Novel resveratrol-based substrates for human hepatic, renal, and intestinal UDP-glucuronosyltransferases

Article abstract of DOI:10.1021/tx400408x

Trans-Resveratrol (tRes) has been shown to have powerful antioxidant, anti-inflammatory, anticarcinogenic, and antiaging properties; however, its use as a therapeutic agent is limited by its rapid metabolism into its conjugated forms by UDP-glucuronosyltransferases (UGTs). The aim of the current study was to test the hypothesis that the limited bioavailability of tRes can be improved by modifying its structure to create analogs which would be glucuronidated at a lower rate than tRes itself. In this work, three synthetic stilbenoids, (E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid (NI-12a), (E)-2,4-dimethoxy-6-(4-methoxystyryl)benzaldehyde oxime (NI-ST-05), and (E)-4-(3,5-dimethoxystyryl)-2,6-dinitrophenol (DNR-1), have been designed based on the structure of tRes and synthesized in our laboratory. UGTs recognize and glucuronidate tRes at each of the 3 hydroxyl groups attached to its aromatic rings. Therefore, each of the above compounds was designed with the majority of the hydroxyl groups blocked by methylation and the addition of other novel functional groups as part of a drug optimization program. The activities of recombinant human UGTs from the 1A and 2B families were examined for their capacity to metabolize these compounds. Glucuronide formation was identified using HPLC and verified by β-glucuronidase hydrolysis and LC-MS/MS analysis. NI-12a was glucuronidated at both the -COOH and -OH functions, NI-ST-05 formed a novel N-O-glucuronide, and no product was observed for DNR-1. NI-12a is primarily metabolized by the hepatic and renal enzyme UGT1A9, whereas NI-ST-05 is primarily metabolized by an extrahepatic enzyme, UGT1A10, with apparent K_m values of 240 and 6.2 μM, respectively. The involvement of hepatic and intestinal UGTs in the metabolism of both compounds was further confirmed using a panel of human liver and intestinal microsomes, and high individual variation in activity was demonstrated between donors. In summary, these studies clearly establish that modified, tRes-based stilbenoids may be preferable alternatives to tRes itself due to increased bioavailability via altered conjugation.

Full text of DOI:10.1021/tx400408x

Article
pubs.acs.org/crt
Novel Resveratrol-Based Substrates for Human Hepatic, Renal, and
Intestinal UDP-Glucuronosyltransferases
Aleksandra K. Greer,† Nikhil R. Madadi,‡ Stacie M. Bratton,† Sarah D. Eddy,† Zofia Mazerska,†,§
Howard P. Hendrickson,‡ Peter A. Crooks,‡ and Anna Radominska-Pandya†,*
‡
†Departments of Biochemistry & Molecular Biology, College of Medicine, and Department of Pharmaceutical Sciences, College of
Pharmacy, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, United States
§Department of Pharmaceutical Technology and Biochemistry, Chemical Faculty, Gdan
́
sk University of Technology, 11/12
́
Narutowicza St., 80-233 Gdansk, Poland
ABSTRACT: Trans-Resveratrol (tRes) has been shown to
have powerful antioxidant, anti-inflammatory, anticarcinogenic,
and antiaging properties; however, its use as a therapeutic
agent is limited by its rapid metabolism into its conjugated
forms by UDP-glucuronosyltransferases (UGTs). The aim of
the current study was to test the hypothesis that the limited
bioavailability of tRes can be improved by modifying its
structure to create analogs which would be glucuronidated at a
lower rate than tRes itself. In this work, three synthetic
stilbenoids, (E)-3-(3-hydroxy-4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylic acid (NI-12a), (E)-2,4-dimethoxy-6-
(4-methoxystyryl)benzaldehyde oxime (NI-ST-05), and (E)-4-
(3,5-dimethoxystyryl)-2,6-dinitrophenol (DNR-1), have been designed based on the structure of tRes and synthesized in our
laboratory. UGTs recognize and glucuronidate tRes at each of the 3 hydroxyl groups attached to its aromatic rings. Therefore,
each of the above compounds was designed with the majority of the hydroxyl groups blocked by methylation and the addition of
other novel functional groups as part of a drug optimization program. The activities of recombinant human UGTs from the 1A
and 2B families were examined for their capacity to metabolize these compounds. Glucuronide formation was identified using
HPLC and verified by β-glucuronidase hydrolysis and LC−MS/MS analysis. NI-12a was glucuronidated at both the −COOH
and −OH functions, NI-ST-05 formed a novel N−O-glucuronide, and no product was observed for DNR-1. NI-12a is primarily
metabolized by the hepatic and renal enzyme UGT1A9, whereas NI-ST-05 is primarily metabolized by an extrahepatic enzyme,
UGT1A10, with apparent Km values of 240 and 6.2 μM, respectively. The involvement of hepatic and intestinal UGTs in the
metabolism of both compounds was further confirmed using a panel of human liver and intestinal microsomes, and high
individual variation in activity was demonstrated between donors. In summary, these studies clearly establish that modified, tRes-
based stilbenoids may be preferable alternatives to tRes itself due to increased bioavailability via altered conjugation.
region- and stereospecific, with a trans-3-O-glucuronide being
the predominant product formed.2 In order to potentially
circumvent the unfavorable pharmacokinetics of tRes, the
search for compounds with enhanced bioavailability, such as
synthetic or naturally occurring structural analogs of tRes that
exhibit more favorable conjugation profiles, is of great
significance.
Natural prenylated tRes analogs, trans-arachidin-1 and -3,
produced in and purified from peanut hairy root culture, were
previously tested toward several UGT1A enzymes and human
hepatic and intestinal microsomes.9 These two derivatives, both
prenylated at position 4, had glucuronidation rates that were
slower in comparison to native tRes (decreased Vmax values).
Therefore, the greater lipophilicity of prenylated tRes analogs
changed the enzyme affinity, ultimately leading to a slower
INTRODUCTION
■
Resveratrol (trans-3,5,4′-trihydroxystilbene; tRes) is a well-
known, natural polyphenol found in grapes, peanuts, red wine,
and other foods.1,2 It has been shown to be a powerful
antioxidant, neuroprotector, anticarcinogenic, and anti-inflam-
matory agent that lacks detectable toxicity.3 In clinical studies,
resveratrol has demonstrated the ability to reduce tumor cell
proliferation in patients with colorectal cancer.4 However,
although initial preclinical data are encouraging, the oral
bioavailability of native tRes is relatively low, due in large part
to its rapid conjugation,5−7 which presents a significant
limitation to future clinical development of this potentially
valuable drug.
The in vitro glucuronidation of tRes has been relatively well
characterized,1,2,5,8,9 and it has been shown that human hepatic
and intestinal UGTs are actively involved in tRes glucur-
onidation and that both tissues contribute significantly to its
biotransformation.2,8 The glucuronidation reactions are very
Received: November 4, 2013
Published: February 26, 2014
© 2014 American Chemical Society
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Figure 1. Synthesis and chemical structures of novel stilbenoids. (a) POCl3, DMF, 0 °C, 69% yield; (b) hydroxylamine, AcONa, methanol, reflux, 4
h, 90% yield; (c) triphenylphosphine, toluene, reflux, 12 h, 95% yield; (d) concentrated H2SO4, fuming HNO3, 0 °C, 55% yield; (e) NaOMe,
methanol, ambient temp., 12 h, 60% yield; (f) TEA, Ac2O, 140 °C, 40% yield. (3) NI-ST-05; (8) DNR-1; (11) NI-12a; (tRes) trans-resveratrol.
metabolism, which is expected to improve bioavailability.
Although the isolation of compounds from medicinal plants
is an exciting avenue for obtaining compounds that could be
developed into drugs, this method is limited to naturally
occurring compounds. Chemical synthesis opens the door for
the production of compounds specifically designed to maximize
effectiveness and improve bioavailability.
A number of nucleophilic functional groups are known to be
targets for glucuronidation. Excluding extraordinary cases,
glucuronides can be formed at carbon atoms with oxygen
derived nucleophiles (O-glucuronides), and upon N-carbamyo-
lation of primary and secondary amino groups (N-glucur-
onides).10 Sulfur- and selenium-linked glucuronides and di- and
bis-glucuronides are less common but also occur.10 Considering
the above, a number of tRes-based stilbenoid derivatives can be
designed that have improved bioavailability due to restricted
glucuronidation while maintaining biological activity.
In the current studies, three new stilbenoids, NI-ST-05,
DNR-1, and NI-12A, have been designed based on the
structure of tRes, with the majority of the hydroxyl groups
blocked by methylation and novel functional groups added to
the stilbene scaffold. It was expected that this would result in
lower levels of UGT-mediated metabolism. The study aimed to
elucidate: (i) whether the newly synthesized stilbene derivatives
are substrates for human recombinant UGTs and (ii) what the
level of glucuronidation activity of selected UGT enzymes is
toward these novel stilbenes in comparison to the native tRes.
Twelve major recombinant human UGT1A and 2B enzymes
have been examined for their capacity to metabolize these
compounds. The formation of glucuronide conjugates has been
identified using HPLC−UV−vis analysis, and the structures of
these metabolites have been elucidated by LC−MS/MS and β-
glucuronidase hydrolysis. We have hypothesized that the
selected UGT enzymes would have lower affinity for these
modified analogs of tRes. Therefore, the synthesized stilbenoids
would represent a useful scaffold for the design of selective and
efficacious resveratrol analogs with improved bioavailability and
which could be developed as chemotherapeutic agents.
EXPERIMENTAL PROCEDURES
■
Materials. All chemicals used in this study were of at least reagent
grade. Recombinant proteins UGT1A1, 1A3, 1A4, and 1A6−1A10
were provided by Dr. Moshe Finel (Drug Discovery and Development
Technology Center, Faculty of Pharmacy, University of Helsinki,
Helsinki, Finland). Recombinant proteins UGT2B4, 2B7, 2B15, and
2B17 were obtained from BD Biosciences (San Diego, CA).
Spectrophotometric grade dimethyl sulfoxide (>99.9% pure) was
purchased from Sigma-Aldrich (St. Louis, MO), and LC−MS grade
methanol and reagent grade acetic acid were from Fisher Scientific
(Pittsburgh, PA). Human hepatic and intestinal microsomes were
obtained as described previously.11,12 UDP-glucuronic acid (-GlcUA)
and all other chemicals and reagents, unless otherwise stated, were
purchased from Sigma-Aldrich.
Chemical Synthesis of (E)-2,4-Dimethoxy-6-(4-
methoxystyryl)benzaldehyde Oxime (NI-ST-05). (E)-1,3-Dime-
thoxy-5-(4-methoxystyryl)benzene (1) (1 mmol) was dissolved in 5
mL of dimethylformamide (DMF) and cooled to 0 °C, and POCl3
(1.3 mmol) was added dropwise over 15 min. The reaction mixture
was stirred for 30 min at ambient temperature. The purple-colored
solution was then added to 20 mL of ice-cold water and the resulting
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mixture extracted three times with 10 mL portions of dichloromethane
(DCM). The organic extracts were combined and washed with water,
dried over anhydrous Na2SO4, and concentrated on a rotary
evaporator to afford the desired crude product. (E)-2,4-Dimethoxy-
6-(4-methoxystyryl)benzaldehyde (2) was recrystallized in DCM from
the crude product in 65% yield. (E)-2,4-Dimethoxy-6-(4-
methoxystyryl)benzaldehyde (2) (1 mmol), hydroxylamine HCl (1.1
mmol), and sodium acetate (AcONa; 1.5 mmol) were dissolved in 10
mL of ethanol (EtOH), and the mixture was heated under reflux for 4
h. A white precipitate was formed, which was filtered off to afford NI-
ST-05 (3) in 80% yield (see Figure 1).
Recombinant proteins UGT1A1, 1A3, 1A4, and 1A6−1A10 were
cloned and expressed in baculovirus-infected insect cells, as described
previously.13,14 Human UGT2B4, 2B7, 2B15, and 2B17 were
purchased from BD Biosciences (Woburn, MA) and assayed according
to manufacturer protocols. Each enzyme tested in this study is known
to be active toward substrates specific for that enzyme.
Screening of Human Microsomes and Recombinant UGTs.
Screening experiments for glucuronidation activity were also
performed with human hepatic and intestinal microsomes from 10
and 13 donors, respectively, one pooled liver sample, and
commercially available hepatosomes (Human Biologics International).
A 250 μM aliquot of substrate was added to the incubation tube
dissolved in ethanol. This was allowed to air dry, and DMSO was
added to solubilize the substrate and to act as an activator for
membrane protein. The samples were sonicated to ensure substrate
solubilization. Reaction buffer and human microsomes (50 mg of total
protein) or recombinant UGT membranes (5 μg of total protein) were
then added. UDP-GlcUA in molar excess was added, and the samples
were incubated at 37 °C for 60 min. Final reaction concentrations
were as follows: 100 μM Tris−HCl (pH 7.4)/5 mM MgCl2/5 mM
saccharolactone/2% DMSO/250 μM substrate/2 mM UDPGA/50 or
5 μg of total protein, respectively. The total reaction volume was 30
μL. Reactions omitting substrates were run under the same conditions
as controls. No additional detergents or activators were used in the
incubations. The rate of glucuronidation with these enzymes has been
shown to be linear for a maximum of 3 h (data not shown). The
reactions were stopped by addition of 30 μL of ethanol. This was
followed by centrifugation of the samples at 14 000 rpm for 8 min to
collect the protein. The supernatants were separated by HPLC using
an HP1050 HPLC system equipped with a UV−vis diode array
detector. Instrument function and data acquisition were evaluated
using Agilent ChemStation software.
Enzyme Kinetics Assays. Kinetic parameters were established by
incubating recombinant UGT protein with varying concentrations of
NI-12a (10−1000 μM) or NI-ST-05 (1−1000 μM) with a molar
excess of UDP-GlcUA for 60 min. All kinetic assays were performed in
duplicate under conditions identical to those utilized for screening
experiments.
Data Analysis. Kinetic data for the glucuronidation of NI-12a and
NI-ST-05 by UGTs were estimated by plotting the measured initial
reaction velocity values as a function of substrate concentration.
Curve-fitting and statistical analyses were conducted utilizing Graph-
Pad Prism v4.0b (GraphPad Software, Inc., San Diego, CA). Kinetic
constants were obtained by fitting the experimental data to the
following kinetic models using the nonlinear regression (Curve Fit)
function
1
C18H19NO4. H NMR (DMSO-d6, ppm): δ 3.77 (s, 3H, −OCH3),
3.81 (s, 3H, −OCH3), 3.85 (s, 3H, −OCH3), 6.54 (s, 1H, −ArH), 6.89
(s, 1H, −ArH), 6.93−6.96 (d, 2H, −ArH, J = 8.4 Hz), 7.11−7.15 (d,
1H, −ArH, J = 16.4 Hz), 7.45−7.47 (d, 2H, −ArH, J = 8 Hz), 7.59−
7.63 (d, 1H, −ArH, J = 16.4 Hz), 8.30 (s, 1H, −ArH), 11.15 (s, 1H,
−OH). 13C NMR (DMSO-d6, ppm): δ 55.6, 55.8, 55.3, 98.0, 102.7,
110.0, 112.6, 114.6, 126.4, 128.3, 130.1, 130.5, 138.7, 145.2, 159.4,
159.7, 161.0.
Chemical Synthesis of (E)-4-(3,5-dimethoxystyryl)-2,6-dini-
trophenol (DNR-1). 1-(Bromomethyl)-3,5-dimethoxybenzene (4)
(1.0 mmol) and triphenylphosphine (1.5 mmol) were dissolved in
toluene and the mixture refluxed overnight to yield (3,5-dimethox-
ybenzyl) triphenylphosphonium bromide (5) as a white precipitate,
which was filtered off. The yield was 95%. In a separate reaction, 4-
hydroxybenzaldehyde (6) (2 g) was dissolved in 12.5 mL of
concentrated H2SO4 and cooled to 0 °C. A mixture of concentrated
H2SO4 (5 mL) and fuming HNO3 (5 mL) was cooled to 0 °C and
added slowly to the above reaction mixture. The resulting mixture was
stirred for 4 h at ambient temperature. The reaction mixture was then
slowly added to 50 mL of ice water and the resulting mixture stirred
for 1 h at 0 °C. At this point, a yellow precipitate was formed, which
was filtered off and purified through silica gel flash column
chromatography with 2:3 ethyl acetate/hexane as the mobile phase,
affording 4-hydroxy-3,5-dinitrobenzaldehyde (7) in 75% yield. (3,5-
Dimethoxybenzyl)triphenylphosphonium bromide (5) (1.0 mmol)
was dissolved in 5 mL of methanol, and 4-hydroxy-3,5-dinitrobenzal-
dehyde (7) (1.0 mmol) and sodium methoxide (NaOMe, 5.0 mmol)
were added. The resulting solution was stirred at room temperature for
5 h. The reaction was monitored by TLC, and after consumption of
starting materials, the reaction mixture was concentrated and extracted
into ethyl acetate followed by concentration on a rotary evaporator
and silica gel flash column chromatographic fractionation using
methanol/DCM as the mobile phase to afford DNR-1 (8) in 45%
yield (see Figure 1).
C16H14N2O7. 1H NMR (DMSO-d6, ppm): δ 3.76 (s, 6H, −OCH3),
6.33 (s, 1H, −ArH), 6.71 (s, 2H, −ArH), 6.83−6.88 (d, 1H, −ArH, J =
16.4 Hz), 7.12−7.16 (d, 1H, −ArH J = 16 Hz), 8.10 (s, 2H, −ArH)
13C NMR (DMSO-d6, ppm): δ 55.5, 99.6, 104.2, 110.0, 113.5, 124.2,
1. Michaelis−Menten (M−M) equation for the one-enzyme
model
Vmax × [S]
v =
128.1, 128.5, 140.7, 143.6, 159.7, 161.0.
Chemical Synthesis of (E)-3-(3-hydroxy-4-methoxyphenyl)-
2-(3,4,5-trimethoxyphenyl)acrylic acid (NI-12a). 3-Hydroxy-4-
methoxybenzaldehyde (9) (1.0 mmol), 2-(3,4,5-trimethoxyphenyl)-
acetic acid (10) (2.0 mmol) and triethylamine (3.2 mmol) were added
to 5 mL of acetic anhydride. The resulting reaction mixture was
refluxed at 140 °C for 4 h and monitored by TLC. When the reaction
was complete, 10 mL of ice water was added and extracted with 10 mL
of ethyl acetate. The organic phase was concentrated on a rotary
evaporator and the desired product purified by silica gel flash column
chromatography using methanol/DCM as mobile phase to afford NI-
12a (11) in 35% yield (see Figure 1).
Km + [S]
2. Hill equation, which describes sigmoidal autoactivation kinetics,
where S50 is the substrate concentration at 50% Vmax (analogous
to Km in M−M kinetics) and n is the Hill coefficient, which can
be considered to be a measure of autoactivation, and reflects
the extent of cooperativity among multiple binding sites15
Vmax × [S]n
v =
n
S50 + [S]n
C18H20O5. 1H NMR (CDCl3-d6, ppm): δ 3.69−3.72 (d, 12H,
−OCH3, J = 15.2 Hz), 6.43−6.44 (d, 2H, −ArH, J = 2.8 Hz), 6.54 (s,
1H, −ArH), 6.61 (s, 1H, −ArH), 6.80 (s, 1H, −ArH), 8.93 (s, 1H,
−ArH), 12.41 (s, 1H, −COOH). 13C NMR (DMSO-d6, ppm): δ 55.9,
56.4, 60.6, 107.2, 110.0, 112.0, 118.0, 123.3, 127.4, 131.0, 132.5, 137.4,
139.5, 146.2, 149.3, 153.5, 169.0.
Source of Human Microsomes and Recombinant UGTs.
Human hepatic microsomes were obtained from 10 donors, and
human intestinal microsomes were obtained from 13 donors.
3. Uncompetitive substrate inhibition (USI) model, where Ki is
the inhibition constant describing the reduction in rate
Vmax
v =
Km
[S]
Ki
1 +
+
[S]
The fit of the data for each model was assessed from the standard
error, 95% confidence intervals, and R2 values. Kinetic curves were also
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Figure 2. Metabolism of NI-12a (A) and NI-ST-05 (B) with human liver microsomes. Representative HPLC analyses are shown from 60 min
incubations of 50 μg of human liver proteins with 0.25 mM substrate and 3 mM UDP-GlcUA. DNR-1 was not metabolized under the studied
conditions.
Figure 3. Extracted ion chromatogram (m/z 361) and (+)-ESI mass spectra of NI-12a and its glucuronide conjugate NH4+ adducts. Spectra of NI-
12a glucuronides (two isomers of tR = 16.0 and 16.3 min) showed an [M + NH4]+ peak (m/z 554) plus a fragment ion resulting from neutral loss of
the glucuronide (m/z 361).
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Figure 4. Mass spectra and structures of NI-ST-05 and its glucuronide conjugates. Spectrum of NI-ST-05 glucuronide (tR = 21.4 min) showed an [M
+ H]+ peak (m/z 490) plus a major peak corresponding to the NI-ST-05 substrate (m/z 314).
Figure 5. Glucuronidation of NI-12a (A) and NI-ST-05 (B) by human recombinant UGTs. UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4,
2B7, 2B15, 2B17 (5 μg of protein) were evaluated for their ability to glucuronidate DNR-1, NI-12a, and NI-ST-05 (250 μm). No activity was
observed toward DNR-1, and UGT2B7, 2B4, 2B15, and 2B17 were not active toward any compound. Activities are expressed in nanomoles per
milligram of protein per minute.
analyzed as Eadie−Hofstee plots to support kinetic models (graphs
not shown). Kinetic constants are reported as the mean standard
error.
Glucuronidation by human hepatic and intestinal microsomes is
presented as the line of median value and the error bars represent the
range of values. Statistical comparisons were performed using the
Kruskal−Wallis nonparametric analysis of variance followed by Dunn’s
multiple comparison test.
Glucuronide Product Analysis. HPLC−UV analysis of the
supernatants was carried out on an HP1050 LC system equipped with
a variable wavelength UV−DAD detector. Instrument control and data
collection was accomplished using Agilent ChemStation software. The
samples were separated on a reversed-phase 5 μm Suplex pKb-100
analytical column (0.46 cm × 25 cm, C18) (Supelco, USA)
maintained at 25 °C. HPLC analyses were carried out at a flow rate
of 1 mL/min with the following elution system: a linear gradient from
15 to 80% methanol in ammonium formate buffer (0.05 M, pH 3.4)
for 25 min followed by a linear gradient from 80 to 100% methanol in
ammonium formate for 3 min. The column was then re-equilibrated to
initial conditions for 10 min between runs. The elution of each
metabolite of NI-12a was monitored at 335 nm, whereas the elution of
the metabolite of NI-ST-05 was monitored at 325 nm.
Analysis of Glucuronide Product Structures by ESI−HPLC−
MS. HPLC−MS analysis of the glucuronide products were conducted
using an Acquity uHPLC system interfaced to a Quattro Premier triple
quadrupole mass analyzer (Waters Corporation, Beverly, MA) with an
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electrospray probe operating in the positive ion mode. LC conditions
were identical to those used for LC−UV analysis with 10 μL of sample
injected onto the column. The mass spectrometer was calibrated using
sodium cesium iodide (NaCsI) over a range of 20−1974 Da. The
electrospray capillary voltage was 3.5 kV, and the cone voltage was 25
V. The desolvation gas (nitrogen) flow rate was 600 L/h and was
maintained at 500 °C. The source temperature was 150 °C.
screened produced any measurable metabolite under our
conditions (data not shown). UGT1A9 (a hepatic enzyme)
had the highest activity toward NI-12a, that is, close to 10
nmol/mg/min.
With NI-ST-05, UGT1A1, 1A9, and 1A10 catalyzed the
formation of the CN−O-glucuronide with the extrahepatic
enzyme, with UGT1A10 having the highest activity. All the
remaining UGTs screened were inactive toward this com-
pound.
Glucuronidation of NI-12a and NI-ST-05 by Human
Hepatic and Intestinal Microsomes. Screening experiments
for glucuronidation activity were also carried out with human
hepatic and intestinal microsomes from 10 and 13 donors,
respectively, one pooled liver sample, and commercially
available hepatosomes (Figure 6). All hepatic samples were
shown to glucuronidate both NI-12a and NI-ST-05 producing
two and one metabolic products, respectively. In assays with
intestinal samples, donors HI27, HI28, HI29, and HI54 failed
to display glucuronidation activity toward NI-12a. Additionally,
RESULTS
■
HPLC−UV−vis and HPLC−MS/MS Analysis of Glucur-
onide Metabolites. Preliminary studies on the glucuronida-
tion of NI-ST-05, DNR-1, and NI-12a were aimed at evaluating
their glucuronidation by UGT1A and UGT2B enzymes. HPLC
analysis of the incubation mixtures demonstrated the formation
of glucuronidation products for NI-ST-05 and NI-12a but not
for DNR-1. Representative HPLC analyses indicates the
formation of two metabolites at tR = 17.6 and tR = 17.8 min
for NI-12a, and one product at tR 23.2 min for NI-ST-05
(Figure 2). Retention time values of these metabolites are lower
than those of their respective parent compounds.
LC−(+)-ESI−MS analysis, presented in Figure 3, indicated
that the two NI-12a glucuronides (tR = 16.0 and 16.3 min) had
an [M + NH4]+ peak m/z 554, and a major peak at tR =18.9
min, (m/z 361, 100%) corresponded to NI-12a. The two
slightly different in-source fragmentation patterns in the spectra
of the NI-12a metabolites indicated the presence of two types
of glucuronides. We suggest that these are COO- and O-
glucuronides. In the studies presented below, β-glucuronidase,
which selectively hydrolyzes only O-glucuronides, was used to
identify which of the two possible glucuronide metabolites was
present. The major fragment observed for both glucuronides
resulted from neutral loss of the glucuronide moiety. The
higher intensity of the m/z 361 peak relative to the m/z 554 is
evidence that the chromatographic peak at tR = 16.0 min is an
acyl glucuronide. The acyl glucuronide should be a better
leaving group than the corresponding O-glucuronide.
Ammonium adducts of NI-ST-05 glucuronides were not
observed in MS/MS spectra, but the proton adduct ([M + H]+)
was observed with a retention time of 23.2 min and an m/z 490
(Figure 4). The parent compound (NI-ST-05) eluted at 25.5
min and had a base peak at m/z 314 [M + H]+. NI-ST-05 has
only one available group for glucuronidation, allowing
unequivocal determination that the product was the CN−
O-glucuronide. In-source fragmentation was not observed for
these compounds. Retention times for LC−MS runs (Figures 3
and 4) were slightly different from LC−UV runs, despite
analyses being run using the same chromatographic column and
mobile phase. The small differences can be attributed to
different LC systems with unique system dead volumes.
Screening of Recombinant UGTs for Activity toward
NI-12a and NI-ST-05. Eight human recombinant UGT1A
enzymes expressed as His-tag proteins in baculovirus-infected
Sf9 insect cells, and UGT2B4, 2B7, 2B15, and 2B17 from BD
Biosciences were evaluated for their ability to glucuronidate NI-
12a and NI-ST-05. Activities at a substrate concentration of 250
μM were determined using HPLC−UV/vis analysis (Figure 5).
Beta-glucuronidase hydrolysis was used to differentiate between
COO-glucuronides and O-glucuronides.
Figure 6. Glucuronidation activities of human hepatic (A) and human
intestinal (B) microsomes toward NI-12a and NI-ST-05. Human liver
microsomes from 10 different donors, a pooled liver sample, and
purchased “hepatosomes” as well as and human intestinal microsomes
from 13 different donors were analyzed. Each substrate concentration
was 0.25 mM, with a molar excess of UDP-GlcUA (2 mM); the
reactions were incubated for 60 min. Activities are expressed in
nanomoles per milligram of protein per minute.
Data for NI-12a indicated that UGT1A3 catalysis led to both
COO- and O-glucuronides. UGT1A1 produced only the
carboxyl metabolite, whereas UGTs 1A7−1A10 produced
only the hydroxyl metabolite. UGT1A4 and 1A6 were not
active toward this compound. None of the UGT2B enzymes
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a
Table 1. Glucuronidation Kinetics for NI-12a and NI-ST-05 Metabolites
UGT1A1
UGT1A7
UGT1A8
UGT1A9
UGT1A10
NI-12a
NI-12a-O-gluc
not produced
Km (μM)
62 43
240 76
6.0 1.3
950 449
25
240 40
500 75
Vmax (nmol/mg/min)
Ks (μM)
2.2 0.77
242 133
35
8.3 0.52
3.5 0.24
CLint or CLmax (μL/mg/min)
kinetic model
R2
35
7
USI
USI
M−M
0.97
M−M
0.98
0.82
0.98
NI-12a-COO-gluc
too low to be assessed
not produced
not produced
not produced
not produced
NI-ST-05
NI-ST-05−N−O-gluc
Km or S50 (μM)
not produced
not produced
b
b
<5
<5
6.2 0.35
0.65 0.017
3.4
b
Vmax (nmol/mg/min)
0.21 0.02
undetermined
n
CLint or CLmax (μL/mg/min)
105
b
kinetic model
R2
M−M
0.61
undetermined
Hill
b
0.87
a
Glucuronidation activities of selected recombinant UGTs were measured by incubating membrane fractions with increasing concentrations of
substrate (see Figures 7 and 8) at a constant concentration of UDP-GlcUA (3 mM). Reactions were centrifuged, supernatants separated by HPLC,
b
and curve fits and kinetic constants determined using GraphPad Prism 4 software.Values estimated based on an incomplete data set. Values
estimated based on an incomplete data set.
Figure 7. Steady state kinetic curves for the glucuronidation of NI-12a by selected human recombinant UGTs. Glucuronidation activities for wild-
type UGT1A7, 1A8, 1A9, and 1A10 were measured by incubating membrane fractions containing recombinant UGTs with increasing concentrations
(shown in figure) of the substrates at a constant concentration of UDP-GlcUA (3 mM) for 60 min at 37 °C. Curve fits and kinetic constants were
determined using GraphPad Prism 4 software. The graphical fits of the data (mean SD) are shown.
donors HI34, HI36, HI40, and HI41 produced only one (the
hydroxyl) metabolite, whereas the remaining donors with
activity produced two (hydroxyl and carboxyl) metabolites. For
NI-ST-05, donors HI27, HI29, and HI30 failed to display
glucuronidation activity, whereas all other donors produced one
metabolite. In general, liver microsomes had significantly higher
O-glucuronidation activity for NI-12a than for COO-glucur-
onidation of this compound or for CN−O-glucuronidation
of NI-ST-05. In contrast, the glucuronidation activity of
intestinal microsomes did not significantly differ with the
type of metabolites.
Kinetic Analysis of Selected Recombinant UGTs with
NI-12a and NI-ST-05. On the basis of the activity screening
data, selected human recombinant UGT enzymes (UGT1A7−
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Chemical Research in Toxicology
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Figure 8. Steady state kinetic curves for the glucuronidation of NI-ST-05 by selected human recombinant UGTs. Glucuronidation activities for wild-
type UGT1A1, 1A9, and 1A10 were measured by incubating membrane fractions containing recombinant UGTs with increasing concentrations
(shown in figure) of the substrates at a constant concentration of UDP-GlcUA (3 mM) for 60 min at 37 °C. Curve fits and kinetic constants were
determined using GraphPad Prism 4 software. The graphical fits of the data (mean SD) are shown.
1A10 for NI-12a and UGT1A1, 1A9, and 1A10 for NI-ST-05)
were subjected to steady state kinetic analysis (Table 1 and
Figures 7 and 8). For NI-12a, UGT1A9 was the most active
recombinant UGT (Vmax = 8.3 nm/mg protein/min) and had a
relatively low affinity, with a Km value of 240 μM. UGT1A7
demonstrated the highest affinity for NI-12a (Km = 62 μM),
and UGT1A10 had the lowest affinity for NI-12a (Km = 500
μM). The catalytic efficiency of UGT1A9 toward NI-12a,
measured by the ratio Vmax/Km (35 μL/min/mg) was also the
highest, likely because of the high Vmax. Both, UGT1A9 and
UGT1A10 fit Michaelis−Menten kinetics. Data generated for
UGT1A7 and 1A8 both fit the uncompetitive substrate
inhibition kinetic model. This model suggests the existence of
multiple binding sites in these enzymes.
The CN−O-glucuronidation of NI-ST-05 resulted in very
little product formation and therefore our ability to perform
kinetic analyses were limited by the sensitivity of our HPLC−
UV/vis method. Each of the active enzymes exhibited high
affinity and relatively low activity for this compound. Because of
this, the data generated with UGT1A1 cannot be used to
estimate Km values because of a lack of measurable product
formation at substrate concentrations below 5 μM. Thus, we
have indicated that the Km value for this reaction is <5 μM.
UGT1A1 activities at concentrations above 5 μM were used to
estimate a Vmax value of 0.2 nmol/mg/min assuming M−M
kinetics. UGT1A10 fit the Hill equation, indicating cooperative
binding. This reaction produced very low S50 and Vmax values of
6.2 μM and 0.65 nm/mg protein/min, respectively. The
catalytic efficiency of this reaction (Vmax/Km = 105 μL/min/
mg) was 3 to 10 times higher than that of NI-12a. Data
generated with UGT1A9 did not fit any of the kinetic models
tested. This assay showed the highest level of activity at the 5
μM concentrations, indicating a high affinity for this enzyme-
compound set, and this activity decreased in a concentration-
dependent manner up to 15 μM when the velocity plateaued
and remained constant up to 75 μM (the highest concentration
assayed).
It is recognized that resveratrols induce a large number of
different downstream effects in vitro and in vivo. However, the
exact mechanism of their action has yet to be revealed.
Moreover, it is documented that the presence of the 3 hydroxyl
groups in its structure may actually decreases the cytotoxicity of
these compounds because they are targets for rapid metabolism
leading to poor bioavailability. In fact, it has been shown that
when these groups are substituted with methoxy groups the
cytotoxic activity of these compounds is much improved.19−21
This phenomenon was the inspiration behind the design of our
compounds.
In designing the structures of the new compounds studied
here, it was essential to decrease the number of free hydroxyl
groups and to modulate the electron density both in the
aromatic rings and in the molecule as a whole. Thus, three new
stilbenoid derivatives were synthesized by replacing hydroxyl
groups with methoxyl groups and by adding N-hydroxyl,
aromatic nitro, and carboxyl substituents to create NI-ST-05,
DNR-1, and NI-12a, respectively. Such chemical structures are
assumed to be less susceptible to glucuronidation, thus
improving their bioavailability, while hopefully preserving
their biological properties. In previous studies carried out in
our laboratory, we have used the same recombinant and
microsomal enzyme preparations to determine tRes glucur-
onidation rates,9 allowing us to directly compare the effects of
these structural changes on the glucuronidation of these
compounds.
We first demonstrated that only two of the synthesized
stilbenoids, NI-ST-05 and NI-12a, were substrates for
recombinant UGT enzymes and human hepatic and intestinal
microsomes. The absence of DNR-1 glucuronidation is not
surprising. Although a phenolic hydroxyl substituent remains in
the structure of DNR-1, this lack of DNR-1 glucuronidation
may result from the presence of two nitro groups at ortho
positions to this phenolic hydroxyl group, which changes its
acid−base and nucleophilic properties. Therefore, the nucleo-
philic attack of the charged oxygen atom on C1 of glucuronic
acid in the presence of the two negatively charged phosphatic
groups of UDP would be limited. This, combined with the large
steric hindrance of the two polar nitro groups limits the
glucuronidation potential of DNR-1.
DISCUSSION
■
The aim of the current study was to test the hypothesis that the
limited bioavailability of tRes, resulting from its rapid UGT-
mediated metabolism, can be improved by modifying its
structure to create analogs which would be glucuronidated at a
lower rate than tRes itself. tRes has been the focus of great
attention because it is a very efficacious antioxidant16 and anti-
inflammatory17 agent with potential therapeutic applications.18
The structures of the NI-12a and NI-ST-05 glucuronides
formed were determined by HPLC−MS/MS analysis. MS
spectra of the molecular proton adduct were not observed in
the case of NI-12a, but the ammonium adduct was detected.
These data suggest that the ammonium ion, which is present in
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Chemical Research in Toxicology
Article
the mobile phase, chelates with the ortho methoxy/hydroxy
pair to form the ammonium adduct observed in the MS
spectrum of the NI-12a glucuronide. This proposed interaction
is supported by the absence of an ammonium adduct in the MS
spectrum of NI-ST-05, which does not contain the ortho
substituted methoxy/hydroxy moiety.
developed as therapeutics. These preliminary enzymatic studies
have provided useful information for a better understanding of
the involvement of conjugative metabolism in the biotransfor-
mation of synthetic stilbenoids in humans and for assessing the
potential utility of these molecules as novel chemotherapeutic
agents.
Glucuronidation assays with NI-12a resulted in multiple
products and atypical reaction kinetics specific for each enzyme
(Table 1), which point to a complex mechanism of metabolism
for this compound. The hepatic enzyme UGT1A9 had the
highest O-glucuronidation activity toward NI-12a, whereas
UGT1A7, 1A8, and 1A10 transformation rates were lower. It is
important to note that in our previous work,9 the enzyme that
was determined to have the highest rate of tRes-3-O-
glucuronidation (the primary metabolite in humans) was
UGT1A1 (Vmax = 31 nmol/mg/min) and that this enzyme
has very little activity toward this compound (too low to
reliably determine kinetic parameters using our system). Steady
state kinetic analyses demonstrated that UGT1A9 catalyzed the
formation of the NI-12a−O-glucuronide at a slightly higher rate
than the tRes-3-O-glucuronide (Vmax = 8.3 and 2.5 nmol/mg/
min, respectively); however, the affinity of this enzymes for NI-
12a (Km = 240 μM) is significantly less than for tRes (Km = 2.5
μM) or for the naturally occurring tRes analogs tA1 (Km = 190
μM) and tA3 (Km = 21 μM).9 Taken together, these
parameters indicate that the modified stilbene structure of
NI-12a most likely will have improved bioavailability compared
to that of tRes and to those of the naturally occurring
prenylated stilbenoids.
Glucuronidation assays with NI-ST-05 resulted in the
formation of a rare CN−O-glucuronide, and the rate of
this reaction was significantly lower than the formation of either
of the tRes or tA glucuronides.9 NI-ST-05 is characterized by
CN−OH group, which may be responsible for the high
affinity of this compound for each of the UGTs assayed, and
the as yet unidentified kinetic mechanism observed with
UGT1A9. NI-ST-05 shows promise for further development as
a stilbenoid with low glucuronidation potential; however, the
resulting atypical reaction kinetics indicate a complex
mechanism for the metabolic pathway for this compound,
which is specific for selected UGT enzymes.
In conclusion, the glucuronidation of the novel stilbenoids
studied in this report revealed that these compounds
demonstrate improved glucuronidation profiles as compared
to tRes. The selected structural modifications have resulted in
them acting as poorer substrates for recombinant and
microsomal UGT enzymes. Thus, these tRes analogs can be
predicted to have better bioavailability in vivo. In addition to
glucuronidation, tRes is also extensively sulfated in vitro and in
vivo.5−7 It is anticipated that we can extrapolate the conclusions
from this work from UGTs to sulfotransferases. It is also
possible that these structural changes could affect the
metabolism of these compounds by other enzymes such as
cytochromes P450s. These additional metabolic pathway
should be the topic of future studies.
These resveratrol analogs have also been evaluated as
anticancer agents against human cancer cell lines MCF-7
(breast), A549 (lung), KB-3-1 (epidermoid carcinoma), and
IGROV (ovarian).22−24 Compound NI-12A was the most
potent analog and was found to be an effective antitubilin
agent.22 On the basis of these data, the synthesized stilbenoids
likely represent a useful scaffold for the design of highly
selective and efficacious analogs which potentially could be
AUTHOR INFORMATION
Corresponding Author
*A. Radominska-Pandya. Phone: (501) 686-5414. Fax: (501)
603-1146. E-mail: radominskaanna@uams.edu. University of
Arkansas for Medical Sciences, 4301 W. Markham, Slot 516,
Little Rock, Arkansas 72205, United States.
■
Funding
This work was funded by a National Institutes of Health Grant
[GM075893] and a Department of Defense grant [X81XWH-
11-1-0795] funded by USAMRMC to A.R.-P., as well as an
Arkansas Research Alliance grant to P.A.C.
Notes
The authors declare no competing financial interest.
Both laboratories at the University of Arkansas for Medicinal
Sciences contributed equally to this work.
ACKNOWLEDGMENTS
■
The authors would like to acknowledge Dr. Moshe Finel for his
generously provided recombinant UGT proteins.
ABBREVIATIONS
■
UGTs, uridine diphosphate glucuronosyltransferases; NI-12a,
( E ) - 3 - ( 3 - h y d r o x y - 4 - m e t h o x y p h e n y l ) - 2 - ( 3 , 4 , 5 -
trimethoxyphenyl)acrylic acid; NI-ST-05, (E)-2,4-dimethoxy-6-
(4-methoxystyryl)benzaldehyde oxime; DNR-1, (E)-4-(3,5-
dimethoxystyryl)-2,6-dinitrophenol
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