Benzotropolone inhibitors of estradiol methylation: Kinetics and in silico modeling studies

Article abstract of DOI:10.1016/j.bmc.2005.01.037

Natural and synthetic benzotropolone compounds were assessed in vitro for their ability to inhibit hydroxyestradiol methylation by catechol-O- methyltransferase (COMT). The compounds were also modeled in silico with a homology model of human COMT. Purpurogallin (1), purpurogallin carboxylic acid (2), and theaflavin-3,3′-digallate (6) were the most potent inhibitors of 2-hydroxy and 4-hydroxyestradiol methylation (IC₅₀ 0.22-0.50 μM). Compounds 1 and 6 decreased the V_max and increased the K_m of COMT, indicating a mixed-type inhibition. Compounds 1 and 2 bound to COMT by inserting the six-membered ring of the benzotropolone into the active site. Decreased acidity of the hydroxyl groups on this ring or increased bulkiness reduced potency. Compound 6 bound by inserting the galloyl ester into the active site, which allowed the compound to overcome increased bulkiness and resulted in restored potency. Further studies are needed to determine the impact in vivo of COMT inhibition by these compounds.

Full text of DOI:10.1016/j.bmc.2005.01.037

Bioorganic & Medicinal Chemistry 13 (2005) 2501–2507
Benzotropolone inhibitors of estradiol methylation: kinetics
and in silico modeling studies
a
Joshua D. Lambert, Dapeng Chen, Ching Y. Wang, Ni Ai, Shengmin Sang,
a
b
b
a
c
Chi-Tang Ho, William J. Welsh and Chung S. Yang
b
a,
*
a
Department of Chemical Biology Rutgers, Ernest Mario School of Pharmacy, The State University of New Jersey,
64 Frelinghuysen Rd, Piscataway, NJ08854, USA
1
b
Department of Pharmacology, Robert Wood Johnson Medical School and The Informatics Institute,
University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA
Department of Food Science, Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA
c
Received 19 November 2004; revised 6 January 2005; accepted 21 January 2005
Abstract—Natural and synthetic benzotropolone compounds were assessed in vitro for their ability to inhibit hydroxyestradiol
methylation by catechol-O-methyltransferase (COMT). The compounds were also modeled in silico with a homology model of
0
human COMT. Purpurogallin (1), purpurogallin carboxylic acid (2), and theaflavin-3,3 -digallate (6) were the most potent inhibitors
of 2-hydroxy and 4-hydroxyestradiol methylation (IC50 0.22–0.50 lM). Compounds 1 and 6 decreased the Vmax and increased the
m
K of COMT, indicating a mixed-type inhibition. Compounds 1 and 2 bound to COMT by inserting the six-membered ring of the
benzotropolone into the active site. Decreased acidity of the hydroxyl groups on this ring or increased bulkiness reduced potency.
Compound 6 bound by inserting the galloyl ester into the active site, which allowed the compound to overcome increased bulkiness
and resulted in restored potency. Further studies are needed to determine the impact in vivo of COMT inhibition by these
compounds.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1
. Introduction
of COMT potentially has both beneficial and deleterious
effects. For example, entacapone, a selective inhibitor of
COMT, is currently included in a regimen to treat Park-
Catechol-O-methyltransferase (COMT) is responsible
for the S-adenosylmethionine (AdoMet)-dependent
methylation of a wide variety of endogenous and cate-
chol-containing xenobiotic compounds. For example,
methylation of dopamine by COMT in the central ner-
vous system contributes to the process which results in
termination of the neurotransmission signal. Methyl-
ation of catechols results in a decrease in the polarity
and limits the redox activity of these potentially toxic
endogenous and exogenous substrates. Both soluble
and membrane-bound forms of COMT have been char-
acterized, and COMT has been found in all mammalian
4
,5
insonÕs disease in humans.
This compound blocks
the methylation, and subsequent inactivation, of 3,4-
dihydroxy-L-phenylalanine (L-dopa) in the periphery.
This improves the clinical efficacy of L-dopa by increas-
ing the amount of L-dopa that crosses the blood–brain
barrier.
1
2
The metabolic pathway of estradiol (E ) includes cyto-
2
1
chrome P450-mediated hydroxylation of estradiol,
resulting in the formation of the catechol intermediates,
2-hydroxy (2-OH E ) and 4-hydroxyestradiol (4-OH
2
3
6
tissues studied. COMT activity is highest in the liver
followed by the kidney and small intestine. Inhibition
E ). These intermediates, which are redox active and
2
have been shown to be carcinogenic (especially 4-OH
E ), are then detoxified by COMT-mediated methyl-
2
ation. Inhibition of this methylation process has been
shown to increase the formation of 8-hydroxy-2-deoxy-
6
guanosine in MCF-7 human breast cancer cells. Treat-
ment of male Syrian hamsters with 3% dietary quercetin,
a flavonoid inhibitor of COMT, resulted in a significant
Keywords: Methylation; Benzotropolone; Estradiol; Theaflavins.
*
Corresponding author. Tel.: +1 732 445 5360; fax: +1 732 445
687; e-mail: csyang@rci.rutgers.edu
0
0
968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.037

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J. D. Lambert et al. / Bioorg. Med. Chem. 13 (2005) 2501–2507
increase in E -mediated kidney tumorigenesis presum-
2
ably by increasing the accumulation of these carcino-
7
genic intermediates.
for compounds 1 and 6, and inhibitory activity was com-
pared with inhibitory activity predicted by molecular
modeling with a homology model of human COMT.
Previously, we have reported the synthesis of a series of
benzotropolone-containing polyphenolic compounds
ranging in structural complexity from purpurogallin
2. Results
0
(
1, mw = 220.2) to theaflavin-3,3 -digallate (6, mw =
2.1. HPLC analysis of estradiol metabolites
878.0) (Fig. 1) by the peroxidase-catalyzed reaction
8
of various polyphenolic starting materials.
HPLC analysis of enzyme reactions with 2-OH E as the
2
substrate revealed a clear peak with retention time
(t ) = 10.2 min corresponding to 2-methoxyestradiol
Purpurogallin is a benzotropolone-containing natural
9
R
product, which occurs in the nut gall of Quercus spp.
Human exposure to this compound occurs mainly
through consumption of edible oils to which purpuro-
(2-OCH E ) (Fig. 2). A similar retention time was ob-
3
2
served for 4-methoxyestradiol when 4-OH E was used
2
as the substrate for the reaction.
1
0
gallin has been added as a stabilizer. Veser has previ-
ously reported that purpurogallin competitively
inhibits COMT isolated from the yeast, Candida tropi-
calis, but the inhibitory effects of this compound on
human COMT and with physiologically-important
Under these HPLC conditions, 2-OH and 4-OH E were
2
eluted in the void volume and could not be quantified.
Additionally, two additional peaks (t ꢀ 12 min) were
R
observed in chromatograms following incubation with
human liver cytosol. We hypothesize that these peaks
represent the minor methylation products, 2-hydroxy-
1
1
substrates have not been studied. Theaflavins, which
also contain a benzotropolone moiety, are present as
3
water extract. This beverage represents nearly 80% of
the world consumption of tea.
–6% of the dry weight of black tea (Camellia sinensis)
3-methoxyestradiol (from 2-OH E ) and 3-methoxy-4-
2
hydroxyestradiol (from 4-OH E ), however, we did not
2
have reference standards of these compounds and could
not therefore confirm this hypothesis nor quantify these
metabolites (Fig. 2). The standard curve was linear over
1
2
In the present study, we report the inhibitory activity of
a series of these benzotropolone-containing compounds
against COMT-mediated methylation of 2-OH E and
the range of 0.5–5 lg/mL for both 2-OCH (y = 6.74x–
3
2
0.09, R = 0.998) and 4-OCH3 (y = 18.28–0.16,
2
2
R = 0.998).
4
-OH E . The kinetics of inhibition were determined
2
OH
g
h
i
f
OH
k
j
O
e
a
OH
d
b
HO
O
c
OR3
8
1
OH
OH
7
6
2
3
3
'
7'
R1
OH
5
h
4
1'
O
f
i
OH
O
OH
HO
g
h
i
OH
f
OH
O
k
j
a
4
)
d
HO
O
e
a
1
OH
d
b
7
c
3
OH
R2
OR3
O
OH
OH
OH
1
)
R = OH, R = H
1 2
2)
)
R = OH, R 2 = CO2
H
5) R = H
3
1
3
R = H, R = CO H
6) R = Galloyl
1
2
2
3
OH
Galloyl group
Figure 1. Structures of benzotropolone-containing inhibitors of catechol-O-methyltransferase.

J. D. Lambert et al. / Bioorg. Med. Chem. 13 (2005) 2501–2507
2503
difference significantly enhanced the potency of com-
pound 6, and resulted in an IC50 value of
0.50 ± 0.01 lM: the higher potency was in spite of the
higher molecular weight and greater steric bulk.
Compounds 1–6 were also shown to inhibit methylation
4-OH E in a dose-dependent manner (Fig. 3). The IC
values were very similar to those observed when 2-OH
2
5
0
E was used as the substrate. The potency of inhibition
2
of the compounds showed the following rank order:
2
> 6 > 1 ꢁ 4 > 5 > 3.
Kinetic analysis of compounds 1 and 6 was conducted
using 2-OH E and 4-OH E as substrates (Fig. 4).
2
2
Increasing concentrations of compound 1 resulted in de-
creased Vmax and increased Km.
Figure 2. HPLC analysis of 2-OH and 4-OH E
liver cytosol. Representative chromatograms of reactions containing
0 lM 2-OH E (A) or 4-OH E (B) as the substrate. Identification of
the main methylated metabolites were performed by comparison with
pure 2-OCH and 4-OCH reference standards.
2
methylation by human
5
2
2
For example, using 2-OH E as the substrate the K val-
m
2
ues after treatment with 0, 1, and 2 lM compound 1
were 17.5 ± 0.7, 39.3 ± 2.6, and 70.0 ± 4.3 lM, respec-
tively: Vmax was decreased from 424.8 ± 9.1 to
3
E
2
3 2
E
192.2 ± 8.3 pmol/mg/min (Table 1). Such changes indi-
cate a mixed-type inhibition for compound 1 with re-
gard to 2- and 4-OH E . Similarly, in the presence of
2
2.2. Inhibition of COMT-mediated methylation of
hydroxylated estradiol metabolites
compound 6 (0–2 lM) the Km was increased from
1
8.2 ± 0.6 to 73.2 ± 3.5 lM and the Vmax was decreased
from 395.1 ± 11.1 to 180.7 ± 4.9 pmol/mg/min when
-OH E was used as the substrate. This again indicates
2
2
2
and 2 displayed relatively high potency with IC50 values
of 0.50 ± 0.04 and 0.30 ± 0.02 lM, respectively. Loss of
the hydroxyl groups at the g-position (g-OH) or in-
creased bulkiness decreased the potency of the molecules
as demonstrated by compounds 3–5, which had IC50
values of 7–15 lM. Compounds 5 and 6 differ in
structure by the presence of two galloyl moieties. This
a mixed-type inhibition. Similarities of the results be-
tween 2-OH E and 4-OH E when compound 1 was
2
2
tested, we did not determine the inhibitory kinetics of
compound 6 with 4-OH E as the substrate.
2
2.3. Molecular modeling and docking studies of benzo-
tropolone inhibitors
To characterize the inhibitory mechanism of compounds
1–6 against COMT, a homology model of human
COMT was prepared by comparison with the published
crystal structure of rat COMT. Docking of compounds
1
1
20
00
8
0
0
0
0
1
–5 revealed that the six-membered ring of these com-
pounds occupied the active site of COMT (Figs. 5 and
).
6
4
2
6
1
3
5
2
4
6
Calculated values of FLEXX scoring function for com-
pounds 1–3 were ꢂ13.05 ± 2.54, ꢂ14.39 ± 0.26, and
ꢂ14.13 ± 0.38 kcal/mol, respectively. A more negative
value of FLEXX scoring function indicates stronger li-
gand–COMT binding affinity, and our results suggest
that compound 2 binds more tightly to human COMT
than compound 1 and 3, however the difference is not
statistically significant. The distance between the hydro-
xyl groups g, h, and i on the six-membered ring of the
0
0
.01
0.1
1
10
100
1
1
20
00
8
0
0
0
0
6
4
2
1
3
5
2
4
6
compound 1 and the N atom of the terminal NH group
2
on the side chain of Lys144 were calculated. The dis-
tance between Lys144 and g-OH, h-OH, and i-OH were
0
˚
.79 ± 0.96, 4.00 ± 0.58, and 3.79 ± 0.92 A, respectively.
4
For compound 2, the average distance between these
0
.01
0.1
1
10
100
Concentration (µM)
˚
groups were 5.63 ± 0.60, 4.09 ± 0.14, and 2.91 ± 0.71 A
for g-OH, h-OH, and i-OH, respectively. Compound
Figure 3. Inhibition of human liver cytosol-mediated methylation of 2-
OH and 4-OH E by benzotropolone inhibitors (0–50 lM). Points
represent n = 3–5. Error bars are omitted for clarity; the standard
3, which lacked the g-OH, had the distance of
˚
4.58 ± 0.25 and 2.64 ± 0.00 A between Lys144 and the
2
deviation was 10% or less for each point.
h- and i-OH groups, respectively.

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J. D. Lambert et al. / Bioorg. Med. Chem. 13 (2005) 2501–2507
Figure 4. Kinetics of COMT-inhibition by compound 1 and 6 (0–2 lM) with respect to 2-OH E
compounds exhibited mixed-type inhibition with respect to hydroxyestradiol concentration. Changes in K
point represents n = 3. Error bars are omitted for clarity; the standard deviation was 10% or less for each point.
2
and 4-OH E
2
concentration (5–200 lM). Both
m
and Vmax are presented in Table 1. Each
0
a
Table 1. Kinetics of inhibition of COMT-mediated methylation of 2-OH and 4-OH E
2
by purpurogallin (1) and theaflavin-3,3 -digallate (6)
Concentration (lM)
Compound 1
Compound 6
2
-OH E
2
4-OH E
2
2-OH E
2
V
max
K
m
V
max
K
m
V
max
K
m
0
1
2
424.8
241.8
192.2
17.5
39.3
70.0
287.8
162.6
113.8
16.1
17.0
24.0
395.1
194.3
180.7
18.2
35.3
73.2
b
b
b
b
b
b
b
b,c
b,c
b,c
b,c
a
b
c
m
Units of Vmax = pmol/mg/min; units of K = lM.
Statistically different from control by one-way ANOVA and TukeyÕs Multiple Comparison Test, p < 0.05.
Statistically different from 1 lM by one-way ANOVA and TukeyÕs Multiple Comparison Test, p < 0.05.
R , was unable to find a consensus binding mode within
3
human COMT.
3. Discussion
In the present report, we describe the inhibition of
COMT by a series of benzotropolone-containing mole-
cules including purpurogallin (1), theaflavin (5), and
0
theaflavin-3,3 -digallate (6). These compounds displayed
IC50 values of 0.22–15 lM: potency appeared to depend
largely on the presence of a vicinal hydroxyl group and
small molecular size. An exception to this observation
0
was theaflavin-3,3 -digallate (6), which had the highest
molecular weight but was also quite potent with an
IC50 of 0.30–0.50 lM. The inhibitory kinetics of com-
pounds 1 and 6 were determined using 2-OH E and 4-
OH E as substrates. In both cases, these compounds
2
2
Figure 5. Molecular modeling of the interaction between compound 2
and hCOMT showing the consensus orientation for compound 2. The
protein is depicted in ribbon representation and colored by secondary
structures (i.e., helix, strand, and loop). Both compound 2 and ligand
contact residues are represented in stick form and colored by atom
type with carbon in gray, oxygen in red, sulfur in yellow, and
magnesium in magenta.
demonstrated a mixed-type inhibition as indicated by
the increasing K and the decreasing Vmax. We hypothe-
m
size that this mixed-type inhibition is due to competitive
inhibition by the test compound and noncompetitive
inhibition by the predicted methylated metabolite of
the test compound. Previously we have observed such a
pattern of inhibition with the tea polyphenol epigalloca-
techin-3-gallate (EGCG, competitive inhibitor) and its
di-methylated metabolite (noncompetitive inhibitor)
(unpublished results). We are currently identifying the
methylated metabolites of compounds 1 and 6; these will
be tested for inhibitory activity against COMT.
Compound 6 docked in a different manner from com-
pounds 1–5: in this case, the galloyl moiety occupied
the active site of COMT (Fig. 6). The FLEXX scoring
function for this compound was ꢂ1.02 kcal/mol: the dis-
0
0
00
00
tance between 3 -OH, 4 -OH, and 5 -OH were
3
5
˚
.20 ± 0.57, 3.12 ± 0.76, and 4.72 ± 0.90 A. Compound
, which resembles 6 but lacks the galloyl moieties at
In order to gain a clearer understanding of the differ-
ences in potency of compounds 1–6, docking studies

J. D. Lambert et al. / Bioorg. Med. Chem. 13 (2005) 2501–2507
2505
AdoMet
S
CH3
Lys 144
OH2
NH2
OH
H
O
Asp 141
O
O
Mg
O
O
H
O
Asp 169
HOOC
O
O
OH
Asn 170
NH2
Compound 2
OH
OH
OH
O
OH
O
OH
OH
AdoMet
HO
HO
O
O
S
OH
O
CH3
OH2
Lys 144 NH2
O
H
O
Asp 141
OH
O
O
Mg
O
O
O
O
Asp 169
OH
H
O
Asn 170
NH2
Compound 6
2+
Figure 6. Compound 2 forms a hexa-coordination complex with Mg and surrounding residues using the six-membered ring moiety. Compound 6
2+
forms a hexa-coordination complex with Mg and surrounding residues using the galloyl moiety instead.
were performed using the FLEXX software and a homo-
logy model of human COMT. These studies indicated
that the six-membered ring of benzotropolone moiety
occupied the binding pocket of COMT and formed a
restored despite the increased bulkiness. The results of
the molecular modeling studies correlate quite well with
what was observed in the in vitro studies.
2
+
stable hexa-coordination complex with Mg metal ion
through the vicinal hydroxyl groups. As the bulkiness
of the molecule increases, this insertion becomes im-
paired and the potency of the inhibitor is decreased. In
the case of compound 6, which has the highest molecu-
lar weight, binding to COMT occurred via the galloyl
moiety instead of the six-membered ring of the benzo-
tropolone pharmacophore. The galloyl moiety had
greater freedom than the benzotropolone ring to insert
into the binding pocket and thus the potency was
The tri-hydroxyl group on the six-membered ring of the
benzotropolone moiety is also important for the potency
of the inhibitor. Compounds 2 and 3 differ only with re-
spect to the presence of this g-hydroxyl group and yet
had a 15-fold difference in potency. The loss of potency
may result from the lower acidity of the i-OH group due
to the absence of the g-OH group. As an analogy, the
overall pKa of catechol is 9.5 ± 0.2 whereas that of pyro-
gallol, which has a tri-hydroxyl structure, is 9.1 ± 0.2
(calculated by SciFinder Scholar, ACS). Modeling

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J. D. Lambert et al. / Bioorg. Med. Chem. 13 (2005) 2501–2507
studies suggest that the i-OH group interacts with
Lys144 in the active site of COMT via a general acid–
base reaction, in which Lys144 acts as the general
base. A decrease in the acidity of this hydroxyl group
would cause compound 3 to interact less strongly within
the binding pocket of COMT and would thus result in
decreased potency.
4.2. HPLC analysis of methylated estradiol metabolites
The methylation of hydroxylated E2 was monitored
using HPLC with electrochemical detection. The HPLC
system consisted of two Waters Model 510 pumps
(Waters Corp., Milford, MA) equipped with an ESA
Coulochem II electrochemical detector (ESA Inc.,
Chelmsford, MA). Samples were injected with a Waters
717plus autoinjector equipped with a 200 lL loop. Sep-
aration was achieved using a Supelcosil C-18 reversed-
phase column (250 mm · 4.6 mm) with a 5 lm pore size
(Supelco Inc., Bellefonte, PA) and an isocratic mobile
phase of sodium phosphate monobasic buffer
(17.1 mM, pH 3.4) containing 33% acetonitrile and 6%
tetrahydrofuran. The flow rate was 1 mL/min and eluant
was monitored at a potential of 300 mV. Data was pro-
1
3
Finally, the carboxylic acid moiety on the d-carbon of
the benzotropolone ring appears to be an important
determinant of potency, although less so than the tri-hy-
droxyl structure. This carboxylic acid group contributes
additional resonance stabilization to the deprotonated
intermediate of the inhibitor and its importance is dem-
onstrated by comparing the potency of 1 and 2
(
IC50 = 0.50 ± 0.04 vs 0.30 ± 0.02 lM).
3
2
cessed using MILLENIUM
software (Waters Corp.,
Inhibition of COMT in vivo may be seen either as bene-
ficial or deleterious depending upon the particular situ-
ation. For example, current clinical treatment of
ParkinsonÕs disease includes COMT inhibitors (e.g., enta-
capone and tolcapone) as adjunct therapy in combina-
tion with the standard regimen of L-dopa and
Milford, MA). Identification of 2-OCH3 E2 and 4-
OCH E was accomplished by comparison of retention
times between experimental samples and pure reference
standards of those metabolites.
3
2
4.3. Inhibition of human liver COMT-mediated methyl-
ation of estradiol metabolites
4
carbidopa. COMT inhibitors prolong the half life of
L-dopa and allow that compound to enter the central
nervous system. By contrast, chronic inhibition of
COMT-mediated methylation of hydroxylated estradiol
metabolites may represent a risk factor for mammary
carcinogenesis since such metabolites are known to be
The IC50 of the purpurogallin derivatives for the inhibi-
tion of human liver cytosol-mediated methylation of 2-
OH E (or 4-OH E ) was determined as follows. Human
2
2
liver cytosol (2 mg/mL), 2-OH E2 (50 lM), DTT
(1 mM), MgCl2 (1 mM), Tris–HCl (10 mM, pH 7.4),
and the compound of interest (0–50 lM) were combined
and pre-incubated for 2 min at 37 ꢁC. AdoMet (60 lM)
was added and the reaction was incubated at 37 ꢁC for
30 min. The reaction was stopped by the addition of
an equal volume of ice-cold methanol containing 1%
ascorbic acid. After centrifugation at 10,000g for
10 min, 50 lL of the supernatant was analyzed by
HPLC with electrochemical detection.
1
4,15
redox active.
Methylation of these compounds is
generally considered to be a detoxification pathway.
For example, Zhu and Liehr have reported that admin-
istration of quercetin, a flavonoid inhibitor of COMT,
in the estradiol-induced hamster kidney tumor model in-
creased the incidence of large tumors and abdominal
metastases relative to animals treated only with estra-
7
–16
diol.
We have found no reports of an increased risk
of estrogen-related malignancies in human beings asso-
ciated with the use of selective COMT inhibitors.
To determine inhibition kinetics, the concentration of
substrate (2-OH E or 4-OH E ) was varied between 5
In summary, we have demonstrated the efficacy of a series
of benzotropolone-containing molecules as inhibitors of
COMT. Using classical enzymology and computer mod-
eling studies, we have characterized these compounds in
terms of the mechanism of inhibition using 2- and 4-
OH estradiol as substrates. Further studies are needed
to determine the potential beneficial or deleterious impli-
cations of the observed COMT inhibition in vivo.
2
2
and 200 lM at saturating AdoMet concentrations
(200 lM) in the presence of 0, 1, or 2 lM test com-
pound. After pre-incubation at 37 ꢁC for 3 min, Ado-
Met was added to start the reaction. The incubation
was carried out at 37 ꢁC for 30 min and terminated as
above. Aliquots (50 lL) were analyzed by HPLC with
electrochemical detection.
4
.4. In silico molecular studies of human COMT
4. Experimental
The protein sequence of human COMT (accession num-
ber BC000419) was retrieved from the NCBI Reference
Sequence (RefSeq) Collection. A structural model of hu-
man COMT was constructed using the Insight II Homol-
ogy Module (Accelrys, Inc., San Diego, CA) from the
published crystal structure of rat COMT (RCSB Protein
Data Bank www.rcsb.org/pdb; PDB ID = 1VID) as the
modeling template. The quality of the model was con-
firmed by the WHATIF-CHECK program (www.cmbi.-
kun.nl/gv/servers/WIWWWI). All molecule modeling
operations for compounds 1–6 were performed using
SYBYL 6.9 (Tripos, Inc., St. Louis, MO), running on a
4
.1. Chemicals and enzymes
Pooled human liver cytosol (20 mg/mL protein concen-
tration) was purchased from BD-Gentest Co. (Woburn,
MA). Purpurogallin and derivatives were prepared as
previously reported, dissolved as 50 mM stock solutions
in DMSO, and stored at ꢂ80 ꢁC (Sang et al., 2004).
Estradiol metabolites (2-OCH E and 4-OCH E ) were
purchased from Steraloids Inc. (New Port, RI), prepared
as a 5 mM stock solution in DMSO and stored at ꢂ80 ꢁC.
All other chemicals were of the highest grade available.
3
2
3
2

J. D. Lambert et al. / Bioorg. Med. Chem. 13 (2005) 2501–2507
2507
Silicon Graphics (SGI) O2 workstation under the IRIX 6.5
operation system. Initial structures of compounds 1–6
were constructed using the Sketch Molecule module in
SYBYL and subsequently energy minimized to yield a sta-
ble conformation using the MMFF94 molecular force
2. Akil, M.; Kolachana, B. S.; Rothmond, D. A.; Hyde, T.
M.; Weinberger, D. R.; Kleinman, J. E. J. Neurosci. 2003,
2
3, 2008.
. Lu, H.; Meng, X.; Yang, C. S. Drug Metab. Dispos. 2003,
1, 572.
. Stocchi, F.; Barbato, L.; Nordera, G.; Bolner, A.; Carac-
eni, T. J. Neural Transm. 2004, 111, 173.
. Blandini, F.; Nappi, G.; Fancellu, R.; Mangiagalli, A.;
Samuele, A.; Riboldazzi, G.; Calandrella, D.; Pacchetti,
C.; Bono, G.; Martignoni, E. J. Neural Transm. 2003, 110,
911.
3
4
5
3
1
7–19
field with a dielectric constant of e = 4.0.
was docked in the active site using the FLEXX software
Each ligand
2
0
implemented in SYBYL (Tripos). FLEXX is a fast flexible
docking method using an incremental construction algo-
rithm that allows maximum rotations and conformation
2
1
of ligands in the binding pocket. Distances between the
terminal NH group of the Lys144 side chain and the hy-
6. Saintot, M.; Malaveille, C.; Hautefeuille, A.; Gerber, M.
Int. J. Cancer 2003, 107, 652.
2
7
8
. Zhu, B. T.; Liehr, J. G. Toxicol. Appl. Pharmacol. 1994,
25, 149.
. Sang, S.; Lambert, J. D.; Tian, S.; Hong, J.; Hou, Z.;
Ryu, J. H.; Stark, R. E.; Rosen, R. T.; Huang, M. T.;
Yang, C. S.; Ho, C. T. Bioorg. Med. Chem. 2004, 12,
droxyl groups were calculated as an indicator of the rate
of deprotonation. FLEXX scoring function (kcal/mol),
was calculated as an indicator of the strength of the pro-
tein–ligand interactions.
1
4
59.
. Abou-Karam, M.; Shier, W. T. Phytother. Res. 1999, 13,
37.
4
.5. Statistical analysis
9
3
Kinetic parameters (Vmax, K ) of methylation were cal-
m
10. Prasad, K.; Kapoor, R.; Lee, P. Mol. Cell. Biochem. 1994,
139, 27.
culated using GRAPHPAD PRISM 3.0 (GraphPad Software,
San Diego, CA). Statistical differences in Vmax, K , and
11. Veser, J. J. Bacteriol. 1987, 169, 3696.
12. Lambert, J. D.; Yang, C. S. J. Nutr. 2003, 133,
m
dose–response curves were assessed using one-way
ANOVA followed by a Tukey Multiple Comparison
Test. Significance was achieved at p < 0.05.
3
262S.
3. Zheng, Y. J.; Bruice, T. C. J. Am. Chem. Soc. 1997, 119,
137.
4. Yoo, K. Y.; Kang, D. Breast Cancer 2003, 10, 289.
1
8
1
Acknowledgements
15. Creveling, C. R. Neurotox. Res. 2003, 5, 473.
1
6. Zhu, B. T.; Taneja, N.; Loder, D. P.; Balentine, D. A.;
Conney, A. H. J. Steroid Biochem. Mol. Biol. 1998, 64,
This work was supported by NIH grant P01 CA 88961
to C.S.Y.).
2
07.
(
1
1
7. Halgren, T. A. J. Am. Chem. Soc. 1990, 112, 4710.
8. Halgren, T. A. J. Comput. Chem. 1996, 17, 490.
References and notes
. Casarett s Toxicology: The Basic Science of Poisons;
19. Halgren, T. A. J. Comput. Chem. 1999, 20, 720.
2
0. Rarey, M.; Wefing, S.; Lengauer, T. J. Comput. Aided
Mol. Des. 1996, 10, 41.
1
Klaassen, C. D., Ed., 5th ed.; McGraw-Hill: New York,
NY, 1996; p 1111.
21. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol.
Biol. 1996, 261, 470.