such effects on this enzyme (Figure S16A), further sup-
porting the evidence that this compound served as a pro-drug,
being converted into the active inhibitor 2 upon entering the cell.
Further experiments established that acid 3 was a competitive
inhibitor of fumarate hydratase with a Ki value of 4.5 µM (Figure
5B), which was fully consistent with antiproliferative activity of
this compound. Similar experiments were conducted to confirm
fumarate hydratase inhibitory activity of compound 4, which was
employed for photoaffinity labeling studies (Figure S18).
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In conclusion, we have developed a novel class of cell-permeable
inhibitors of fumarate hydratase. This work was enabled by the
initial observation of nutrient-dependent cytotoxicity of such
compounds, followed by target identification using an effective
photoaffinity labeling strategy. Such compounds display an inter-
esting structure-activity profile and provide useful chemical
probes for modulating the activity of fumarate hydratase in live
cells. Chemical inhibition of fumarate hydratase renders cells
highly dependent on glucose metabolism for survival. In the field
of cancer biology, recent interest has focused on the identification
of genetic disruptions in metabolism that render tumor cells selec-
tively dependent on alternative pathways for survival.22 Humans
carrying mutations in fumarate hydratase are predisposed to the
development of leiomyomatosis and renal cancers, in cells that
undergo loss of heterozygosity. The increases in fumarate and
succinate caused by loss of fumarate hydratase can then promote
tumor progression through the activation of the hypoxia-inducible
transcription factor.23-26 Hence, inhibition of fumarate hydratase
can contribute to tumorigenicity in some cells. However, many
tumor cells exhibit high basal levels of oxidative stress, making
them vulnerable to therapies that augment the generation of reac-
tive oxygen species or that undermine endogenous antioxidant
mechanisms.27 In that regard, loss of fumarate hydratase results
in the accumulation of fumarate that reacts with reduced glutathi-
one, a critical component of the cellular antioxidant defense sys-
tem, to form succinated glutathione.28 Subsequent metabolism by
glutathione reductase depletes NADPH, a proximal substrate for
the maintenance of cellular redox balance and reductive biosyn-
thesis.29 Hence, fumarate hydratase inhibition may have thera-
peutic potential arising from the disruption of cellular redox bal-
ance and by promoting absolute dependence on glycolysis.
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ASSOCIATED CONTENT
Supporting Information
Experimental details and data. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
skozmin@uchicago.edu
(26) Mullen, A.R.; Wheaton, W.W.; Jin, E.S.; Chen, P.-H.; Sullivan,
L.B.; Cheng, T.; Yang, Y.; Linehan, W.M.; Chandel, N.S.; DeBerardinis,
R.J. Nature 2012, 481, 385–388.
(27) Schumacker, P. T. Cancer Cell 2006, 10, 175–176.
(28) Sullivan, L. B.; Martinez-Garcia, E.; Nguyen, H.; Mullen, A. R.;
Dufour, E.; Sudarshan, S.; Licht, J. D.; Deberardinis, R. J.; Chandel, N. S.
Mol. Cell 2013, 51, 236–248.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We are grateful for financial support to the National Institutes of
Health (P50 GM086145), the Chicago Biomedical Consortium
with support from the Searle Funds at the Chicago Community
Trust.
(29) Fan, J.; Ye, J.; Kamphorst, J. J.; Shlomi, T.; Thompson, C. B.;
Rabinowitz, J. D. Nature 2014, 510, 298–302.
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50
51
52
53
54
55
56
57
58
59
60
5
ACS Paragon Plus Environment