Structure-activity relationship of flavonoids as potent inhibitors of carbonyl reductase 1 (CBR1)

Article abstract of DOI:10.1016/j.fitote.2014.12.010

Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase superfamily, reduces a variety of carbonyl compounds including therapeutic drugs. CBR1 is involved in the reduction of the anthracycline anticancer drugs to their less anticancer C-13 hydroxy metabolites, which are cardiotoxic. CBR1 inhibitors are thought to be promising agents for adjuvant therapy with twofold beneficial effect in prolonging the anticancer efficacy of the anthracyclines while decreasing cardiotoxicity, a side effect of the drugs. In this study, we evaluated 27 flavonoids for their inhibitory activities of CBR1 in order to explore the structure-activity relationship (SAR). Among them, luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one) showed the most potent inhibition (IC₅₀95 nM), which is also more potent compared to all known classes of CBR1 inhibitors. The inhibition of luteolin was noncompetitive with respect to the substrate in the NADPH-dependent reduction direction, but CBR1 exhibited moderate NADP⁺-dependent dehydrogenase activity for some alicyclic alcohols, in which the luteolin inhibition was competitive with respect to the alcohol substrate (K_i59 nM). The SAR of the flavonoids indicated that the 7-hydroxy group of luteolin was responsible for the potent inhibition of CBR1. The molecular docking of luteolin in CBR1-NADPH complex showed that theflavonoid binds to the substrate-binding cleft, in which its 7-hydroxy group formed a H-bond with main-chain oxygen of Met234, in addition to H-bond interactions (of its 5-hydroxy and 4-carbonyl groups with catalytically important residues Tyr193 and/or Ser139) and a π-stacking interaction (between its phenyl ring and Trp229).

Full text of DOI:10.1016/j.fitote.2014.12.010

Structure-activity relationship of flavonoids as potent inhibitors of carbonyl
reductase 1 (CBR1)
Yuki Arai, Satoshi Endo, Namiki Miyagi, Naohito Abe, Takeshi Miura,
Toru Nishinaka, Tomoyuki Terada, Masayoshi Oyama, Hiroaki Goda, Ossama
El-Kabbani, Akira Hara, Toshiyuki Matsunaga, Akira Ikari
PII:
DOI:
Reference:
S0367-326X(14)00332-3
doi: 10.1016/j.fitote.2014.12.010
FITOTE 3092
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
15 November 2014
20 December 2014
22 December 2014
Please cite this article as: Arai Yuki, Endo Satoshi, Miyagi Namiki, Abe Naohito,
Miura Takeshi, Nishinaka Toru, Terada Tomoyuki, Oyama Masayoshi, Goda Hiroaki,
El-Kabbani Ossama, Hara Akira, Matsunaga Toshiyuki, Ikari Akira, Structure-activity
relationship of flavonoids as potent inhibitors of carbonyl reductase 1 (CBR1), Fitoterapia
(2014), doi: 10.1016/j.fitote.2014.12.010
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ACCEPTED MANUSCRIPT
Structure-activity relationship of flavonoids as potent inhibitors of carbonyl
reductase 1 (CBR1)
Yuki Araia, Satoshi Endoa,*, Namiki Miyagia, Naohito Abeb, Takeshi Miurac,1, Toru Nishinakac,
Tomoyuki Teradac, Masayoshi Oyamab, Hiroaki Godad, Ossama El-Kabbanie, Akira Haraf,
Toshiyuki Matsunagaa, Akira Ikaria
a Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
b Laboratory of Pharmacognosy, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
c Laboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University, Osaka
584-8540, Japan.
d School of Pharmacy, Showa University, Tokyo 142-8555, Japan.
e Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052,
Australia.
f Faculty of Engineering, Gifu University, Gifu 501–1193, Japan.
* Corresponding author
Tel: +81 58 230 8100
Fax: +81 58 230 8105
E-mail: sendo@gifu-pu.ac.jp (S. Endo).

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Key Words
Carbonyl reductase; CBR1; flavonoid; luteolin; structure-activity relationship.
Chemical compounds
Acacetin (PubChem CID: 5280442); Apigenin (PubChem CID: 5280443); Chrysin
(PubChem CID: 5281607); Isatin (PubChem CID: 7054); Kaempferide (PubChem CID:
5281666); Kaempferol (PubChem CID: 5280863); Luteolin (PubChem CID: 5280445);
Quercetin (PubChem CID: 5280343); Quercitrin (PubChem CID: 5280459);
(S)-(+)-1,2,3,4-Tetrahydro-1-naphthol (PubChem CID: 7058029)
Abbreviations:
CBR,
carbonyl
reductase;
DOX,
doxorubicin;
Hydroxy-PP,
3-(1-tert-butyl-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol; SAR, structure-activity
relationship; S-Tetralol, (S)-(+)-1,2,3,4-tetrahydro-1-naphthol.
1Present address: School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's
University, Hyogo 663-8179, Japan.

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Abstract
Human carbonyl reductase
1
(CBR1),
a
member of the short-chain
dehydrogenase/reductase superfamily, reduces a variety of carbonyl compounds including
therapeutic drugs. CBR1 is involved in the reduction of the anthracycline anticancer drugs to
their less anticancer C-13 hydroxy metabolites, which are cardiotoxic. CBR1 inhibitors are
thought to be promising agents for adjuvant therapy with twofold beneficial effect in
prolonging the anticancer efficacy of the anthracyclines while decreasing cardiotoxicity, a
side effect of the drugs. In this study, we evaluated 27 flavonoids for their inhibitory activities
of CBR1 in order to explore the structure-activity relationship (SAR). Among them, luteolin
(2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one) showed the most potent
inhibition (IC50 95 nM), which is also more potent compared to all known classes of CBR1
inhibitors. The inhibition of luteolin was noncompetitive with respect to the substrate in the
NADPH-dependent reduction direction, but CBR1 exhibited moderate NADP+-dependent
dehydrogenase activity for some alicyclic alcohols, in which the luteolin inhibition was
competitive with respect to the alcohol substrate (Ki 59 nM). The SAR of the flavonoids
indicated that the 7-hydroxy group of luteolin was responsible for the potent inhibition of
CBR1. The molecular docking of luteolin in CBR1-NADPH complex showed that the
flavonoid binds to the substrate-binding cleft, in which its 7-hydroxy group formed a H-bond
with main-chain oxygen of Met234, in addition to H-bond interactions (of its 5-hydroxy and
4-carbonyl groups with catalytically important residues Tyr193 and/or Ser139) and a
π-stacking interaction (between its phenyl ring and Trp229).

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1. Introduction
Anthracyclines including doxorubicin (DOX) and daunorubicin are chemotherapeutic
agents against a broad-spectrum of human cancers. DOX is clinically used for breast cancer,
childhood solid tumors, soft tissue sarcomas and malignant lymphoma, while daunorubicin
is important in the treatment of acute leukemias [1]. The major anticancer actions of the
drugs are well-known to be mediated by intercalating into the double-stranded DNA,
inhibiting topoisomerase II and DNA/RNA polymerases, and producing free radicals to kill
cancer cells [2]. Although the anthracyclines are highly effective anticancer drugs, their
dosages are strictly limited due to increased risk for congestive heart failure depending on
the cumulative dose [3]. It is suggested that metabolic reduction of the C-13 carbonyl group
of the anthracycline drugs plays a crucial role in the cardiotoxicity and anticancer efficacy of
the drugs, because the produced alcohol metabolites accumulate in the heart and show
much less anticancer activity compared to the parent drugs [4-6]. The reduction of the C-13
carbonyl group of the drugs is mediated by several reductases belonging to the aldo-keto
reductase superfamily and short-chain dehydrogenase/reductase superfamily [7-9], of which
carbonyl reductase 1 (CBR1) is reported to be the predominant DOX reductase in human
tissues [10]. The involvement of this enzyme in DOX-triggered cardiotoxicity was also
supported by experiments in mice [11,12]. Therefore, potent and selective inhibitors for
CBR1 are thought to be promising agents for adjuvant therapy with twofold beneficial effect
in prolonging the anticancer efficacy of the anthracyclines while decreasing the cardiotoxic
side effect. CBR1 is inhibited by structurally different compounds, which are divided into
synthetic and natural inhibitors. Representative synthetic inhibitors are ethacrynic acid,
indomethacin
[13],
3-(1-tert-butyl-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol
(hydroxy-PP) [14], triclosan and zearalenone analogues [15], of which a zearalenone
analogue shows the most potent inhibition (IC50 0.21 μM). The natural inhibitors are
resveratrol [16] and flavonoids [15,17-20], of which some flavonoids were reported to act as
protectors against in vitro and in vivo cardiotoxicity [21,22]. However, the IC50 values of the
inhibitory flavonoids range from 68 to 0.67 μM [15,17-20], and a molecular docking study of
four flavonoids in CBR1 suggested their different orientations in its active site [18]. Thus, the
structure-activity relationship (SAR) of the inhibitory flavonoids remains unclear.
CBR1 reduces a variety of xenobiotic and endogenous carbonyl compounds using
NADPH as a coenzyme, and its NADP+-dependent dehydrogenase activity has not been
detected [9,13,23,24]. In addition to CBR1, its isoform, CBR3, is expressed in human
tissues, but its reductase activity towards carbonyl compounds is much lower than that of
CBR1 [24,25]. Therefore, CBR1 is thought to act as a major drug metabolizing enzyme in

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the Phase-I metabolism of carbonyl-containing drugs [9,23,24]. Although the physiological
role of CBR1 has not been fully elucidated, isatin, prostaglandin E2, 4-oxonon-2-enal and
S-nitrosoglutathione are reported as its efficient endogenous substrates [13,26-28].
In this study, we evaluated 27 structurally divergent flavonoids for the enzyme inhibitory
activity to explore SAR of flavonoids as potent inhibitors of CBR1. In addition, we found that
CBR1
moderately
catalyzes
NADP+-dependent
oxidation
of
(S)-(+)-1,2,3,4-tetrahydro-1-naphthol (S-tetralol), and examined the inhibition patterns of the
most potent inhibitor, luteolin in both reduction and oxidation directions. Furthermore, the
binding mode of luteolin in CBR1 was investigated by molecular docking.
2. Materials and Methods
2.1. Materials and Reagents
Flavonoids were purchased from Wako Pure Chemical Industries (Osaka, Japan), Tokyo
Chemical Industry (Tokyo, Japan), INDOFINE Chemical Company (Hillsborough, NJ) and
Extrasynthese (Genay, France). Isatin and NADP(H) were obtained from Nacalai Tesque
(Kyoto, Japan) and Oriental Yeast Co. (Osaka, Japan), respectively. trans-Benzene
dihydrodiol was synthesized as described previously [29]. Daunorubicin was supplied from
Meiji Seika Pharma Co. (Tokyo, Japan). All other chemicals were of the highest grade that
could be obtained commercially.
2.2. Preparation of recombinant CBR1
The recombinant His-tagged CBR1 was expressed in Escherichia coli BL21(DE3)pLysS
cells transformed with the expression plasmid harboring its cDNA, and purified to
homogeneity as described previously [25].
2.3. Assay of enzyme activity
The reductase and dehydrogenase activities of His-tagged CBR1 were assayed at 25 ˚C
by measuring the rate of change in NADPH absorbance (at 340 nm) and fluorescence (at
455 nm with an excitation wavelength of 340 nm), respectively. The IC50 values for inhibitors
were determined in the reaction mixture that consisted of 0.1 M potassium phosphate buffer,
pH 7.0, 0.1 mM NADPH, 50 μM isatin and CBR1 (4 μg) in a total volume of 2.0 mL [26]. In
the assay for dehydrogenase activity of CBR1, 0.25 mM NADP+ and an appropriate amount

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of alcohol were employed as the coenzyme and substrate, respectively, in the above
reaction mixture. The kinetic studies in the presence of inhibitors were carried out in both
isatin reduction and S-tetralol oxidation over a range of five or six substrate concentrations
at a saturating concentration of coenzyme. The inhibition patterns were judged from the
Lineweaver-Burk plots, and inhibition constants, Ki (slope effect) and Ki’ (intercept effect),
were determined from replots of the slopes and intercepts, respectively, versus inhibitor
concentration [30]. The IC50 and Ki values are expressed as the means ± SD of at least
three determinations.
2.4. Molecular modeling
The coordinates for CBR1-NADPH-hydroxy-PP complex (PDB code: 1WMA) [14] were
obtained from the RCSB Protein Data Bank. All bound waters and the inhibitor were
removed from the protein, and optimization for protein and ligand was performed. The
performance of the docking method was evaluated by re-docking the ligand into the active
site of CBR1. The molecular docking calculation was performed using the software ICM
version 3.0 (Molsoft, La Jolla, CA) [31], and the figure of the docked model shown in Figure
1 was generated using PyMOL (DeLano Scientific, San Carlos, CA).
2.5. Synthesis of 3,5,7-trimethoxyflavone
A solution of galangin (4.5 mg) in DMF (10 mL) was heated at 80 ˚C with CH3I (0.1 mL)
and K2CO3 (1.0 g) for 5 h. After cooling, the reaction mixture was poured into water and
extracted with ethyl acetate. The ethyl acetate extract was dried and purified by preparative
TLC using CHCl3-ethyl acetate (4:1) affording 3,5,7-trimethoxyflavone (5.1 mg).
3,5,7-Trimethoxyflavone: white powder; 1H NMR (500 MHz, CDCl3): δ 3.89 (3H, s), 3.90
(3H, s), 3.97 (3H, s), 6.35 (1H, d, J = 2.5 Hz), 6.52 (1H, d, J = 2.5 Hz), 7.49 (3H, m), 8.08 (1H,
dd, J = 8.0 1.5 Hz); HRESIMS: m/z [M + Na]+ 335.0861 (Calcd for C18H16O5Na, 335.0890).
3. Results and Discussion
3.1. Inhibitory potencies of flavonoids
The flavonoids evaluated in this study are divided into four groups:
monohydroxyflavones (Table 1), 7-hydroxyflavone derivatives with additional hydroxy and/or
methoxy groups (Table 2), 7-methoxyflavones (Table 3) and other types (flavanones,

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isoflavones, anthocyanin and catechin, Table 4). Among monohydroxyflavones,
7-hydroxyflavone showed prominent inhibitory activity for CBR1, in contrast to no or low
inhibition by flavone itself and other monohydroxyflavones (Table 1). This suggests that the
7-hydroxy group is an important structural prerequisite for the potent inhibition of CBR1.
When the inhibitory activities of 7-hydroxyflavone derivatives were examined, all of
them inhibited more potently than 7-hydroxyflavone (Table 2). Among them, luteolin was the
most potent inhibitor (IC50 0.095 µM), followed by apigenin, kaempferol, quercitrin and
quercetin, whose IC50 values were identical within experimental error. Compared to the
luteolin structure (3’,4’,5,7-tetrahydroxyflavone), the lack of the 3’-hydroxy group (apigenin)
and addition of 3-hydroxy group (quercetin) slightly decreased the inhibitory potency, and
the lack of two hydroxy groups at positions 3’ and 4’ (chrysin) and positions 3’ and 5
(4’,7-dihydroxyflavone) resulted in further decreases in inhibitory potency. This suggests
that, among the substituents of luteolin, the 3’-, 4’- and/or 5-hydroxy groups are the ones
involved in its potent inhibition. The importance of the 5-hydroxy group of 7-hydroxyflavones
for the inhibitory potency is supported by comparison of inhibitory potencies between
structurally similar pairs with and without the 5-hydroxy group: The three 7-hydroxyflavones
with the 5-hydroxy group (apigenin, acacetin and chrysin) showed higher inhibition than their
corresponding 7-hydroxyflavones without the 5-hydroxy group (4’,7-dihydroxyflavone, pratol
and 7-hydroxyflavone). However, clear SAR for the 3’- and 4’-hydroxy groups were not
observed when comparing inhibitory potencies between structurally similar pairs with and
without the 3’- or 4’-hydroxy group. Quercetin (with the 3’-hydroxy group) and kaempferol
(without the 3’-hydroxy group) showed almost the same IC50 values, in contrast to the SAR
of luteolin versus apigenin described above. Apigenin, kaempferol and
4’,7-dihydroxyflavone are 4’-hydroxy derivatives of chrysin, galangin and 7-hydroxyflavone,
respectively. The three 4’-hydroxy derivatives showed lower IC50 values than their
corresponding 4’-unsubstituted flavones, but their inhibitory potencies were similar to those
of their corresponding 4’-methoxylated flavones (acacetin, kaempferide and pratol). Thus,
the 3’- and 4’-hydroxy groups might contribute to its binding to CBR1, but are not essential
for the binding of flavonoid aglycones.
The 7-hydroxyflavone derivatives investigated in this study include previously known
inhibitory flavonoids, chrysin, quercetin and quercitrin, of which chrysin was reported to be
the most potent with an IC50 of 0.67 μM [15,18]. This value is higher than the present result,
which may be due to difference of the activity assay conditions. Even considering this, we
concluded that luteolin is the most potent CBR1 inhibitor, and its IC50 value is also lower
than that (0.21 μM) of the most potent synthetic inhibitor, the zearalenone analogue [15]. It
should be noted that luteolin inhibited the reductase activity of CBR1 toward 0.1 mM

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daunorubicin, showing an IC50 value of 0.34 ± 14 µM. Although the value is higher than that
assayed with isatin as the substrate (Table 2), it is significantly lower than the values (2.1 -
6.5 µM) of flavonoids previously determined in the daunorubicin reduction [18].
To further verify the importance of the 7-hydroxy group of flavonoids for their tight binding
to CBR1, we examined the inhibitory activities of 7-methoxyflavone, tectochrysin and 3,5,7-
trimethoxyflavone that are 7-methoxylated derivatives of 7-hydroxyflavone, chrysin and
galangin, respectively (Table 3). The three 7-methoxylated derivatives did not exhibit
significant inhibition of CBR1 at a high concentration of 10 μM. The results further support
that the 7-hydroxy group is the most important structural requisite fot the CBR1 inhibition by
flavones.
Since naringenin, genistein, catechin and anthocyanines, belonging to other types of
flavonoids, were reported to inhibit CBR1 [13,15,20,26], the Inhibitory activities of
flavanones, isoflavones, anthocyanin and catechin were determined in order to compare
their inhibitory potencies with those of the flavones under the same assay conditions (Table
4). Among them, isosakuranetin and naringenin exhibited relatively low IC50 of
approximately 1 μM, but were much less potent compared to the flavones hydroxylated at
the corresponding positions (acacetin and apigenin). In addition, the isoflavones, genistein
and daidzein, were less potent inhibitors. The results suggest that the flavone-type
configuration involving the fusion of the chroman and phenyl rings is required for the tight
binding to the enzyme. Cyanidin, an anthocyanine, also showed relatively potent inhibition
with an IC50 of 0.34 μM, which is much lower than the value (16 μM) previously determined
using human liver cytosol as the enzyme [20]. The value is higher than that of quercetin, a
flavone with the same hydroxy substituents as cyanidin, suggesting that the 4-carbonyl
moiety of flavones contributes to its tight binding to the enzyme.
3.2. Kinetics of luteolin inhibition
The inhibition patterns of luteolin in the reduction of isatin by CBR1 were noncompetitive
with respect to the substrate, showing Ki and Ki’ values of 59 ± 4 nM and 95 ± 1 nM,
respectively. Similar inhibition patterns of flavonoids [18,19] and resveratrol [16] in the
reduction direction were reported previously. In some NADP(H)-dependent reductases, the
inhibition patterns of inhibitors have been reported to be different between the forward and
reverse directions, in which noncompetitive or uncompetitive inhibitors in the reduction
direction show competitive inhibition in the oxidation direction [32,33]. In fact, such inhibitors
bind to the substrate-binding site in the crystals of the enzymes [34,35]. When various
alcohols were tested as the substrates in the dehydrogenase activity by CBR1, we found

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that the enzyme oxidized S-tetralol (Km = 2.2 mM, kcat = 3.0 s-1) and S-1-indanol (Km = 2.8
mM, kcat = 0.57 s-1) that are known as representative alcohol substrates of several aldo-keto
reductases [23]. It should be noted that CBR1 showed no significant dehydrogenase
activities for R-isomers of the above alcohols and other alicyclic alcohols, such as
cyclohexanol, trans-benzene dihydrodiol, geraniol, and benzyl alcohol, most of which are
substrates of the aldo-keto reductases [23,32,33]. In the NADP+-linked S-tetralol oxidation
catalyzed by CBR1, luteolin inhibited competitively with respect to the substrate, showing
the Ki value of 59 ± 6 nM (Figure 1). This suggests that luteolin binds to the
substrate-binding site of CBR1. The use of S-tetralol as the alcohol substrate may be
kinetically useful not only to judge whether inhibition by a CBR1 inhibitor is competitive or
not, but also to determine the Ki value that implies the dissociation constant of the
competitive inhibitor.
3.3. Molecular docking
Carlquist et al. [18] reported docked models of 7-hydroxyflavones such as quercetin and
quercitrin in CBR1-NADPH complex, in which the 3-hydroxy, 4-carbonyl and 3’-hydroxy
groups of quercetin formed H-bond interactions with the side chains of Ser139 and Tyr193
and the main chain of Met234 of CBR1, respectively. The authors suggested the importance
of the three moieties for tight binding of flavonoids to CBR1, which is not in agreement with
our SAR study. In particular, the inhibitory potencies of luteolin and kaempferol (3- and
3’-deshydroxy analogues of quercetin, respectively) were superior and comparable to that of
quercetin (Table 2). To examine critical interactions supporting the present SAR study, we
constructed a docked model of the most potent inhibitor luteolin in the CBR1–NADPH
complex (Figure 2). In this model, luteolin was orientated in the substrate-binding site of the
enzyme, in which its 7-hydroxy group forming H-bond interaction with the main chain of
Met234, and its 4-carbonyl and 5-hydroxy groups forming an H-bonding network with the
catalytically important residues, Ser139 and Tyr193. In contrast, no H-bond interactions
were observed between the 3-, 3’- and 4’-hydroxy groups of luteolin and residues of CBR1.
Although the binding mode of luteolin in this docked model is different from that of quercetin
reported previously [18], it is in agreement with the present SAR results, indicating that the
7-hydroxy group of luteolin is the important structural requisite for its tight binding to the
enzyme. In addition to the interactions of the hydroxyl groups of flavonoids, the orientations
of the chroman and phenyl rings of luteolin also differ from those of quercetin [18]. In this
luteolin-docked model, its phenyl ring formed a π-stacking interaction with the side chain of
Trp229, while the resorcinol moiety of the chroman ring was reported to be interacting with
the side chain of Trp229 in the quercetin-docked model [18]. This π-stacking interaction may

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explain why 7-hydroxyflavones exhibit more potent inhibition of CBR1 than
7-hydroxyflavanones and 7-hydroxyisoflavones. Reasons for the above disparities between
the previous [18] and present studies may be differences in methodologies used in the
molecular docking, e.g. the software, program, parameters, etc.
4. Conclusion
The present study evaluated 27 flavonoids as CBR1 inhibitors, and showed that luteolin is
the most potent inhibitor among not only the flavonoids, but also the previously reported
CBR1 inhibitors. In addition, our study provides a kinetic method to determine the inhibition
pattern and Ki value of the inhibitor by assaying the oxidation of S-tetralol. Luteolin was a
competitive inhibitor with respect to S-tetralol, and showed a Ki value of 59 nM. The SAR
and molecular docking studies reveal that flavones bind to the enzymes more tightly than
other types of flavonoids, and their 5- and 7-hydroxy groups and 4-carbonyl groups are
important for the binding to the enzyme. Among these groups of flavonoids, the 7-hydroxy
group is suggested to be critical for the potent inhibition. These findings may contribute to
the development of CBR1 inhibitors as novel anti-cancer agents for adjuvant therapy
together with the anthracycline drugs.
Acknowledgements
This work is supported in part by JSPS Grant-in-Aid for Scientific Research (C) from the
Japan Society for the Promotion of Science Grant Number 26460149.
Conflict of interest
The authors have no conflict of interests.
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Table 1. Inhibitory effects of flavone and its monohydroxy derivatives on CBR1.
Compound
IC50 (μM) a
Flavone
> 10
3-Hydroxyflavone
6-Hydroxyflavone
7-Hydroxyflavone
4’-Hydroxyflavone
> 10
> 10
0.60 ± 0.089
19 ± 1.2
a Less than 27% inhibition at 10 μM.

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Table 2. Inhibition of CBR1 by 7-hydroxyflavones with additional hydroxy and/or methoxy
groups
a
a
a
a
Compound
Luteolin
R1
R2
R3
R4
IC50 (μM)
0.095 ± 0.0071
0.13 ± 0.0035
0.13 ± 0.011
0.14 ± 0.018
0.15 ± 0.014
0.18 ± 0.021
0.19 ± 0.021
0.24 ± 0.018
0.27 ± 0.040
0.28 ± 0.014
0.34 ± 0.028
0.37 ± 0.040
H
OH
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
OMe
OMe
H
OH
H
Apigenin
H
Kaempferol
Quercitrin
OH
ORhab
OH
OH
H
H
OH
OH
H
Quercetin
Kaempferide
Acacetin
H
Chrysin
H
H
Pratol
H
OMe
OH
H
H
4’,7-Dihydroxyflavone
Galangin
H
H
H
OH
H
OH
H
Apigenin-4’-O-β-glucoside
a
OH OGlcb
H
b Rha; α-L-rhamonpyranose, Glc; β-D-glucopyranose

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Table 3. Inhibition of CBR1 by 7-methoxyflavones
a
a
Compound
R1
R2
% inhibition
at 10 μM
7-Methoxyflavone
Tectochrysin
H
H
H
37 ± 2.3
9.6 ± 0.46
No inhibition
OH
3,5,7-Trimethoxyflavone
OMe
OMe
a

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Table 4. Inhibition of CBR1 by flavanones, isoflavones, anthocyanin and catechin
a
a
a
a
Compound
Flavanonesa
Isosakuranetin
Naringenin
Dihydrokaempferide
Isoflavonesa
Genistein
R1
R2
R3
R4
IC50 (μM)
H
H
OH OMe
OH OH
OH OMe
H
H
H
0.94 ± 0.021
1.1 ± 0.035
7.4 ± 0.90
OH
H
H
OH
H
OH
OH
H
H
2.1 ± 0.35
11 ± 2.1
Daidzein
Others
Cyanidina
OH
OH
OH
OH
OH
OH
OH
OH
0.34 ± 0.037
5.9 ± 0.85
(+)-Catechina
a
Flavanones
Isoflavones
Anthocyanin
Catechin

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Figure Legends
Figure 1. Inhibition of S-tetralol dehydrogenase activity of CBR1 by luteolin. The initial
velocities (munit/mL) determined in the absence (○) and presence of luteolin were
double-reciprocally plotted as a function of concentration of S-tetralol. Luteolin
concentrations: 25 nM (●), 50 nM (△) and 100 nM (▲).
Figure 2. Docking model of luteolin with the crystal structure of CBR1. (A) Schematic
representation of the overall structure of the CBR1-NADPH-luteolin complex. The ribbon
structure of the protein, NADPH and luteolin are shown in green, yellow and magenta,
respectively. (B) Binding mode of luteolin in the CBR1-NADPH complex. NADPH (yellow)
and residues (green) within 4.0 Å from luteolin (magenta) are depicted with possible H-bond
interactions (dotted line), and the H-bond distances are shown in Å.

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