Development of chromanes as novel inhibitors of the uncoupling proteins

Article abstract of DOI:10.1016/j.chembiol.2010.12.012

The uncoupling proteins (UCPs) are mitochondrial carriers that modulate the energetic efficiency and, as a result, can lower superoxide levels. Here, we describe the discovery of a small-molecule inhibitor of the UCPs. Screening of potential UCP1 regulators led to the identification of chromane derivatives that inhibit its proton conductance. Members of the UCP family can act as a defense against oxidative stress and, thus, UCP2 plays a protective role in tumor cells. High UCP2 levels have been associated with chemoresistance. We demonstrate that chromanes also inhibit UCP2 and, in HT-29 human carcinoma cells, cause oxidative stress. The chromane derivatives can act synergistically with chemotherapeutic agents; for instance, they increase the toxicity of arsenic trioxide in HT-29 cells. These findings open a promising line in the development of novel anticancer agents.

Full text of DOI:10.1016/j.chembiol.2010.12.012

Chemistry & Biology
Article
Development of Chromanes as Novel Inhibitors
of the Uncoupling Proteins
1
1
1
1
Eduardo Rial,1, Leonor Rodrı´guez-Sanchez, Patricio Aller, Arancha Guisado, M. Mar Gonzalez-Barroso,
*
´
´
1
1
2
2
´
Eunate Gallardo-Vara, Mariano Redondo-Horcajo, Esther Castellanos, Roberto Fernandez de la Pradilla,
and Alma Viso2
1
´
Centro de Investigaciones Biologicas—CSIC, 28040 Madrid, Spain
2Instituto de Qu´ımica Orga´ nica General—CSIC, 28006 Madrid, Spain
*Correspondence: rial@cib.csic.es
DOI 10.1016/j.chembiol.2010.12.012
SUMMARY
the UCPs presumably increase the rate of mitochondrial respira-
tion, an increase in their activity would also lead to a reduction in
The uncoupling proteins (UCPs) are mitochondrial the mitochondrial production of superoxide. In fact, UCPs are
up-regulated in many physiological situations where there is
oxidative stress, and there are data showing that their overex-
pression reduces ROS damage. As a consequence, UCPs are
carriers that modulate the energetic efficiency and,
as a result, can lower superoxide levels. Here, we
describe the discovery of a small-molecule inhibitor
of the UCPs. Screening of potential UCP1 regulators
led to the identification of chromane derivatives that
inhibit its proton conductance. Members of the UCP
family can act as a defense against oxidative stress
and, thus, UCP2 plays a protective role in tumor cells.
widely considered as an additional element of antioxidant
defense (Brand and Esteves, 2005; Nu¨ bel and Ricquier, 2006;
Echtay, 2007; Baffy, 2010). Although the acceleration of respira-
tion due to UCP-mediated uncoupling would lead to a reduction
in ROS production, it has also been proposed that UCPs could
induce a metabolic shift that would promote glycolysis and
therefore indirectly lower ROS production (Bouillaud, 2009).
The mitochondrial activity of the UCPs is regulated by different
High UCP2 levels have been associated with chemo-
resistance. We demonstrate that chromanes also
inhibit UCP2 and, in HT-29 human carcinoma cells, ligands, although the physiological relevance of some of them is
unclear. Only the physiological regulation of the uncoupling
cause oxidative stress. The chromane derivatives
protein from BAT, UCP1, is well understood. Cytosolic purine
nucleotides inhibit UCP1, while fatty acids override the inhibition
can act synergistically with chemotherapeutic
agents; for instance, they increase the toxicity of
arsenic trioxide in HT-29 cells. These findings open
a promising line in the development of novel anti-
cancer agents.
´
and increase its proton conductance (Rial and Gonzalez-
Barroso, 2001). This regulatory mechanism fits precisely with
its physiological context: noradrenaline binds to b-receptors,
initiating a cAMP-dependent lipolytic cascade where the fatty
acids liberated are the substrates that mitochondria will oxidize
´
and the activators of UCP1 (Rial and Gonzalez-Barroso, 2001).
INTRODUCTION
Although nucleotides and fatty acids are the more prominent
regulators, there are other ligands that modulate the proton
The uncoupling proteins (UCPs) are carriers that belong to the conductance in UCP1. Thus, all-trans-retinoic acid (ATRA) is
protein superfamily constituted by the metabolite transporters a high-affinity activator of the protein (Rial et al., 1999) and, inter-
of the mitochondrial inner membrane. In mammals, five genes estingly, also is a powerful transcriptional activator of the ucp1
encode proteins considered UCPs (Ledesma et al., 2002a; gene (Alvarez et al., 1995). Additionally, reduced ubiquinone
Krauss et al., 2005). UCP1 is present in brown adipose tissue lowers the affinity of UCP1 for nucleotides and, as a result, facil-
(BAT), UCP2 is ubiquitous, and UCP3 is expressed in heart, itates the activation by fatty acids (Swida-Barteczka et al., 2009).
´
skeletal muscle, and brown adipose tissue. UCP4 and BMCP1 Ubiquinone also increases the affinity of UCP1 for ATRA (Tomas
(UCP5) are predominantly expressed in the nervous system. et al., 2002).
Genes homologous to the mammalian UCPs have been
The regulation of the rest of the members of the UCP family is
described not only in most phyla of the animal kingdom but not yet understood (Brand and Esteves, 2005; Cannon et al.,
also in plants and unicellular eukaryotes (Ledesma et al., 2006; Echtay, 2007; Azzu and Brand, 2010). The interpretation
2002a). The function of the different members of the UCP protein of most data is complicated by the discovery that the adenine
family is not fully established. Their biological activity appears to nucleotide translocator (ANT) could be responsible for some of
be linked to the modulation of the efficiency of the oxidative the effects ascribed to the UCPs. Furthermore, the ANT has
phosphorylation, although the actual molecular mechanism been shown to contribute significantly to the basal proton
may vary among the different family members. This energy-dissi- conductance (Brand et al., 2005). The inhibition of proton trans-
pating mechanism is used in functions as diverse as the elimina- port by GDP has often been used as diagnostic for the involve-
tion of an excess calorie intake, regulation of insulin secretion, or ment of a UCP in the modulation of the efficiency of the oxidative
maintenance of body temperature (Krauss et al., 2005). Because phosphorylation, but GDP, although not translocated by the
264 Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Uncoupling Protein Inhibitors
ANT, does inhibit the proton conductance via ANT (Khailova of NADH oxidation in control mitochondria (Figure 1B), an
et al., 2006). The involvement of the UCPs in the defense against uncoupling effect that was also evident in UCP1-containing
oxidative stress led to the investigation of possible regulators mitochondria. However, at low concentrations, this retinoid
that could be generated when ROS levels rise. Thus, superoxide decreased the NADH oxidation rate in UCP1 mitochondria,
and lipid peroxidation products such as hydroxynonenal have thus suggesting the inhibition of the basal proton conductance
been proposed to activate the UCPs (Echtay et al., 2002, of UCP1. The reason behind this mixed behavior could be the
2003). These peroxidation products would set off a negative ester hydrolysis under aqueous conditions that generates two
feedback mechanism by which the activation of the UCPs would new organic acids, 2,2,4,4-tetramethylchroman-6-carboxylic
increase respiration, lower ROS production, and thereby protect acid and (E)-3-(4-hydroxyphenyl)acrylic acid, from the starting
against oxidative stress. Although this hypothesis is very attrac- retinoid. Because small organic acids can permeate across the
tive, the activation by ROS-derived products has not been lipid bilayer, the two resulting compounds could act as protono-
confirmed by some laboratories, and, as previously stated, the phores and thus explain the observed nonspecific uncoupling
ANT could account for the ROS-induced changes in proton activity.
conductance (Couplan et al., 2002; Shabalina et al., 2010). Nucleotides inhibit UCP1 by binding deep inside the a-helical
The development of specific inhibitors of the UCPs would bundle core with the purine ring interacting with the matrix loops
´
greatly facilitate the investigation of their transport activity and (Gonzalez-Barroso et al., 1999; Ledesma et al., 2002b). The
regulation. Here we describe the identification and synthesis of chromane moiety of the retinoid AGN199108 could act as
a family of inhibitors of the uncoupling proteins. We demonstrate a surrogate of the purine ring, and we thus hypothesized that
that certain chromane derivatives inhibit the fatty acid–induced this moiety would be responsible for the inhibition. To test the
proton conductance of UCP1 without affecting other compo- hypothesis, two chromane derivatives, 6-bromo-8-isopropyl-
nents of the oxidative phosphorylation (OXPHOS) system. 2,2-dimethylchromane (CSIC-E183) and 8-isopropyl-2,2-dime-
Furthermore, the chromane derivatives also inhibit UCP2 in yeast thylchroman-6-carboxylic acid (CSIC-E137), were synthesized
mitochondria. Finally, we show that in HT-29 tumor cells, which and their activity tested. The chromane CSIC-E137 retained
express UCP2, chromanes inhibit respiration and increase ROS the carboxylic moiety, whereas in the chromane CSIC-E183,
levels. Interestingly, the inhibitor lowers the viability of HT-29 the position C-6 at the chromane ring was substituted by
cells, increasing the efficacy of antitumor drugs that cause a bromine to remove the protonable residue. Experiments with
oxidative stress, such as arsenic trioxide or doxorubicin.
control mitochondria confirmed the essential role of the carbox-
ylate for the protonophoric properties because the bromine-
substituted compound had no significant effect on respiration
(Figure 1C). The inhibitory properties of CSIC-E183 on UCP1
containing mitochondria were tested in the presence of
palmitate to include a physiological activator. Data clearly
RESULTS AND DISCUSSION
Chromane Derivatives Inhibit the Uncoupling
Protein UCP1
All-trans retinoic acid (ATRA) up-regulates the expression of the demonstrate that CSIC-E183 was able to inhibit both the palmi-
uncoupling proteins UCP1 and UCP2 (Alvarez et al., 1995; Hata- tate-induced and the basal proton conductance of UCP1
keyama and Scarpace, 2001). We have previously shown that because the rate of NADH oxidation was decreased to the level
ATRA also activates the proton conductance of UCP1 and of control mitochondria (Figure 1D).
UCP2 (Rial et al., 1999). Because
a large collection of
compounds structurally related to ATRA has been synthesized Characterization of the Active Compounds
as specific ligands for retinoid receptors (Altucci et al., 2007), To determine the structural requirements of the inhibitory
we screened a set of retinoids to get an insight into the specificity compounds, we designed and synthesized a series of chromane
´
of the UCP1 ligand (Tomas et al., 2004). The screening protocol derivatives of general structure I, and some of them proved to be
was based on the analysis of the effect of the compounds on the valuable inhibitors of UCP1. In these derivatives, we analyzed the
rate of NADH oxidation by mitochondria isolated from Saccharo- effect of the substituents in four different positions (R1–R4) thus
´
myces cerevisiae that expressed UCP1 (Tomas et al., 2004). allowing us to establish the first basis of their influence on the
Under our experimental conditions, in the absence of added inhibitory activity of these chromanes. Table 1 gathers the new
regulators, yeast mitochondria expressing UCP1 present a respi- series of compounds examined and presents the data obtained
ratory activity higher than that of control mitochondria (Fig- when tested in control yeast mitochondria and mitochondria
ure 1A). This higher respiration rate can be inhibited by purine containing UCP1. The EC50 was calculated from the difference
nucleotides and represents the basal proton conductance of in inhibition kinetics (see Figures S1 and S2 available online).
´
´
UCP1 (Gonzalez-Barroso et al., 1998; Jimenez-Jimenez et al.,
´
One of the most marked structural features for inhibition of
UCP1, which arises upon inspection of Table 1, is the crucial
2006).
Figure 1A shows the effect of ATRA in control mitochondria and presence of a substituent at C-8 of the chromane skeleton.
mitochondria containing UCP1. The low concentrations of ATRA Thus, lower EC50 values are obtained when R4 is an alkyl group
used only stimulate respiration (NADH oxidation) when UCP1 is than when R4 is a proton (entries 1–9 vs. entries 12–15). Linear
present. We have previously shown that the increase can be in- and branched alkyl groups are well-tolerated at the C-8 position
´
hibited by GDP (Rial et al., 1999; Tomas et al., 2004). We have increasing the potency of the inhibitors, mainly for the ethyl
also described that other retinoids, which must possess a free group (entries 1–4); however, a methyl ketone at this position
carboxylate group, are also activators of UCP1. Interestingly, precludes the inhibition (entry 10). We also evaluated the influ-
the retinoid AGN199108 displayed a nonspecific stimulation ence of the nature of the group R3. The presence of an acidic
Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 265

Chemistry & Biology
Uncoupling Protein Inhibitors
Figure 1. Chromane Derivatives Inhibit the Proton
Conductance of UCP1
UCP1
Control
80
70
60
50
40
30
20
10
80
70
60
50
40
30
20
10
The activity of UCP1 is determined from the rate of NADH
oxidation of mitochondria from yeast that express UCP1
recombinantly (filled circles) and compared with control
yeast mitochondria (empty circles). Rates of respiration
are expressed as a percentage of the maximal rate of
respiration that is determined in the presence of 1 mM
FCCP. Data are means SEM of 4–6 independent exper-
iments each performed in triplicate.
B
A
(A) Activation of respiration in UCP1 containing mitochon-
dria by ATRA and lack of effect on control mitochondria.
(B) Activation by the retinoid AGN-199108.
0
1
2
3
4
0
1
2
3
4
(C) Effect of the chromanes CSIC-E137 and CSIC-E183 on
control yeast mitochondria.
Molar Ratio Retinoic acid : Albumin
Molar Ratio Retinoid : Albumin
O
(D) Effect of the compound CSIC-E183 on control yeast
mitochondria and UCP1-containing yeast mitochondria.
O
OH
O
O
OH
O
CSIC-E379 (8-isopropyl-2,2-dimethyl-6-phe-
nylchromane; EC50, 20 mM), was used as inhib-
itor for the remaining of the work. Similarly, the
chromane CSIC-E167 was subsequently used
as inactive control because of its lack of effect
on UCP1.
all-trans Retinoic acid
AGN-199108
80
70
60
50
40
30
20
10
60
50
40
30
20
10
E137
C
D
E183
Chromane Derivatives Inhibit
the Uncoupling Protein UCP2
We have previously reported that, when the un-
coupling protein UCP2 is expressed recombi-
nantly in S. cerevisiae, the isolated mitochondria
present a higher state 4, whereas the phosphor-
ylation rate or the maximal respiratory activity
remain unchanged (Rial et al., 1999). The effect
of the chromanes on UCP2 was also tested
using the NADH-based screening protocol.
Under these experimental conditions, UCP2-
containing mitochondria presented a higher
basal respiratory activity, although the differ-
ence with control mitochondria accounted for
only 9% of the maximal respiratory capacity
(Figure 2; Figure S3). The active chromane
CSIC-E379 caused the inhibition of the UCP2-
dependent respiratory activity that was evident
0.1
2
20
125250
0.1
2
20
125
Concentration (micromolar)
O
Concentration (micromolar)
Br
OH
O
O
CSIC-E137
CSIC-E183
moiety deprives the compound of any inhibitory capacity when the nonspecific effects on control mitochondria were taken
(EC50, >200 mM) that is recovered, however, upon esterification into account (Figure 2). The estimated EC50 was close to 30 mM
(EC50, 27 mM) (entries 16 vs. 6). The absence of a substituent and thus similar to the one obtained with UCP1 (Table 1). As ex-
(R3 = H) and the substitution by a bromine allow for moderate pected, when the UCP1-inactive compound CSIC-E167 was
inhibitory activities (entries 9 and 3). Alternatively, the introduc- tested on mitochondria containing UCP2, the NADH oxidation
tion of an aromatic group (R3 = Ph) leads to the most potent rate was not modified (Figure 2; Figure S3).
inhibitor in the series (entry 5); in contrast, a vinyl group in this
position hampers the inhibitory activity (entry 11). Finally, we Chromane Do Not Affect OXPHOS in Liver Mitochondria
examined the nature of the substitution at the oxygenated ring, The screening protocol used to characterize the inhibitory pro-
and no significant differences have been found between the perties of the chromane derivatives is performed under nonphos-
chromane skeletons examined, 2,2-dialkylated and 4,4-dialky- phorylating conditions. Because we had hypothesized that the
lated (R1 = Me vs. R2 = Me, entries 6/7 and 8/9); however, the inhibition of the proton transport activity of UCP1 by chromane
need for the chromane ring has been proven with the lack of inhi- moiety was likely due to its resemblance with the purine ring, it
bition of CSIC-E243 (4-bromo-2-isopropyl-1-methoxybenzene), was necessary to assess whether the chromane derivatives were
a monocyclic analog to CSIC-E183 and CSIC-E179 (entries 17, interfering with the exchange of ADP/ATP or the phosphorylation
3, and 8). Consequently, the chromane with the highest affinity, of ADP at the F0F1-ATPase. Figure 3 presents the effect of the
266 Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Uncoupling Protein Inhibitors
Table 1. Chemical Structure, Nomenclature, and Activity of the Chromane Derivatives Synthesized and Characterized in the Present
Study
R2
R2
R3
R1
R1
O
R4
EC50(μM)
Entry
Compound
R1
R2
R3
R4
1
2
CSIC-E225
CSIC-E221
CSIC-E183
CSIC-E231
CSIC-E379
CSIC-E1127
CSIC-E1131B
CSIC-E179
CSIC-E155
CSIC-E161
CSIC-E393
CSIC-E1125
CSIC-E375
CSIC-E167
CSIC-E1113
CSIC-E137
CSIC-E243
Me
H
Br
Et
34
54
Me
Me
Me
Me
Me
H
H
H
Pr
i-Pr
Br
Br
3
111
39
4
H
t-Bu
i-Pr
Br
5
H
20
Ph
6
H
i-Pr
27
CO2Et
CO2Et
Br
7
Me
Me
H
i-Pr
32
8
H
i-Pr
132
98
9
Me
H
i-Pr
H
COCH3
10
11
12
13
14
15
16
17
Me
H
>200
>200
135
109
>200
>200
>200
>250
Br
Me
Me
Me
Me
H
i-Pr
H
CH=CH2
CO2Et
Ph
H
H
H
H
H
Br
Me
H
H
CO2Et
CO2H
Me
i-Pr
4-bromo-2-isopropyl-1-methoxybenzene (see text)
The concentration that causes a 50% inhibition of transport activity of UCP1 (EC50) was calculated from the difference between the NADH oxidation
rates in UCP1-containing mitochondria and control mitochondria. Experimental data for each compound are included in Figures S1 and S2.
Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 267

Chemistry & Biology
Uncoupling Protein Inhibitors
G/M
CSIC-E379
CSIC-E167
A
E379 60 µM
12
10
8
ADP
20
DMSO
170
19
6
FCCP
176
4
2
100
nmol ‘O’
0
0.1
2
20 40 100 200
Concentration (µM)
3 min
Figure 2. Chromane Derivatives Inhibit Respiration in UCP2-Con-
taining Yeast Mitochondria
n.s.
Respiration was determined from the rate of NADH oxidation in control mito-
chondria and mitochondria isolated from yeasts expressing UCP2 recombi-
nantly, either in the presence of CSIC-E379 (circles) or CSIC-E167 (triangles).
Data are expressed as the difference in the NADH oxidation rate of UCP2
minus control mitochondria (see also Figure S3). Data points are means
SEM of 3 independent experiments each performed in triplicate.
B
State 4
n.s.
State 3
175
150
125
100
chromane CSIC-E379 on rat liver mitochondria. The compound
CSIC-E379 had no effect on the basal respiratory rate (state 4),
thus ruling out a direct effect on the respiratory complexes or the
membrane permeability. Although this chromane had no statisti-
cally significant effect on the phosphorylation of added ADP
(state 3), a trend is apparent showing a 10% inhibition when the
concentration was raised to 60 mM. Therefore, the chromane
CSIC-E379 does not significantly affect the OXPHOS system.
20
10
0
0
20
60
Concentration CSIC-E379 (µM)
Chromane Derivatives Inhibit the Cellular Respiration
of the Cancer Cell Line HT-29
The biochemical activity of the UCPs is to lower the efficiency of
the oxidative phosphorylation, presumably by increasing the
membrane proton conductance. This mild uncoupling would
lead to an increase in respiration, which results in a lower gener-
ation of superoxide due to the decrease in proton-motive force.
This uncoupling effect could constitute a mechanism of defense
against oxidative stress. Indeed, UCPs are up-regulated in phys-
iological situations where there is oxidative stress and, further-
more, their overexpression reduces ROS damage (Echtay,
2007; Azzu and Brand, 2010; Baffy, 2010; Rial et al., 2010).
The effect of the chromane CSIC-E379 on the cellular respira-
tion was investigated using the cell line HT-29, a colon cancer
Figure 3. Effect of the Chromane CSIC-E379 on the Oxidative Phos-
phorylation of Isolated Rat Liver Mitochondria
(A) Representative traces with the measurement of the oxygen consumption in
the presence and absence of the compound CSIC-E379. Respiration was initi-
ated with the addition of glutamate 5 mM plus malate 5 mM (G/M); the state 3
respiration rate was determined after the addition of ADP 0.2 mM. FCCP 1 mM
was added at the end of the run. Rates are expressed as nmol ‘‘O’’ per minute
and milligrams of protein.
(B) Effect of CSIC-E379 on the rate of respiration in the basal state 4 (filled bars)
and in state 3 (empty bars). Bars represent the mean SEM of 4–5 indepen-
dent experiments. There are no significance differences (n.s.) between the
rates in the presence and absence of the chromane.
cell line that expresses the uncoupling protein UCP2 (Figure S4). among other diseases, type 2 diabetes. Zhang et al. (2006) re-
Cells were incubated for 1 hr in the presence of the chromane, ported that genipin inhibited UCP2 in b-cells, raising ATP levels
and then the respiratory activity was determined on an XF24 and increasing insulin secretion while reducing insulin-stimu-
Extracellular Flux Analyzer. Figure 4A shows that the chromane lated glucose uptake in 3T3-L1 adipocytes (Zhou et al., 2009).
causes a 22% decrease in the basal rate of respiration, in agree- Mailloux et al. (2010) have recently reported that genipin also
ment with the data obtained with UCP2-containing yeast mito- inhibits respiration in UCP2 overexpressing leukemia cells.
chondria. Genipin, an iridoid glucoside, has also been described
Bouillaud (2009) has proposed a ‘‘metabolic hypothesis,’’
to inhibit UCP2. Genipin is extracted from the fruits of Gardenia whereby UCP2 (and UCP3) has a transport activity that does
jasminoides and is used in traditional Chinese medicine to treat, not involve proton translocation. The working model proposes
268 Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Uncoupling Protein Inhibitors
Figure 4. The chromane CSIC-E379 inhibits mito-
chondrial respiration in HT-29 colon cancer cells
causing oxidative stress and decreasing their
viability
*
A
2.0
(A) Basal rate of oxygen consumption of HT-29 cells after
subtraction of the rate obtained in the presence of antimy-
cin A and rotenone. Cells were incubated for 1 hr in the
presence (empty bar) or in the absence (filled bar) of
20 mM CSIC-E379. Data are the mean SEM of three inde-
pendent experiments.
1.5
1.0
0.5
(B) Representative flow-cytometry histograms of the ROS
levels determined with the fluorescent probe H2DCFDA in
control HT-29 cells (left panel) or cells treated for 24 hr with
CSIC-E379.
0.0
Control
E379
(C) Histograms summarizing the effect of the chromanes
CSIC-E379 and CSIC-E167 on ROS levels in HT-29 cells
after a treatment for 24 hr. Treatment with the respiration
inhibitor rotenone for 1 hr is used as positive control for
ROS generation. Bars represent the mean SEM of 5-6
independent experiments.
B
Control
CSIC-E379
(D) Effect of the compound CSIC-E379 on the viability of
HT-29 cells after a 24 hr treatment determined from the
rate of reduction of the reagent WST-1. Data points repre-
sent the mean SEM of 8–10 independent experiments
each performed in triplicate.
See also Figure S4.
H DCFDA Fluorescence
H DCFDA Fluorescence
2
2
C
D
a determinant for oncogenic transformation
(Trachootham et al., 2009). Cells adapt to
increased ROS levels with defense mechanisms
that include the up-regulation of antioxidant
enzymes (superoxide dismutase, catalase, and
glutathione peroxidase) and enzymes that
regenerate nonenzymatic antioxidants, such
as gluthatione reductase or the thioredoxin
system. In addition, tumor cells defend them-
selves against the deleterious effects of oxida-
tive stress by promoting the expression of cell-
survival proteins, such as Bcl-2, or by activating
survival pathways such as PI3K/Akt or MEK/
ERK (Trachootham et al., 2009; Pani et al.,
2010). In this regard, UCP2 has been shown to
be expressed in cancer cells (Carretero et al.,
300
1.0
***
***
250
200
150
100
50
0.8
0.6
0.4
0.2
0.0
0
0
10
20
30
40
50
Concentration CSIC-E379 (µM)
that UCP2/3 would catalyze the export of pyruvate from the 1998; Harper et al., 2002; Horimoto et al., 2004; Samudio
matrix, thus limiting substrate supply to mitochondria, and that et al., 2008; Santandreu et al., 2009, 2010; Mailloux et al.,
would also lead to a metabolic shift that would promote glycol- 2010; Sastre-Serra et al., 2010; Baffy, 2010), and it has been
ysis. The lower redox pressure on the mitochondrial respiratory proposed that UCP2 could be a metabolic strategy used by
chain would indirectly lead to a decreased ROS production. the tumor cells to develop resistance against radio- or chemo-
The observed effects of genipin or the chromane derivatives therapy (Harper et al., 2002; Mailloux et al., 2010; Baffy, 2010).
would not be compatible with this hypothetical transport activity
The capacity of the chromane derivatives to inhibit UCP2 and
because the predicted effect of the inhibition of UCP2 would have influence cell viability was first tested by analyzing their effect on
been a higher (or an unchanged) rate of respiration. The inconsis- ROS levels in HT-29 cells. Figure 4B presents representative
tency is even more evident when the effect of the chromanes on histograms of ROS levels determined with fluorescent probe
UCP2-containing yeast mitochondria is taken into consideration. H2DCFDA in control cells and cells treated for 24 hr with the
chromane CSIC-E379. Data show that the active chromane
Chromane Derivatives Increase the Production
of Superoxide and Decrease the Viability
of the Cancer Cell Line HT-29
CSIC-E379 causes an increase in ROS levels, similar to that
obtained with rotenone, which is not evoked by the inactive
compound CSIC-E167 used as a control (Figure 4C). The
Compared with their nontransformed counterparts, cancer cells observed chromane-induced increase in ROS resembles those
often present high intrinsic oxidative stress, which is probably reported when tumor cells are treated with genipin (Mailloux
Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 269

Chemistry & Biology
Uncoupling Protein Inhibitors
Figure 5. The compound CSIC-E379 sensi-
tizes the cancer cell line HT-29 to arsenic
trioxide
Control
A
B
CSIC-E379 20 µM
CSIC-E167 20 µM
1.0
1.0
0.8
0.6
0.4
0.2
0.0
(A) Titration of the effect of ATO on the viability of
the cells treated for 24 hr in the absence (filled
circles) or in the presence of 20 mM CSIC-E379
(empty circles).
0.8
0.6
0.4
0.2
0.0
(B) Effect of ATO on the viability of the cells treated
for 48 hr in the absence of chromanes (filled
circles), in the presence of 20 mM CSIC-E379
(empty circles), or in the presence of 20 mM
CSIC-E167 (gray diamonds). In panels (A) and
(B), data points represent the mean
SEM of
0
10
20
30
40
80
0
10
20
30
40
80
8–16 independent experiments each performed
in triplicate. Difference between control values
and those from CSIC-E379 treated cells are signif-
icant, with p < 0.001.
ATO Concentration (µM)
ATO Concentration (µM)
ATO 10 µM +
CSIC-E379 20 µM
Control
ATO 10 µM
(C) Representative flow cytometry profiles of the
effect of ATO and CSIC-E379 on HT-29 cells. Cells
were treated for 24 hr, stained with Annexin-V and
PI, and apoptosis subsequently was analyzed by
flow cytometry. Left panel, control cells; central
panel, cells treated with 10 mM ATO; right panel,
cells treated with 10 mM ATO plus 20 mM CSIC-
E379.
C
(D) Histogram summarizing the analysis of the
effect of the chromane derivatives CSIC-E379
and CSIC-E167 on the apoptosis of HT-29 cells.
Empty bars represent the viable cells and are
those that are not stained with Annexin-V or PI;
hatched bars represent the cells in the early
apoptotic phase that are Annexin-V positive and
PI negative; and black bars represent the cells in
late apoptosis plus necrotic cells and therefore
include all PI positive cells. Bars represent the
Propidium Iodide
D
Viable
80
60
40
20
0
Early Apoptosis
PI positive
mean
SEM of 3–8 independent experiments
each performed in duplicate.
Chromane Derivatives Potentiate
the Efficacy of the Cytotoxic Drug
Arsenic Trioxide
As indicated above, the elevated intrinsic
oxidative stress of tumor cells is behind
the efficacy of therapies with some anti-
tumor drugs or radiation. The increase in
oxidative stress can be due to a direct
et al., 2010; Santandreu et al., 2010). It is worth recalling that the generation of ROS and/or to an interference with the antioxidant
overexpression of UCP2 in the colon cancer cell line HCT116 has defenses. Arsenical compounds, such as arsenic trioxide (ATO),
been shown to decrease the ROS levels (Derdak et al., 2008).
have been shown to be effective in the treatment of acute pro-
The intrinsic elevated level of oxidative stress makes tumor myelocytic leukemia and other human cancers (Lau et al.,
cells more susceptible to the action of ROS-inducing cytotoxic 2008). Generation of ROS is one of the mechanisms explaining
agents, a property that has been satisfactorily exploited in anti- the induction of apoptosis by ATO. Interestingly, the ATO effi-
tumor therapies (Lau et al., 2008; Trachootham et al., 2009). cacy is determined, at least in part, by the intracellular redox
We therefore tested the effect of the chromane CSIC-E379 on status. Thus, it is known that its cytotoxicity is greatly dependent
the cell viability and observed that the concentration (20 mM) on the intracellular content of reduced glutathione and that, for
and treatment time (24 hr) that were shown to increase the example, depletion of glutathione stores sensitizes cells to
ROS level caused a moderate, although significant, decrease ATO (Yi et al., 2002; Lau et al., 2008). In the same context, we
in viability (Figure 4D). When the concentration of CSIC-E379 have recently shown that genistein, a soy-derived isoflavone,
was raised to 50 mM, the decrease in viability was close to behaves as a pro-oxidant agent potentiating ATO-induced
50%. Again, genipin has also been shown to reduce the viability apoptosis in human leukemia cells via ROS overproduction
of other tumor cells lines that express UCP2 (Mailloux et al., and the activation of the ROS-inducible protein kinases p38-
´
MAPK and AMPK (Sanchez et al., 2008).
2010; Santandreu et al., 2010).
270 Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Uncoupling Protein Inhibitors
Figure 6. The chromane derivative CSIC-
E379 sensitizes HT-29 cancer cells to doxo-
rubicin and cisplatin
Drug
Drug + 20 µM CSIC-E379
A
1.0
B
Cell viability was determined from the reduction of
WST-1. In the two panels, data points represent
the mean SEM of at least 9 independent experi-
ments each performed in triplicate. The difference
between the control values and those from CSIC-
E379 treated cells are significant, with p < 0.001.
(A) Effect of doxorubicin on the viability of the cells
treated for 48 hr in the absence (filled circles) or in
the presence of 20 mM CSIC-E379 (empty circles).
(B) Effect of CDDP on the viability of the cells
treated for 48 hr in the absence (filled circles) or
in the presence of 20 mM CSIC-E379 (empty
circles).
1.0
0.8
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
0.0
0.0
0.5
1.0
2.0
0
5
10
20
50
Doxorubicin Concentration (µM)
CDDP Concentration (µM)
Because we have shown that the chromane CSIC-E379 mitochondrial carriers (Nury et al., 2006). The basic fold is a barrel
increases ROS levels in the cancer cell line HT-29, it was of formed by the six transmembrane a helices that is closed at the
interest to determine whether its combination with ATO could matrix side. The a helices define a hydrophilic cavity opened to
also potentiate the effect of the antitumor drug. Figure 5A pres- the intermembrane space and that enters deeply into the protein,
ents the effect of increasing ATO concentrations on the viability constituting the translocation pathway. The nucleotide-binding
of HT-29 cells incubated for 24 hr either in the absence or in the site in UCP1 is probably the best-known structural feature of
presence of 20 mM CSIC-E379. Data revealed that the decreased the protein. Photoaffinity labeling and site-directed mutagenesis
in viability was markedly pronounced in the presence of the experiments have shown that the nucleotide enters the protein
UCP2 inhibitor but the effect appeared as additive. However, through its cytosolic side and gets deep inside the cavity to
when the treatments were maintained for 48 hr (Figure 5B), the interact with the loops at the bottom of the barrel. We have
ATO toxicity was clearly potentiated, being more evident at proposed that the purine ring would fit in a hydrophobic pocket
very low doses. Thus, 5 mM ATO (concentration close to the and that its stabilization would occur through stacking interac-
´
useful therapeutic dose) caused a 10% reduction in viability, tions, as it happens in other nucleotide binding proteins (Gonza-
whereas in the presence of CSIC-E379, the reduction was close lez-Barroso et al., 1999; Ledesma et al., 2002b). The hydropho-
to 35%. As expected, the inactive chromane CSIC-E167 had no bicity and planar structure of the chromane derivatives identified
potentiating effect. in the present report suggest that the chromane could probably
The cell viability is often inferred from the metabolic activity and, occupy the same site than the purine ring.
thus, we used a protocol based on the reduction of the reagent
As we have previously stated, GDP inhibition of the mito-
WST-1 that is dependent on the rate of NAD(P)H formation. To chondrial proton conductance was considered for many
ascertain the effect of the chromanes on cell death, we analyzed years as diagnostic of the involvement of a UCP. However, it
the cellular apoptosis and necrosis using annexin-V labeling has become clear that the activity of the ANT, being the most
combined with propidium iodide (PI) staining. Figure 5D summa- abundant carrier, overlaps with that of the UCPs and can also
rizes the results that revealed that, as expected, 10 mM ATO be inhibited by GDP. As a result, doubts have been shaded
decreases the number of viable cells and causes a marked on some biological activities attributed to the UCPs. The
increaseinannexin-V–positivecellsthatrepresentcellsintheearly discovery that the effect of genipin in b-cells is mediated by
apoptotic phase. When the inhibitor was present, the reduction in UCP2 has led to its use as a ‘‘specific’’ UCP2 inhibitor.
viable cells was not significantly different, but the number of However, genipin is a natural product used in traditional
PI-positive cells (late apoptotic or necrotic cells) was greatly Chinese medicine to treat a variety of diseases. Thus, it exhibits
increased. It appears that UCP2 inhibition exacerbates cell death. anti-inflammatory and anti-angiogenic activities, inhibits lipid
The potentiating effect of the chromane CSC-E379 was also peroxidation, and suppresses cell proliferation and migration
testedwiththeantitumordrugscisplatin(CDDP)anddoxorubicin. (Koo et al., 2004; Yamazaki and Chiba, 2008; Kim et al.,
The two drugs are known to induce oxidative stress, and previous 2010). Recent studies have also reported that genipin protects
studies have shown that UCP2 could influence their toxicity (Der- against D-galactosamine/lipopolysaccharide-induced hepatic
dak et al., 2008; Mailloux et al., 2010; Santandreu et al., 2010). apoptosis and liver failure by preventing oxidative stress,
Figure 6 presents the effect of CSIC-E379 on the viability after apoptosis, NF-kB nuclear translocation, and nuclear c-Jun
the treatment for 48 hr with varying concentrations of the two expression (Kim et al., 2010). Finally, it should be borne in
drugs. The inhibitor cooperates in more than additive manner mind that genipin is a highly reactive nonspecific cross-linking
with the two agents, thus confirming that UCP2 inhibition sensi- agent that, for example, is used to generate biocompatible
tizes cancer cells to agents commonly used in chemotherapy.
polymers (Slusarewicz et al., 2010). Therefore, the possible
side effects imply that caution should be exerted when using
genipin as a ‘‘specific’’ UCP2 inhibitor. The above facts
Conclusion
The 3D-structure of the UCPs is not available, but the structure of evidence the need for new and selective inhibitors of the uncou-
the ANT has revealed the common structural scaffold of the pling proteins. Further optimization of the chromane derivatives
Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 271

Chemistry & Biology
Uncoupling Protein Inhibitors
for the isolation of mitochondria from yeasts have been described previously
(Arechaga et al., 1993). The control strain contained the same vector but
with the UCP1 cDNA in the inverse orientation.
described here should lead to a new generation of such high-
affinity inhibitors. Data presented point to their application as
tools for uncoupling protein research and even as new chemo-
therapeutic agents.
The rate of yeast mitochondrial respiration was determined from the
´
decrease in NADH fluorescence as previously described (Tomas et al.,
2004). Mitochondria (0.1 mg protein/ml) were incubated at 20ꢀC in medium
containing 0.65 M mannitol, 0.5 mM EGTA, 10 mM phosphate, 2 mM MgCl2,
3 mM essentially fatty-acid-free albumin, and 10 mM Tris/maleate (pH 6.8).
When required, the medium also contained 9 mM palmitate (3:1 molar ratio
to albumin). Respiration was initiated by the addition of 0.3 mM NADH. Exper-
iments were performed on standard 96-well microtiter plates, and fluores-
cence was measured in a POLARstar Galaxy reader (BMG Labtechnologies).
Excitation was set at 340 nm and emission at 470 nm.
SIGNIFICANCE
Mitochondria play a key role in cellular processes that range
from energy production to redox or calcium homeostasis,
participating in a wide range of metabolic pathways. How-
ever, although mitochondria are essential for cell survival,
a variety of death signals converge on mitochondria making
this organelle also central to cell death. Mitochondria are
also one of the main sites of ROS production, and thus mech-
anisms that prevent ROS damage are not only critical for the
preservation of their function but also for cell survival. The
uncoupling proteins (UCPs) control the energetic efficiency,
and one of the consequences is that they participate in the
control of mitochondrial ROS production.
Isolation of Liver Mitochondria and Determination
of Respiration Rates
Mitochondria from rat liver were isolated as previously described (Heaton and
Nicholls, 1976). Final pellet was resuspended in a buffer containing 250 mM
sucrose, 5 mg/ml albumin, and 10 mM Tes (pH 7.4). Protein concentration
was determined by the BCA method. The respiration experiments were carried
out at 25ꢀC in a Hansa-Tech oxygen electrode. Mitochondria (0.5 mg/ml) were
suspended in medium containing 75 mM NaCl, 10 mM Tes, 1 mM EGTA,
1 mg/ml albumin, and 2 mM sodium phosphate (pH 7.0). Substrate was
5 mM glutamate plus 5 mM malate.
The search for new regulators of the uncoupling activity of
UCP1 has led to the discovery of a small-molecule inhibitor
of the UCPs that we have presented. UCP2 appears as one
of the most interesting pharmacological targets of the chro-
mane derivatives, and, thus, we have explored the applica-
tion of the inhibitor to increase oxidative stress in tumor
cells with the aim of generating an imbalance in ROS status
and thus make the cells more susceptible to other chemo-
therapeutic drugs. The use of selective combinations of
drugs to enhance their therapeutic efficacy is a common
approach undertaken in cancer treatment. Because UCP2
appears to be linked to the antioxidant defense of tumor
cells and has even been shown to promote chemoresistance
(Harper et al., 2002; Derdak et al., 2008; Mailloux et al., 2010;
Baffy, 2010), the development of small-molecule UCP2 inhib-
itors is a promising new step in the development of novel
anticancer strategies.
Cell Line and Measurements of Cellular Respiration
HT-29 colon carcinoma cells were obtained from the American Type Culture
Collection. Cells were cultured at 37ꢀC in a humidified 5% CO2 atmosphere
in DMEM (GIBCO) supplemented with 10% heat-inactivated FBS (Sigma),
2 mM L-glutamine, and 100 U/ml of penicillin/streptomycin (Invitrogen).
An XF24 Seahorse Bioscience instrument was used to measure the rate of
oxygen consumption of HT-29 cells; 60,000 cells per well were seeded in
XF24 cell culture plates in complete DMEM growth media and incubated at
37ꢀC, 5% CO2 for 24 hr prior to beginning the assay. For the XF24 assay,
DMEM growth media was replaced by unbuffered DMEM supplemented
with 25 mM glucose, 1 mM pyruvate, 2 mM L-glutamine, and 20 mM CSIC-
E379 when indicated, and cells were incubated at 37ꢀC in a CO2-free incubator
for 1 hr. Cells were then placed in the instrument, and basal oxygen consump-
tion was recorded for 24 min. At the end of the run, 1 mM rotenone and 1 mM
antimycin A were added to determine the mitochondria-independent oxygen
consumption. Cells were lysed in 50 mM Tris, 150 mM NaCl, 2 mM EDTA,
1% NP40, 0.1% SDS, and 1% deoxycholate (pH 7.5), and the protein concen-
tration was determined with the BCA method.
EXPERIMENTAL PROCEDURES
Cell Viability and Apoptosis Assays
Synthesis of Chromane Derivatives
Cell viability was evaluated with the Cell Proliferation Reagent WST-1 (Roche
Diagnostics GmbH, Mannheim, Germany) following the manufacturer’s
instructions. For cell viability assays, 7000 cells were plated in 96-well plates
and grown for 24 or 48 hr in the absence of serum and in the presence of
the drugs. Ten microliters of WST-1 reagent was added to each well, cells
were incubated for 4 hr, and the dye absorbance at 480 nm was measured
using a Varioskan Flash (Thermo Fisher Scientific) plate reader. The absor-
bance at 690 nm was used as background.
The syntheses of 4-bromo-2-isopropyl-1-methoxybenzene (Burnel and
Caron, 1992), 6-bromo-2,2-dimethylchromane (Bernard et al., 1998), ethyl
2,2-dimethylchroman-6-carboxylate (Teng et al., 1997), and 6-phenyl-2,2-di-
methylchromane (Ishino et al., 2001) were previously reported. 2,2-Dimethyl
chromanes were prepared by cyclization of phenols with 3-methyl-2-buten-
1-ol (Ishino et al., 2001). 4,4-Dimethyl chromanes were prepared by
a sequence that entails O-alkylation of phenols with 4-bromo-2-methyl-2-
butene, acetoxymercuriation/reduction, and cyclization mediated by AlCl3
(Janusz et al., 1998). 6-Phenyl and 6-vinyl chromanes were prepared by
a Stille coupling from 6-bromo chromanes and vinyl or phenyl tributylstan-
nanes (McKean et al., 1987). 6-Carboxyethyl chromanes were obtained
from 6-bromo chromanes with t-BuLi and ethyl chloroformate. Further
saponification with NaOH yielded the carboxylic acid. Finally, 8-acetyl chro-
mane was prepared from the chromane via Friedel-Crafts acylation with
acetyl chloride and AlCl3. All the compounds were purified and characterized
prior to submission to biological essays (see details in Supplemental Exper-
imental Procedures).
Apoptosis was measured using Annexin-V and PI staining using the Vybrant
Apoptosis Assay Kit (Invitrogen) and following the manufacturer’s instructions;
300,000 cells were plated in 6-well plates and exposed to the different agents
in absence of serum for 48 hr. Floating and adherent cells were collected and
stained for 15 min at room temperature in the dark and were fluorescence
analyzed using an EPICS XL flow cytometer.
Measurement of ROS
ROS levels were determined by incubating the cells with 20,70-dichloro-dihy-
dro-fluorescein diacetate (H2-DCFDA, Molecular Probes). A total of 300,000
cells were plated in 6-well plates and exposed to the different agents in
absence of serum for 24 hr. Cells were incubated in the presence of 5 mM
H2-DCFDA for 1 hr at 37ꢀC and a humidified 5% CO2 atmosphere in the
dark. Floating and adherent cells were harvested in the same medium, and
Yeast Strains, Isolation of Yeast Mitochondria, and Determination
of Mitochondrial NADH Oxidation
The procedures for recombinant expression of the rat uncoupling protein
UCP1 or the mouse UCP2 in the S. cerevisiae strain W303 and the protocol
272 Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Uncoupling Protein Inhibitors
the fluorescence was analyzed using an EPICS XL flow cytometer. Cells
Carretero, M.V., Torres, L., Latasa, U., Garcı´a-Trevijano, E.R., Prieto, J., Mato,
J.M., and Avila, M.A. (1998). Transformed but not normal hepatocytes express
UCP2. FEBS Lett. 439, 55–58.
treated for 1 hr with 10 mM rotenone were used as control.
Couplan, E., Gonzalez-Barroso, M.M., Alves-Guerra, M.C., Ricquier, D.,
Goubern, M., and Bouillaud, F. (2002). No evidence for a basal, retinoic, or
superoxide-induced uncoupling activity of the uncoupling protein 2 present
in spleen or lung mitochondria. J. Biol. Chem. 277, 26268–26275.
Statistical Analysis
All values are expressed as mean SEM of at least three independent exper-
iments performed in triplicate. Differences between groups were determined
using either two-tailed unpaired Student’s t tests or the one-way ANOVA
test using the SigmaPlot software. Significant differences between groups
are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.
Derdak, Z., Mark, N.M., Beldi, G., Robson, S.C., Wands, J.R., and Baffy, G.
(2008). The mitochondrial uncoupling protein-2 promotes chemoresistance
in cancer cells. Cancer Res. 68, 2813–2819.
Echtay, K.S. (2007). Mitochondrial uncoupling proteins—what is their physio-
SUPPLEMENTAL INFORMATION
logical role? Free Radic. Biol. Med. 43, 1351–1371.
Supplemental Information includes Supplemental Experimental Procedures
and four figures and can be found with this article online at doi:10.1016/j.
chembiol.2010.12.012.
Echtay, K.S., Roussel, D., St-Pierre, J., Jekabsons, M.B., Cadenas, S., Stuart,
J.A., Harper, J.A., Roebuck, S.J., Morrison, A., Pickering, S., et al. (2002).
Superoxide activates mitochondrial uncoupling proteins. Nature 415, 96–99.
Echtay, K.S., Esteves, T.C., Pakay, J.L., Jekabsons, M.B., Lambert, A.J.,
Portero-Otı´n, M., Pamplona, R., Vidal-Puig, A.J., Wang, S., Roebuck, S.J.,
and Brand, M.D. (2003). A signalling role for 4-hydroxy-2-nonenal in regulation
of mitochondrial uncoupling. EMBO J. 22, 4103–4110.
ACKNOWLEDGMENTS
This work was supported by project grants of the Spanish Ministry of Science
and Innovation (SAF2009-07126, Consolider-Ingenio 2010 CSD2007-00020,
and CTQ 2009-07752), CSIC (2004 2 0E 238), and Comunidad de Madrid
(S-SAL-0249-2006). We also thank the Ministry of Education for a doctoral
Gonza´ lez-Barroso, M.M., Fleury, C., Bouillaud, F., Nicholls, D.G., and Rial, E.
(1998). The uncoupling protein UCP1 does not increase the proton conduc-
tance of the inner mitochondrial membrane by functioning as a fatty acid anion
transporter. J. Biol. Chem. 273, 15528–15532.
´
fellowship to E.C. M.M.G.B. was supported by the ‘‘Ramon y Cajal’’ program.
The expert technical assistance of Pilar Zaragoza is gratefully acknowledged.
´
´
Gonzalez-Barroso, M.M., Fleury, C., Jimenez, M.A., Sanz, J.M., Romero, A.,
Bouillaud, F., and Rial, E. (1999). Structural and functional study of a conserved
region in the uncoupling protein UCP1: the three matrix loops are involved in
the control of transport. J. Mol. Biol. 292, 137–149.
Received: July 30, 2010
Revised: November 15, 2010
Accepted: December 6, 2010
Published: February 24, 2011
Harper, M.E., Antoniou, A., Villalobos-Menuey, E., Russo, A., Trauger, R.,
Vendemelio, M., George, A., Bartholomew, R., Carlo, D., Shaikh, A., et al.
(2002). Characterization of a novel metabolic strategy used by drug-resistant
tumor cells. FASEB J. 16, 1550–1557.
REFERENCES
Hatakeyama, Y., and Scarpace, P.J. (2001). Transcriptional regulation of un-
coupling protein-2 gene expression in L6 myotubes. Int. J. Obes. Relat.
Metab. Disord. 25, 1619–1624.
Altucci, L., Leibowitz, M.D., Ogilvie, K.M., de Lera, A.R., and Gronemeyer, H.
(2007). RAR and RXR modulation in cancer and metabolic disease. Nat.
Rev. Drug Discov. 6, 793–810.
Heaton, G.M., and Nicholls, D.G. (1976). The calcium conductance of the inner
membrane and the determination of the calcium electrochemical gradient.
Biochem. J. 156, 635–646.
´
˜
Alvarez, R., de Andres, J., Yubero, P., Vinas, O., Mampel, T., Iglesias, R., Giralt,
M., and Villarroya, F. (1995). A novel regulatory pathway of brown fat thermo-
genesis: retinoic acid is a transcriptional activator of the mitochondrial uncou-
pling protein gene. J. Biol. Chem. 270, 5666–5673.
Horimoto, M., Resnick, M.B., Konkin, T.A., Routhier, J., Wands, J.R., and
Baffy, G. (2004). Expression of uncoupling protein-2 in human colon cancer.
Clin. Cancer Res. 10, 6203–6207.
Arechaga, I., Raimbault, S., Prieto, S., Levi-Meyrueis, C., Zaragoza, P., Miroux,
B., Ricquier, D., Bouillaud, F., and Rial, E. (1993). Cysteine residues are not
essential for uncoupling protein function. Biochem. J. 296, 693–700.
Ishino, Y., Mihara, M., Hayakawa, N., Miyata, T., Kaneko, Y., and Miyata, T.
(2001). An improved method for synthesis of 1-benzopyrans from unsaturated
alcohols and phenols using a catalytic amount of acids. Synth. Commun. 31,
439–448.
Azzu, V., and Brand, M.D. (2010). The on-off switches of the mitochondrial un-
coupling proteins. Trends Biochem. Sci. 35, 298–307.
Baffy, G. (2010). Uncoupling protein-2 and cancer. Mitochondrion 10,
Janusz, J.M., Young, P.A., Scherz, M.W., Enzweiler, K., Wu, L.I., Gan, L., Pikul,
S., McDow-Dunham, K.L., Johnson, C.R., Senanayake, C.B., et al. (1998).
New cyclooxygenase-2/5-lipoxygenase inhibitors. 2. 7-tert-Butyl-2,3-dihy-
dro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflamma-
tory and analgesic agents: variations of the dihydrobenzofuran ring. J. Med.
Chem. 41, 1124–1137.
243–252.
Bernard, A.M., Cocco, M.T., Onnis, V., and Piras, P.P. (1998). Facile synthesis
of 2,2-dimethylchromans by Mo(CO)6 catalyzed reaction of aryl prenyl ethers.
Synthesis 3, 256–258.
Bouillaud, F. (2009). UCP2, not a physiologically relevant uncoupler but
a glucose sparing switch impacting ROS production and glucose sensing.
Biochim. Biophys. Acta 1787, 377–383.
´
´
´
Jimenez-Jimenez, J., Zardoya, R., Ledesma, A., Garcıa de Lacoba, M.,
Zaragoza, P., Gonza´ lez-Barroso, M.M., and Rial, E. (2006). Evolutionarily
distinct residues in the uncoupling protein UCP1 are essential for its character-
istic basal proton conductance. J. Mol. Biol. 359, 1010–1022.
Brand, M.D., and Esteves, T.C. (2005). Physiological functions of the mito-
chondrial uncoupling proteins UCP2 and UCP3. Cell Metab. 2, 85–93.
Brand, M.D., Pakay, J.L., Ocloo, A., Kokoszka, J., Wallace, D.C., Brookes,
P.S., and Cornwall, E.J. (2005). The basal proton conductance of mitochondria
depends on adenine nucleotide translocase content. Biochem. J. 392,
353–362.
Khailova, L.S., Prikhodko, E.A., Dedukhova, V.I., Mokhova, E.N., Popov, V.N.,
and Skulachev, V.P. (2006). Participation of ATP/ADP antiporter in oleate- and
oleate hydroperoxide-induced uncoupling suppressed by GDP and carboxya-
tractylate. Biochim. Biophys. Acta 1757, 1324–1329.
Burnel, R.H., and Caron, S. (1992). Approach to the synthesis of candelabrone
and synthesis of 3,7-diketo-12-hydroxyabieta-8,11,13-triene. Can. J. Chem.
70, 1446–1454.
Kim, S.J., Kim, J.K., Lee, D.U., Kwak, J.H., and Lee, S.M. (2010). Genipin
protects lipopolysaccharide-induced apoptotic liver damage in D-galactos-
amine-sensitized mice. Eur. J. Pharmacol. 635, 188–193.
Cannon, B., Shabalina, I.G., Kramarova, T.V., Petrovic, N., and Nedergaard, J.
(2006). Uncoupling proteins: a role in protection against reactive oxygen
species—or not? Biochim. Biophys. Acta 1757, 449–458.
Koo, H.J., Song, Y.S., Kim, H.J., and Park, E.H. (2004). Antiinflammatory
effects of genipin, an active principle of gardenia. Eur. J. Pharmacol. 495,
201–208.
Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 273

Chemistry & Biology
Uncoupling Protein Inhibitors
Krauss, S., Zhang, Z.Y., and Lowell, B.B. (2005). The mitochondrial uncou-
Santandreu, F.M., Roca, P., and Oliver, J. (2010). Uncoupling protein-2 knock-
down mediates the cytotoxic effects of cisplatin. Free Radic. Biol. Med. 49,
658–666.
pling-protein homologues. Nat. Rev. Mol. Cell Biol. 6, 248–261.
Lau, A.T., Wang, Y., and Chiu, J.F. (2008). Reactive oxygen species: current
knowledge and applications in cancer research and therapeutic. J. Cell.
Biochem. 104, 657–667.
Sastre-Serra, J., Valle, A., Company, M.M., Garau, I., Oliver, J., and Roca, P.
(2010). Estrogen down-regulates uncoupling proteins and increases oxidative
stress in breast cancer. Free Radic. Biol. Med. 48, 506–512.
Ledesma, A., Garc´ıa de Lacoba, M., and Rial, E. (2002a). The mitochondrial un-
coupling proteins. Genome Biol. 3, 3015.1–3015.9.
Shabalina, I.G., Hoeks, J., Kramarova, T.V., Schrauwen, P., Cannon, B., and
Nedergaard, J. (2010). Cold tolerance of UCP1-ablated mice: a skeletal
muscle mitochondria switch toward lipid oxidation with marked UCP3 up-
regulation not associated with increased basal, fatty acid- or ROS-induced un-
coupling or enhanced GDP effects. Biochim. Biophys. Acta 1797, 968–980.
Ledesma, A., Garcı´a de Lacoba, M., Arechaga, I., and Rial, E. (2002b).
Modeling the transmembrane arrangement of the uncoupling protein UCP1
and topological considerations of the nucleotide-binding site. J. Bioenerg.
Biomembr. 34, 473–486.
Mailloux, R.J., Adjeitey, C.N., and Harper, M.E. (2010). Genipin-induced inhibi-
tion of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic
agents. PLoS ONE 5, e13289.
Slusarewicz, P., Zhu, K., and Hedman, T. (2010). Kinetic characterization and
comparison of various protein crosslinking reagents for matrix modification.
J. Mater. Sci. Mater. Med. 21, 1175–1181.
McKean, D.R., Parrinello, G., Renaldo, A.F., and Stille, J.K. (1987). Synthesis of
functionalized styrenes via palladium-catalyzed coupling of aryl bromides with
vinyl tin reagents. J. Org. Chem. 52, 422–424.
Swida-Barteczka, A., Woyda-Ploszczyca, A., Sluse, F.E., and Jarmuszkiewicz,
W. (2009). Uncoupling protein 1 inhibition by purine nucleotides is under the
control of the endogenous ubiquinone redox state. Biochem. J. 424, 297–306.
Nu¨ bel, T., and Ricquier, D. (2006). Respiration under control of UCPs: clinical
perspective. Horm. Res. 65, 300–310.
Teng, M., Duong, T.T., Johnson, A.T., Klein, E.S., Wang, L., Khalifa, B., and
Chandraratna, R.A.S. (1997). Identification of highly potent retinoic acid
receptor a-selective antagonists. J. Med. Chem. 40, 2445–2451.
´
´
Nury, H., Dahout-Gonzalez, C., Trezeguet, V., Lauquin, G.J., Brandolin, G.,
and Pebay-Peyroula, E. (2006). Relations between structure and function of
the mitochondrial ADP/ATP carrier. Annu. Rev. Biochem. 75, 713–741.
Toma´ s, P., Jime´ nez-Jime´ nez, J., Zaragoza, P., Vuligonda, V., Chandraratna,
R.A.S., and Rial, E. (2004). Activation by retinoids of the uncoupling protein
UCP1. Biochim. Biophys. Acta. 1658, 157–164.
Pani, G., Galeotti, T., and Chiarugi, P. (2010). Metastasis: cancer cell’s escape
from oxidative stress. Cancer Metastasis Rev. 29, 351–378.
´
Rial, E., and Gonzalez-Barroso, M.M. (2001). Physiological regulation of the
´
Tomas, P., Ledesma, A., and Rial, E. (2002). Photoaffinity labeling of the un-
transport activity in the uncoupling proteins UCP1 and UCP2. Biochim.
coupling protein UCP1 with retinoic acid: ubiquinone favors binding. FEBS
Biophys. Acta 1504, 70–81.
Lett. 526, 63–65.
´
Rial, E., Gonzalez-Barroso, M.M., Fleury, C., Iturrizaga, S., Sanchis, D.,
Trachootham, D., Alexandre, J., and Huang, P. (2009). Targeting cancer cells
by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev.
Drug Discov. 8, 579–591.
´
´
Jimenez-Jimenez, J., Ricquier, D., Goubern, M., and Bouillaud, F. (1999).
Retinoids activate proton transport by the uncoupling proteins UCP1 and
UCP2. EMBO J. 18, 5827–5833.
´
Rial, E., Rodrı´guez-Sanchez, L., Gallardo-Vara, E., Zaragoza, P., Moyano, E.,
Yamazaki, M., and Chiba, K. (2008). Genipin exhibits neurotrophic effects
through a common signaling pathway in nitric oxide synthase-expressing
cells. Eur. J. Pharmacol. 581, 255–261.
´
and Gonzalez-Barroso, M.M. (2010). Lipotoxicity, fatty acid uncoupling and
mitochondrial carrier function. Biochim. Biophys. Acta 1797, 800–806.
Samudio, I., Fiegl, M., McQueen, T., Clise-Dwyer, K., and Andreeff, M. (2008).
The warburg effect in leukemia-stroma cocultures is mediated by mitochon-
drial uncoupling associated with uncoupling protein 2 activation. Cancer
Res. 68, 5198–5205.
Yi, J., Gao, F., Shi, G., Li, H., Wang, Z., Shi, X., and Tang, X. (2002). The
inherent cellular level of reactive oxygen species: one of the mechanisms
determining apoptotic susceptibility of leukemic cells to arsenic trioxide.
Apoptosis 7, 209–215.
´
´
´
Sanchez, Y., Amran, D., Fernandez, C., de Blas, E., and Aller, P. (2008).
Genistein selectively potentiates arsenic trioxide-induced apoptosis in human
leukemia cells via reactive oxygen species generation and activation of reac-
tive oxygen species-inducible protein kinases (p38-MAPK, AMPK). Int. J.
Cancer 123, 1205–1214.
Zhang, C.Y., Parton, L.E., Ye, C.P., Krauss, S., Shen, R., Lin, C.T., Porco, J.A.,
Jr., and Lowell, B.B. (2006). Genipin inhibits UCP2-mediated proton leak and
acutely reverses obesity- and high glucose-induced beta cell dysfunction in
isolated pancreatic islets. Cell Metab. 3, 417–427.
´
Santandreu, F.M., Valle, A., Fernandez de Mattos, S., Roca, P., and Oliver, J.
Zhou, H., Zhao, J., and Zhang, X. (2009). Inhibition of uncoupling protein 2 by
genipin reduces insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Arch.
Biochem. Biophys. 486, 88–93.
(2009). Hydrogen peroxide regulates the mitochondrial content of uncoupling
protein 5 in colon cancer cells. Cell. Physiol. Biochem. 24, 379–390.
274 Chemistry & Biology 18, 264–274, February 25, 2011 ª2011 Elsevier Ltd All rights reserved