Post on 26-Aug-2020
DMD #46862
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ATP Serves as an Endogenous Inhibitor of
UDP-Glucuronosyltransferase (UGT): A New Insight into
the ‘Latency’ of UGT
Yuji Ishii, Kie An, Yoshio Nishimura, and Hideyuki Yamada
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka,
Japan
DMD Fast Forward. Published on July 30, 2012 as doi:10.1124/dmd.112.046862
Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: Novel Mechanism of UGT ‘latency’
Correspondence author:
Yuji Ishii, Ph.D.
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka 812-8582, Japan,
Phone: +81-92-642-6586
Fax:+81-92-642-6588
E-mail: ishii@phar.kyushu-u.ac.jp
Number of text pages is 45.
Number of tables is 6.
Number of figures is 6.
Number of references is 45.
Number of words in abstract is 250 (words).
Number of words in introduction is 750 (words)
Number of words in discussion is 1311 (words)
Number of supplemental materials is 3.
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Abbreviations: UGT, UDP-glucuronosyltransferase; ER, endoplasmic reticulum;
RLM, rat liver microsomes; HLM, human liver microsomes; GRP,
glucose-regulated protein; 4-MU, 4-metylumbelliferone; 4-MUG,
4-Methylumbelliferyl-β-D-glucuronide; Brij-58, polyoxyethylene cetyl alcohol
ether; PDI, protein disulfide isomerase; EGFR, epidermal growth factor receptor
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Abstract:
We have suggested that adenine-related compounds are allosteric inhibitors of
UGT in rat liver microsomes (RLM) treated with detergent. To clarify whether the
same occurs with a pore-forming peptide, alamethicin, the effects of
adenine-related compounds on 4-metylumbelliferone (4-MU) glucuronidation
were examined using RLM and human liver microsomes (HLM). ATP inhibited
4-MU glucuronidation when Brij-58-treated RLM were used (IC50= around 70
µM). However, alamethicin-treated RLM exhibited a lower susceptibility (IC50=
around 460 µM) than Brij-58-treated RLM. A similar phenomenon was observed
when pooled HLM were used. Then, the endogenous ATP content of RLM was
determined in the presence and absence of alamethicin or detergent. While no
ATP remained in the microsomal pellets after Brij-58-treatment, more than half
the microsomal ATP remained even after treatment with alamethicin. Further, the
Vmax in the absence of an adenine-related compound was approximately three
times higher in Brij-58-treated than in alamethicin-treated RLM. The difference in
the inhibitory potency observed would be due to the difference in remaining
endogenous ATP and the accessibility of exogenous ATP to the luminal side of
the endoplasmic reticulum (ER) where the active site of UGT is located. Gefitinib,
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a protein tyrosine kinase inhibitor, markedly inhibited human UGT1A9 activity.
Interestingly, AMP antagonized Gefitinib-provoked inhibition of UGT1A9 and ATP
exhibited an additive inhibitory effect at a lower concentration. Therefore,
Gefitinib inhibits UGT1A9 at the common ATP binding site shared with ATP and
AMP. Releasing adenine nucleotide from the ER is suggested to be one of the
mechanisms to explain the ‘latency’ of UGT.
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Introduction:
Glucuronidation is one of the most important steps in drug metabolism.
Many endo- and xeno-biotics and/or their metabolites are converted to
water-soluble glucuronides (Dutton, 1980; Bock et al, 1987; Ritter, 2000). This
step is catalyzed by UDP-glucuronosyltransferase (UGT) which is expressed
mainly in the liver and gastro-intestinal tract (Tukey and Strassburg, 2000). UGT
is a family of enzymes which transfer the glucuronic acid moiety of the
co-substrate, UDP-glucuronic acid (UDP-GlcUA), to a substrate (Mackenzie et al,
2005). This enzyme is localized in the endoplasmic reticulum (ER) and
experimental evidence has suggested that the most important domain of UGT is
located within the ER (Shepherd et al., 1989; Radominska-Pandya et al, 2005).
A membrane-spanning domain of UGT is located near the carboxy-terminus
which extrudes cytosolic tail consisting of about 20 amino acid residues (Meech
and Mackenzie, 1997). In agreement with the topology of UGT in the ER
membrane, it has been suggested that a UDP-GlcUA transporter is expressed
on the ER membrane, and this protein translocates UDP-GlcUA from the cytosol
to the lumen of the ER (Hauser et al., 1988). Muraoka et al. (2007) have cloned
a UDP-GlcUA transporter, UDP-galactose transporter-related protein 7
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(UGTrel7; solute carrier family 35, SLC35D1), which is involved in the above
process.
It has long been known that UGT activity is increased following treatment
of microsomes with detergent (Winsnes, 1969), and this phenomenon is referred
to as the ‘latency’ of UGT. The same is also observed when cells were treated
with a pore-forming peptide, alamethicin (Bánhegyi et al, 1983). The reason why
this latency is observed is thought to be due to the presence of a membrane (ER
membrane) obstructing the free access of substrates to the active site of UGT. It
is generally accepted that the latency of UGT is due to the limited transport of
UDP-GlcUA from cytosol to the lumen of ER, the process of which is mediated
by a UDP-GlcUA transporter(s)(Muraoka et al. 2007). This explanation sounds
reasonable. If this is true, the kinetic Michaellis constant (Km) should be lower
under detergent-treating conditions than under conditions not involving
detergent treatment. However, the Km of the substrate (aglycone) is not often
reduced even after detergent treatment (Puig and Tephly, 1986). It is, therefore,
unlikely that the removal of the membrane obstruction is the sole reason for the
latency of UGT.
We have reported previously that adenine nucleotides, such as ATP,
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NADP+ and NAD+, are endogenous inhibitors of UGT (Nishimura et al., 2007).
These adenine nucleotides inhibit UGT in a non-competitive manner, and they
exhibited the effect only when microsomes are disrupted by detergent. On the
basis of a structure-inhibitory effect relationship, while NADPH and NADH
exhibit no or only a minor inhibitory effect on UGT (IC50>1 mM), their oxidized
forms, NADP+ and NAD+, are potent UGT inhibitors. There are
NAD(P)+-dependent dehydrogenases, such as 11β-hydroxysteroid
dehydrogenase and hexose-6-phosphate dehydrogenase, in the lumen of the
ER (Hewitt et al, 2005). Furthermore, molecular chaperones, such as
glucose-regulated protein 78 (GRP78) and GRP94, are expressed within the ER,
and they bind to ATP and hydrolyze it (Dierks et al., 1996; Rosser et al., 2004).
Therefore, significant amounts of ATP, NAD+ and NADP+ should be present in
the lumen of the ER, and they are needed for the oxidoreductase and chaperone
functions. If adenine nucleotides work as endogenous inhibitors of UGT, their
concentration in the ER is considered to be a determinant regulating the action
of certain hormones which are UGT substrates. However, the actual
concentration of adenine nucleotides within the ER has not been accurately
determined.
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Previously, we observed the nucleotide-dependent inhibition of UGT
using detergent-treated microsomes. To clarify whether the same occurs with a
pore-forming peptide, alamethicin, the effects of adenine-related compounds on
4-metylumbelliferone (4-MU) glucuronidation were examined using liver
microsomes from rats (RLM) and humans (HLM). In addition, we measured the
ATP content in the ER with and without alamethicin/detergent treatment.
Through these experiments, we investigated the hypothesis that a change in the
concentration of adenine nucleotides within the ER is the reason for the latency
of UGT. On the other hand, agents such as Gefitinib inhibiting epidermal growth
factor receptor (EGFR)-protein tyrosine kinase are UGT inhibitors (Liu et al.,
2010; Fujita et al., 2011; Piu et al., 2011). However, the reason why Gefinitib and
related compounds exhibit an inhibitory effect on UGT has not been elucidated.
Since their pharmacological target site is the ATP binding domain of the protein
tyrosine kinase, we also examined whether Gefitinib inhibits UGT through the
ATP binding site on UGT.
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Materials and Methods
Materials. ATP, NADH, NADPH, 4-MU, D-saccharic acid-1,4-lactone, and
L-α-phosphatidylcholine (egg yolk, type XI-E) were purchased from
Sigma-Aldrich (St. Louis, MO). NAD+, NADP+, and UDP-GlcUA trisodium salt
were obtained from Wako Pure Chemicals, Co. Ltd. (Osaka, Japan).
4-Methylumbelliferyl-β-D-glucuronide (4-MUG) and polyoxyethylene cetyl
alcohol ether (Brij-58) were purchased from Nakalai Tesque (Kyoto, Japan).
ENLITEN® rLuciferase/Luciferin reagent and 5xPassive lysis buffer were
purchased from Promega (Madison, WI). Gefitinib (Iressa®) was purchased
from Tocris Bioscience (Bristol, UK). All other reagents were of the highest grade
commercially available.
Animals and treatment. Animal experiments in this study were conducted
following the approval of the Ethics Committee for Animal Experiments of
Kyushu University. Male Sprague-Dawley rats (5 weeks-old) were purchased
from Kyudo (Kumamoto, Japan) and they were maintained for one week with
free access to water and a standard diet under a 7 am to 7 pm light/dark cycle.
The liver was removed and perfused with ice-cold saline, and then the
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microsomes were prepared by differential centrifugation as described previously
(Oguri et al., 1996). The microsomes were thenre-suspended in 0.25 M sucrose.
Protein assay. The protein concentration was determined by the method of
Lowry et al. (1951) using bovine serum albumin as a standard.
Determination of UGT activity. The activity of 4-MU glucuronidation was
determined by the published methods (Hanioka et al., 2001) with slight
modifications (Nishimura et al., 2007). Human UGT1A9 SupersomeTM and
human liver microsomes (pooled microsomes from 150 donors) were purchased
from BDGentest (Woburn, MA).
Determination of ATP by luciferin and luciferase. Rat liver microsomes
were diluted to give a protein concentration of 5 mg/mL and then treated with
Brij-58 (0.25 mg/mg protein) or alamethicin (0.05 mg/mg protein). The mixture
was kept on ice for 1 h, then centrifuged at 105,000 xg (4˚C) for 60 min, and the
supernatant and pellets were separated. Both fractions were mixed/diluted with
20-volume relative to the original microsome amount (protein base) of 1xPassive
Lysis Buffer (Promega, Madison, WI). The mixture was kept at room temperature
for 30 min then a 5 μL aliquot of the diluted sample was mixed with 50 μL
Luciferase/Luciferin reagent. The emission of ATP-dependent
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chemiluminescence was measured in a Turner Biosystems Luminometer (Model
TD-20/20)(Promega, Madison, WI). The detection limit was 0.1 nmol ATP/5 μL
sample.
Data analyses. Statistical significance, kinetic paramerers and IC50 were
calculated using the computer software, GraphPadPrism5 (GraphPad, La Jolla,
CA).
Prediction of the three-dimentional structure and ligand binding sites of
UGT1A9. The amino acid sequence of UGT1A9 (GenBank: AAG30418.1) was
subjected to a modeling system on the Web linking to a server installed with
Phyre2 (Protein Homology/analogY Recognition Engine V 2.0)
(http://www.sbg.bio.ic.ac.uk/phyre2/html/; Kelly and Sternberg, 2009). The
following six proteins were selected as the templates based on the Structural
Classification of Proteins and ASTRAL release 1.75A (March 2012): flavonoid
3-O-glucosyltransferases (d2c1xa1, Offen et al, 2006; and c3hbjA, Modolo et al.,
2009), hydroquinone glucosyltransferase (d2vcha1, Brazier-Hicks et al, 2007),
(iso)flavonoid glycosyltransferase (d2pq6a1, Li et al, 2007),
vancosaminyltransferase (Gtf glycosyltransferase) (d1rrva, Mulichak et al, 2004),
and triterpene/flavonoid glycosyltransferase (d2acva1, Shao et al, 2005). This
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selection was also based on a heuristics to maximize the confidence,
percentage identity and alignment coverage. In the modeling, the
three-dimentional structure of about 87% of whole UGT1A9 body could be
constructed by overlapping with templates. The confidence of this modeling was
expected to be over 90%. Because the similar way could not be applicable to the
structural simulation of the other region involving the highly variable C-terminal
domain, the structure of this region was tentatively predicted by ab initio
calculation. The construct obtained was then submitted to a 3DLigandSite server
(http://www.sbg.bio.ic.ac.uk/~3dligandsite/; Wass et al, 2010) for the estimation
of ligand binding site.
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Results
inhibitory effect of adenine-related compounds on glucuronidation by
Brij-58- and alamethicin-treated RLM. The possibility whether
adenine-related compounds inhibit UGT activity both in RLM treated with a
pore-forming peptide, alamethicin, as well as a detergent, Brij-58, was examined
using 4-MU as a substrate. To clarify the specificity of nucleotides and related
substances, the inhibitory potential of 12 substances was assessed. Table 1
shows the IC50 values of the compounds examined. Of the substances tested,
ATP exhibited the strongest inhibitory effect in both Brij-58 and
alamethicin-treated RLM. However, the IC50 was 7-times higher in
alamethicin-treated RLM than in a Brij-50-treated preparation: the IC50 of ATP
was approximately 70 and 460 µM in Brij-58- and alamethicin-treated RLM,
respectively. Although ADP also exhibited an inhibitory effect in both
preparations, its IC50 was 11- and 3-times higher than ATP in Brij-58- and
alamethicin-treated microsomes. However, the inhibitory effect of NADP+ was
stronger than ADP, and NAD+ exhibited a stronger inhibitory effect than ADP only
in Brij-58-treated RLM. CTP had an inhibitory effect close to that of ADP in
Brij-58-treated RLM but not in alamethicin-treated RLM and no inhibitory effect
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was observed for adenosine, NADH, NADPH, GTP and UTP in RLM treated with
Brij-58 and alamethicin. Although the relative IC50 (alamethicin/Brij-58) varied
from 1.6 with ADP to 6.9 with ATP, alamethicin-treated microsomes were always
less sensitive to nucleotides than Brij-58-treated preparations. Of the nucleotide
triphosphates tested, only CTP had an inhibitory effect close to that of ADP in
Brij-58-treated RLM. The inhibitory potential of adenine-nucleotides also
depends on the number of phosphate groups [ATP >> ADP > AMP (= almost no
effect)]. Thus, the number of phosphates at the 5’-position of ribose is thought to
be one of the determinants of the inhibitory effect of the adenine nucleotides.
Finally, neither NADH nor NADPH exhibited any inhibitory effect on UGT activity,
while NAD+ and NADP+ did have an inhibitory effect. Therefore, the oxidized
status of the nicotinamide moiety seems to be another factor affecting the
inhibitory effect.
Previously, we have confirmed that the mode of inhibition of 4-MU
glucuronidation catalyzed by Brij-58-treated RLM by adenine-related compounds
was non-competitive. In this study, we examined whether the same is also true
for the glucuronidation catalyzed by alamethicin-treated RLM. For this purpose,
a kinetic experiment was performed by varying the 4-MU concentration (Fig. 1).
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Even addition of ATP, or NADP+ to the reaction mixture did not change the
Michaelis-Menten kinetics, and increased the concentrations of nucleotides
producing more pronounced inhibition (Fig. 1A-D). These plots strongly
suggest that 4-MU UGT in both Brij-58- and alamethicin-treated RLM is inhibited
by ATP and NADP+ in a non-competitive manner. This assumption was
supported by re-evaluation of the Eadie-Hofstee plots (Fig. 1E-H); that is,
regression lines with different concentrations of inhibitor run parallel to each
other. Taken together, these results suggest that ATP and NADP+ reduce 4-MU
UGT function in both Brij-58- and alamethicin-treated RLM by the same
mechanism and by interacting with the enzyme(s) at a site distinct from
substrate-binding sites. In agreement with the data shown in Table 1, the
inhibitory constants (Ki) of ATP and NADP+ were smaller in Brij-58-treated than
alamethicin-treated RLM (Table 2).
Effect of AMP on the inhibition caused by ATP and NADP+. As described
before, the 4-MU UGT activity in both Brij-58- and alamethicin-treated RLM is
barely affected by AMP (Table 1). However, we have shown that AMP has an
ability to modify the inhibitory effects of other nucleotides in Brij-58-treated RLM
(Nishimura et al, 2007). To investigate whether the same is also true for
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alamethicin-treated RLM, the effect of AMP on the 4-MU glucuronidation activity
was examined. In both Brij-58- and alamethicin-treated RLM, the inhibitory effect
of ATP on 4-MU UGT activity was antagonized by AMP (Fig. 2A-B). Such an
antagonistic effect became stronger along with an increase in AMP
concentration. AMP also antagonized the inhibitory effect of NADP+ (Fig. 2C-D).
These results suggest that AMP competes with adenine-related inhibitors at the
binding site of 4-MU UGT in both Brij-58- and alamethicin-treated RLM, although
AMP itself lacks any inhibitory potential.
Mechanism underlying the UGT latency caused by detergent- and
alamethicin-treatment. Our preliminary study showed that Brij-58-treatment
causes a 17-fold increase in the Vmax of 4-MU glucuronidation in RLM
(Supplemental Table S1). The Michaelis constant, Km, for 4-MU was slightly
increased by Brij-58, while the Km for co-substrate, UDP-GlcUA, tended to be
reduced by the treatment. Therefore, it is likely that an increase in the affinity for
UDP-GlcUA may contribute to the enhancement of Vmax following detergent
treatment. However, an increase in the Km for 4-MU does not agree with the
hypothesis that removal of membrane obstruction is the sole reason for the
latency of UGT. Table 3 shows the kinetic parameters for 4-MU glucuronidation
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which were compared for Brij-58- and alamethicin-treated RLM. The Km values
for 4-MU and UDP-GlcUA were slightly higher in alamethicin-treated than
Brij-58-treated RLM. When the kinetics were studied with different 4-MU
concentrations, the Vmax in alamethethin-treated RLM was approximately one
third that of Brij-58-treated RLM. As far as the activating conditions with Brij-58
(0.25 mg/mg protein) and alamethicin (0.05 mg/mg protein) were concerned, the
Vmax obtained after varying the 4-MU/UDP-GlcUA concentrations was higher in
Brij-58-treated than in alamethicin-treated RLM.
Although the ER appears to contain a substantial concentration of adenine
nucleotides for molecular chaperons and dehydrogenases, it is not known
whether the luminal adenine nucleotides are released by treating microsomes
with detergent or alamethicin. If this is true, the inhibitory effect should be
abolished or weakened following detergent- or alamethicin-treatment of
microsomes. To examine this hypothesis, we determined the ATP content in the
ER with and without Brij/alamethicin treatment. Table 4 shows the ATP content in
microsomal pellets and supernatant after treatment with Brij-58 or alamethicin,
the concentrations of which were the same as those used for determining UGT
activity. ATP was released by Brij-58 treatment and no detectable ATP remained
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in the detergent-treated microsomes. However, ATP remained in the
microsomes in the control sample. Part of the microsomal ATP was also
released by alamethicin treatment, but almost 60% of ATP remained unreleased
even after alamethicin treatment. The lower Vmax in alamethicin-treated than in
Brij-58-treated RLM (Table 3) seems to correlate with the extent of ATP
remaining in the microsomes. For example, it is suggested that an ATP
concentration sufficient for UGT inhibition still remains in microsomes even after
alamethicin treatment. Conversely, it would be reasonable to believe that the
release of ATP from microsomes after detergent treatment results in the abolition
of its inhibitory effect on UGT. Therefore, it is suggested that the presence of
inhibitory adenine nucleotides is one possible mechanism for the latency of UGT.
The combined effect of adenine-related compounds on UGT activity. Under
normal conditions, the cellular ATP concentration is several mM (Schwiebert,
2000) and this is 5- to 10-fold higher than the ADP level (Taylor et al., 1975). The
proportion of ATP, ADP and AMP concentrations has been assumed to satisfy
the following equation (Hadie et al., 1998; 2001).
[AMP]/[ATP] = ([ADP]/[ATP])2
The concentration of adenine nucleotides in the ER lumen of living cells has
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not been accurately determined. However, on the basis of the above equation,
three ratios (ATP:ADP:AMP=1:1:1, 25:5:1 and 100:10:1) at four total
concentrations (ATP+ADP+AMP) from 250 to 1500 µM were examined under
UGT inhibition. In Brij-58-treated RLM, the inhibitory effect on UGT was
increased depending on an increase in the ATP ratio (Fig. 3). Therefore, the
inhibitory effect of ATP can be negatively modulated by its metabolites ADP and
AMP. Although a similar trend was observed in alamethicin-treated RLM, the
effect of combined nucleotides was more marked in Brij-58-treated RLM. Then,
we examined the effect of more complicated mixtures on UGT activity. For this,
all inhibitory substances listed in Table 1 were mixed at their respective IC50 and
half IC50 concentrations and the effects of these cocktails on UGT activity were
examined. In Brij-58-treated RLM, the ‘IC50/2’ cocktail reduced 4-MU
glucuronidation to approximately 40% of the control, while the effect was almost
abolished when alamethicin-treated RLM were used (Fig. 4). It is, therefore,
likely that the access of inhibitory adenine-related compounds to the target site
of UGT is limited in alamethicin-treated RLM.
Inhibition of glucuronidation in HLM and human UGT1A9 by ATP. The
effect of AMP and ADP as well as ATP on UGT activity was examined in Brij-58
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and alamethicin-treated HLM. ATP exhibited the strongest inhibitory effect in
both Brij-58 and alamethicin-treated HLM (Table 5). However, the IC50 was
27-times higher in alamethicin-treated HLM. No inhibitory effect was observed
for ADP and AMP in HLM treated with Brij-58 or alamethicin. We then examined
the effect of adenine nucleotides on human UGT1A9 which is one of the major
UGT isoforms involved in 4-MU glucuronidation. In this case, we used
alamethicin-treated UGT1A9 Supersome™ which is used widely for research
purposes. As shown in Table 5, ATP inhibited UGT1A9-catalyzed 4-MU
glucuronidation with an IC50 lower than that obtained in alamethicin-treated HLM.
The IC50 of ADP and AMP was greater than 1000 µM (Table 5).
The mechanism of Gefitinib-induced inhibition of UGT1A9. As shown in Fig.
5, Gefitinib significantly inhibited 4-MU UGT activity mediated by UGT1A9 as
well as Brij-58- and alamethicin-treated HLM. The IC50 of Gefitinib for
UGT1A9-catalyzed 4-MU glucuronidation was about 20 µM (Table 6). To
investigate whether Gefitinib inhibits UGT through the putative ATP binding site
on UGT, the inhibitory effect of Gefitinib on UGT1A9 was examined in the
presence or absence of ATP at its IC50 concentration. Although ATP exhibited an
additive inhibitory effect when the Gefitinib concentration was less than 50 µM,
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no such effect was observed in the presence of Gefitinib at a concentration of 75
µM or more (Fig. 6). Furthermore, the Gefitinib-induced inhibition of
UGT1A9-catalyzed 4-MU glucuronidation was antagonized in the presence of
AMP which is a non inhibitory adenine nucleotide but a negative modulator of
ATP-dependent inhibition (Fig. 6). From these observations, Gefitinib and
adenine nucleotides are thought to share the binding site on UGT. Therefore, it is
suggested that Gefitinib inhibits UGT1A9 through the putative ATP binding site
on UGT.
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Discussion
The inhibitory effect of adenine nucleotides and related compounds on
4-MU UGT was studied in RLM and HLM. ATP exhibited a strong inhibitory effect
in HLM as well as RLM. On comparing the inhibitory effect of ATP on UGT
between microsomes pre-treated with Brij-58 and alamethicin, the inhibitory
effect was more marked in Brij-58-treated RLM than in alamethicin-treated RLM.
As the mode of inhibition of 4-MU UGT was non-competitive in both Brij-58- and
alamethicin-treated RLM, ATP is considered to be an allosteric inhibitor of UGT.
The data suggest the poor ability of adenine-related compounds to gain access
to the target site on UGT in alamethicin-treated microsomes (Fig. 4). Therefore,
the IC50 observed in the detergent-treated microsomes is considered as a
relevant index for the inhibitory potency of the adenine-related compounds.
It is suggested that there is an ATP transporter on the microsomal
membrane which draws ATP into the lumen (Clairmont et al, 1992; Kim et al.,
1996). ATP uptake into microsomes is saturable with a Km of 3 to 4 µM and a
Vmax of 3 to 7 pmol/min/mg protein (Clairmont et al, 1992). When
firefly-luciferase, which contains a signal sequence and a sequence for ER
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targeting, was expressed in Chinese hamster ovary (CHO) cells, ATP-dependent
chemiluminescence was observed by microscopy after addition of luciferin
(Dorner and Kaufman, 1994). Thus, it is likely that the luminal side of the ER
contains a substantial level of ATP, although its concentration remains unknown.
In addition, the ER lumen expresses proteins, such as protein disulfide
isomerase (PDI), UGT1A1 and UGT1A7, which are sensitive to phosphorylation
(Quéméneur et al, 1994; Basu et al., 2005). Furthermore, molecular chaperones,
such as PDI, calreticulin, GRP78 and GRP94, capable of binding to ATP are
present within the ER (Guthapfel et al., 1996; Dierks et al., 1996; Rosser et al.,
2004). The dissociation constant of ATP and the Km for ATPase activity of
GRP78 are reported to be 5.4 and 28.6 µM, respectively (Wearsch and Nicchitta,
1997). For PDI, the corresponding values are 9.7 and 7.1 µM, respectively
(Guthapfel et al., 1996). Accordingly, the concentration of ATP needed for the
functions of the above series of proteins is assumed to be around 30 µM, which
is close to the IC50 determined in this study. Since these ATP-binding proteins
are ATPases, it would be possible that they modulate UGT activity by
hydrolyzing inhibitory ATP to less-inhibitory ADP and non-inhibitory AMP in the
ER.
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As shown in Fig. 3, the inhibitory effect of adenine nucleotides on UGT
activity varied depending on the ratio of ATP:ADP:AMP. If the concentration of
ATP in the ER is kept much higher than its IC50, the UGT activity should be
completely suppressed under such conditions. However, it is reasonable to
assume that the concentration of ATP and antagonistic AMP varies dynamically
in the lumen of the ER to switch the glucuronidation reaction on/off. Our
preliminary study indicated that the ATP level within the hepatic ER is higher in
the evening than in the morning (Supplemental Figure S1). The concentration of
nocturnal ATP was close to 30 µM. Also, the extracellular production of
adenosine from ATP in the retina exhibits a circadian variation (Ribelayga and
Mangel, 2005), and we also found an intra-day variation in the ATP level in the
lumen of the ER.
Although it is assumed that the limited transport of UDP-GlcUA from
cytosol to the ER lumen renders UGT latent, the Vmax of UGT was markedly
increased by Brij-58 treatment, and the Km values for 4-MU and UDP-GlcUA
showed only a minor change (Supplemental Table S1). Thus, although it cannot
be excluded that reducing the Km for UDP-GlcUA may make some contribution
to an apparent increase in UGT function produced by detergent, increasing the
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Km for substrates (glucuronic acid acceptors) seems to disagree with the idea
that the removal of interference by the membrane is the absolute mechanism
explaining the latency of UGT.
On the basis of the inhibitory effect of ATP and its level in the lumen of
the hepatic ER determined in this study, we propose a new hypothesis for the
latency of UGT. As mentioned before, in untreated microsomes, the luminal
concentration of ATP is around 30 µM (Supplemental Figure S1), which is
comparable with its IC50 value for UGT inhibition. This observation suggests that
UGT function is partially suppressed by ATP under normal physiological
conditions. However, when microsomes are treated with detergent or
alamethicin, the enclosure of adenine nucleotides in the ER is disrupted, and the
concentration of the nucleotides is markedly reduced. Therefore, the
concentration of adenine nucleotides, especially in Brij-58-treated microsomes,
becomes too low to inhibit UGT. This possibility seems to be consistent with the
fact that the inhibition mode of adenine nucleotides is non-competitive and the
Vmax of UGT is increased by detergent treatment (Supplemental Table S1,
Table 2, Fig. 1). Furthermore, when RLM were treated with Brij-58, ATP did not
remain in the pellets obtained after ultra-centrifugation, while this nucleotide
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remained in the microsomal pellets in untreated microsomes. Taking all above
findings into consideration, it is suggested that a detergent/alamethicin-caused
decrease in adenine nucleotides in the ER is one possible mechanism
explaining the latency of UGT.
The inhibitors of EGFR-protein tyrosine kinase have an undesired
inhibitory effect on UGT (Liu et al., 2010; Fujita et al., 2011; Piu et al., 2011). This
may cause a drug-drug interaction in UGT-dependent metabolism (Fujita et al,
2011). However, it has not been discovered why these EGFR-protein tyrosine
kinase inhibitors suppress UGT activity. In this study, we suggest that Gefitinib
inhibits UGT1A9 by binding to the allosteric site shared with ATP and AMP. ATP
at its IC50 concentration additively enhanced the inhibition of UGT catalysis with
Gefinitib at low concentrations, and AMP antagonized the inhibitory effect of
Gefitnib. The inhibitory potency of Gefitinib was far stronger than that of ATP.
Although ATP at IC50 showed an additive effect on the inhibition of UGT1A9 by
Gefitinib at a low concentration, the effect was diminished by increasing the
concentration of Gefitinib. Therefore, Gefitinib and ATP seem to inhibit UGT1A9
through binding to a common site. No additive effect of ATP at the high
concentration of Gefitinib would be owing to the occupation of the binding site
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with Gefitinib. On the other hand, the Gefitinib-induced inhibitory effect on
UGT1A9 was partially antagonized by 1 mM AMP, and the suppressive effect
remained even in the presence of high concentration Gefinitib. Although AMP at
a concentration more than 1 mM may completely eject Gefitinib from the
competitive binding site an alternative possibility that AMP exerts its antagonistic
effect against Gefinitib through an allosteric mechanism cannot be excluded. It is
considered that the ATP and AMP contents of cells largely depend on the
mitochondrial function which varies in response to a change in physiological
conditions. However, many more studies will be needed to fully understand how
disease conditions affect the ATP concentration within the ER, hormone
metabolism and drug-drug interaction with EGFR-protein tyrosine kinase
inhibitors. The three-dimensional structure of UGT1A9 has already been
simulated using the crystal structure of TDP-epi-vancosaminyltransferase (PDB
ID: 1PN3) as a template (Fujiwara et al, 2009). In their study, the prediction was
carried out using procedures reported by Ogata and Ueyama (2000). On the
contrary, in Phyre2 system used in this study, multiple templates described in the
Materials and Methods were used to predict the UGT1A9 structure. The
templates used were the enzymes utilizing UDP-sugar as the co-substrate
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belonging to UDP-glycosyltransferase/glycogen phosphorylase superfamily and
a flavonoid 3-O-glucosyltransferase (crystal structure of UGT78G1 with binding
UDP as a ligand). Interestingly, we have predicted the binding site of UGT for
ADP in a 3D structural model, using an algorithm “3DLigandSite using similar
structures” and multiple (six) templates substituted for UGT (Kelly and Sternberg,
2009; Wass et al., 2010)(Supplemental Figure S2).
Further studies are needed to confirm which residues are relevant for
the modulation of UGT activity by adenine nucleotides.
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Authorship Contributions
Participated in research design: Ishii, An, Nishimura, Yamada
Conducted experiments: Ishii, An, Nishimura
Performed data analysis: Ishii, An, Nishimura
Wrote or contributed to the writing of the manuscript: Ishii, An, Yamada
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Footnotes
This work was supported in part by Grants-in-Aid for Scientific Research (C)
from the Japan Society for the Promotion of Science (JSPS) [Research
No.19590147], [No.21590164].
Address correspondence to: Yuji Ishii, Ph.D., Graduate School of
Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan, E-mail: ishii@phar.kyushu-u.ac.jp
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Legends for figures:
Fig. 1 Michaelis-Menten and Eadie-Hofstee plots for the inhibition by ATP
and NADP+ of 4-MU glucuronidation catalyzed by RLM pretreated
with Brij-58 or alamethicin: kinetics by varying 4-MU concentration.
Effect of ATP (A, E) and NADP+ (B, F) on 4-MU glucuronide formation in
Brij-58-treated microsomes was estimated at seven concentrations of
4-MU (10 to 1000 µM). The effect of ATP (C, G) and NADP+ (D, H) in
alamethicin-treated microsomes was also determined. The
UDP-GlcUA concentration was fixed at 2 mM.
Fig. 2. Antagonist effect of AMP on an ATP- and NADP+-evoked reduction
in 4-MU glucuronidation catalyzed by Brij-58- or alamethicin-treated
RLM. Effects of ATP (A, B) and NADP+ (C, D) (10 to 2500 μM) on the
4-MU glucuronide formation were assayed in the presence or absence
of AMP (closed circle, 0 μM; open square, 200 μM; closed triangle, 1000
μM). Microsomes were treated with Brij-58 (0.25 mg/mg protein) (A, C)
or alamethicin (0.05 mg/mg protein) (B, D). 4-MU and UDP-GlcUA
concentrations were fixed at 100 µM and 2 mM, respectively. Each plot
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represents the average of a triplicate assay.
Fig. 3. Effect of the ratio of exogeneously added adenine nucleotides on
the UGT activity in detergent- or alamethicin-treated RLM. Effects of
the ratio of adenine nucleotides (ATP:ADP:AMP=100:10:1, 25:5:1, and
1:1:1) on 4-MU glucuronide formation were examined by varying the
total concentration of ATP, ADP and AMP (250, 500, 1000 and 1500 μM).
Microsomes were treated with Brij-58 (0.25 mg/mg protein) or
alamethicin (0.05 mg/mg protein). 4-MU and UDP-GlcUA concentrations
were fixed at 100 µM and 2 mM, respectively. Each bar represents the
mean ± S.E. of a triplicate assay. Significantly different from the activity
in the absence of adenine nucleotides (*, p<0.05; **, p<0.01; ***,
p<0.001).
Fig. 4. Effect of the mixture of inhibitory adenine-related compounds on
the UGT activity in Brij-58- or alamethicin-treated RLM. Effects of a
mixture of inhibitory compounds [ATP, ADP, adenine, NAD+, NADP+ and
CTP (Brij-58); and ATP, ADP, adenine, NAD+ and NADP+ (alamethicin)]
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on 4-MU glucuronide formation were examined. The 'IC50' and 'IC50/2'
cocktails contain the inhibitory compounds described above at a IC50
concentration and half the IC50 concentration (see Table 1), respectively.
Microsomes were treated with Brij-58 (0.25 mg/mg protein) or
alamethicin (0.05 mg/mg protein). 4-MU and UDP-GlcUA concentrations
were fixed at 100 µM and 2 mM, respectively. Significantly different from
the activity in the absence of inhibitory compounds (**, p<0.01; ***,
p<0.001).
Fig. 5. Effects of Gefitinib on pooled human liver microsome- and human
UGT1A9 microsome-catalyzed 4-MU glucuronidation. Effects of
Gefitinib on 4-MU glucuronidation were assayed. Microsomal protein
(HLMs, 20 μg; UGT1A9 supersomes, 10 μg) was used in each assay.
The 4-MU concentration was fixed at 100 and 30 µM for HLM and
UGT1A9 supersomes, respectively. Each bar represents the mean ± S.E.
of triplicate assays. Abbreviation: HLMs, human liver microsomes.
Significantly different from the activity in the absence of Gefitinib (*,
p<0.05; **, p<0.01; ***, p<0.001).
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Fig. 6. Effects of AMP and ATP on a Gefitinib-evoked reduction in 4-MU
glucuronidation catalyzed by humen UGT1A9. Effect of Gefitinib on
the 4-MU glucuronide formation was assayed in the presence of 500 μM
ATP (A) and 1 mM AMP (B). UGT1A9 supersomes were treated with
alamethicin (0.05 mg/mg protein). The 4-MU concentration was fixed at
30 µM. Each plot represents the average of triplicate assays.
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Table 1. IC50 value of nucletotides and adenine-related substances toward
4-MU UGT in RLM pretreated with either Brij-58 or alamethicin.
IC50 (μM)
Compounds Brij-58 Alamethicin
ATP 66.8±2.2 463±36 [6.9]
ADP 769±251 1258±54 [1.6]
Adenine <1000 1890±89 [-]
AMP >1000 >2000 [-]
Adenosine >1000 >2000 [-]
NADP+ 385±65 1079±65 [2.8]
NAD+ 334±116 1594±167 [4.8]
NADPH >1000 >2000 [-]
NADH >1000 >2000 [-]
CTP 853±47 >2000 [-]
GTP >1000 >2000 [-]
UTP >1000 >2000 [-]
RLM pretreated with Brij-58 (0.25 mg/mg protein) or alamethicin (0.05 mg/mg
protein) were used as the enzyme sources. 4-MU and UDP-GlcUA
concentrations were fixed at 100 µM and 2 mM, respectively. Each value
represents the estimated IC50 ± S.E. Value in bracket is the IC50 relative to that
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determined using Brij-58-treated microsomes (=1.0). Control activities in the
absence of inhibitor was 11.2 ± 0.3 and 4.63 ± 0.16 for Brij-58- and
alamethicin-treated RLM, respectively.
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Table 2. Ki values of ATP and NADP+ in terms of 4-MU glucuronidation
activity in RLM.
Compounds Ki (μM)
Brij-58 Alamethicin
ATP 204 ± 17 635 ± 42
NADP+ 298 ± 30 607 ± 23
Each value represents the estimated Ki ± S.D. Details are described in Fig. 1
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Table 3. Kinetic parameters of 4-MU glucuronidation catalyzed by RLM
pretreated either with Brij-58 or alamethicin: kinetics by varying the
UDP-GlcUA concentration
Activator
Km Vmax Vmax/Km
(μM) (nmol/min/mg protein)
Kinetics by varying 4-MUa
Brij-58 93.6 ± 0.3 26.3 ± 0.3 0.28
alamethicin 163 ± 9 8.32 ± 0.15 0.05
Kinetics by varying UDP-GlcUA b
Brij-58 348 ± 22 19.0 ± 0.3 0.05
Alamethicin 586 ± 49 4.73 ± 0.11 0.01
RLM pretreated with Brij-58 (0.25 mg/mg protein) and alamethicin (0.05 mg/mg
protein) were used as the enzyme source. a4-MU glucuronide formation was
estimated at ten concentrations of 4-MU (10 to 5000 µM). The concentration of
UDP-GlcUA was set at 2 mM in this assay. b4-MU glucuronide formation was
estimated at ten concentrations of UDP-GlcUA (50 to 7500 µM). The
concentration of 4-MU was set at 100 µM in this assay. Each value represents
the optimized value ± S.E.
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Table 4. Release of ATP from RLM by Brij-58- and alamethicin-treatment.
ATP (pmol)
Pellet Sup. Total [%]
Intact 36.8 ± 1.0 4.83 ± 0.73 41.6 [100]
Brij-58 N. D. 13.4 ± 0.1 13.4 [32.2]
Alamethicin 21.1 ± 0.5 11.5 ± 2.0 32.6 [78.4]
Each value represents the mean ± S.D. of three determinations. Value in bracket
is the percentage of the sum of the ATP recovered in the pellet and sup. relative
to that determined using intact microsomes (=100). Abbreviation: sup.,
supernatant; N.D., not detected.
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Table 5. IC50 of adenine nucleotides for 4-MU glucuronidation catalyzed
by pooled HLM and UGT1A9.
compounds
HLM UGT1A9
IC50 (µM) Ratio IC50 (µM)
Brij-58 alamethicin (Brij-58=1.0) alamethicin
ATP 33.8±5.2 935±162 27.7 491±22
ADP >1000 >2000 - >1000
AMP >1000 >2000 - >1000
Microsomes (20 μg) were pretreated with Brij-58 (0.25 mg/mg protein) or
alamethicin (0.05 mg/mg protein). Values represent the mean ± S.E. of
triplicate assays. ‘Ratio’ is the IC50 relative to that determined using
Brij-58-treated microsomes (=1.0). For UGT1A9 supersomes, microsomal
protein (10 µg) was pretreated with alamethicin (0.05 mg/mg protein).
Values represent the mean ± S.E. of triplicate assays.
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Table 6. IC50 of Gefitinib for 4-MU glucuronidation catalyzed by pooled
HLM and human UGT1A9 microsomes.
IC50 (μM)
HLMs (Brij-58-treated) >250
HLMs (alamethicin-treated) >250
UGT1A9 (alamethicin-treated) 19.4±6.6
Human microsomal protein (20 μg) was pretreated with either Brij-58 (0.25
mg/protein) or alamethicin (0.05 mg/mg protein). UGT1A9 microsomes (10 μg
protein) were pretreated with alamethicin (0.05 mg/mg protein). Values
represent the mean ±S.E. of triplicate assays.
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A ATP (Brij-58)
B NADP+ (Brij-58)
D NADP+ (alamethicin)
C ATP (alamethicin)
E ATP (Brij-58)
F NADP+ (Brij-58)
H NADP+ (alamethicin)
G ATP (alamethicin)
Figure 1
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ATP (μM)
Rel
ativ
e ac
tivi
ty(%
of
con
tro
l)
0 200 400 600 800 1000
0
20
40
60
80
100
120
1000 μM
250 μM
0 μM
NADP+ (μM)
Rel
ativ
e ac
tivi
ty(%
of
con
tro
l)
0 500 1000 1500 2000
0
20
40
60
80
100
120
1000 μM
250 μM0 μM
NADP+ (μM)
Rel
ativ
e ac
tivi
ty(%
of
con
tro
l)
0 500 1000 1500 2000 2500
0
20
40
60
80
100
120
1000 μM
250 μM
0 μM
ATP (μM)
Rel
ativ
e ac
tivit
y(%
of
con
tro
l)
0 500 1000 1500 2000
0
20
40
60
80
100
120
1000 μM
250 μM
0 μM
A
B
C
D
Figure 2
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Brij-58-treated microsomes
Alamethicin-treated microsomes
Figure 3
*** ***
*** *** *** ***
*** ***
**
**
**
******
***
******
**
******
******
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14
12
10
8
6
4
2
0control IC50/2 IC50
4-M
UG
fo
rmed
(nm
ol/m
in/m
g p
rote
in)
5
4
3
2
1
0control IC50/2 IC50
4-M
UG
fo
rmed
(nm
ol/m
in/m
g p
rote
in)
Brij-58-trated microsomes
Alamethicin-trated microsomes
Figure 4
***
***
***
**
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Relative activity
(% of control)
120
100
80
60
40
20
0
0 2.5 25 250
Gefitinib (μM)
*
*
**
120
100
80
60
40
20
0
Relative activity
(% of control)
0 2.5 25 250
Gefitinib (μM)
**
120
100
80
60
40
20
0
Relative activity
(% of control)
0 2.5 25 250
Gefitinib (μM)
*
*
**
Alamethicin-treated HLM
Brij-58-treated HLM
Alamethicin-treated UGT1A9
Figure 5
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Gefitinib +ATP 500 μM
Gefitinib (μM)
Rel
ativ
e ac
tivi
ty(%
of
con
tro
l)
0 100 200 300
0
20
40
60
80
100
Gefitinib
Gefitinib (μM)
Rel
ativ
e ac
tivi
ty(%
of
con
tro
l)
0 100 200 300
0
20
40
60
80
100
GefitinibGefitinib +AMP 1000 μM
Figure 6
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