DMD Fast Forward. Published on July 30, 2012 as DOI: 10.1124/dmd.112.046862 DMD ThisFast article Forward. has not been Published copyedited andon formatted.July 30, The 2012 final asversion doi:10.1124/dmd.112.046862 may differ from this version.
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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 Downloaded from
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, dmd.aspetjournals.org
Japan
at ASPET Journals on September 29, 2021
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Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics. DMD Fast Forward. Published on July 30, 2012 as DOI: 10.1124/dmd.112.046862 This article has not been copyedited and formatted. The final version may differ from this version.
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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 Downloaded from
Maidashi, Higashi-ku, Fukuoka 812-8582, Japan,
Phone: +81-92-642-6586 dmd.aspetjournals.org
Fax:+81-92-642-6588
E-mail: [email protected] at ASPET Journals on September 29, 2021
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 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2021
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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 Downloaded from were examined using RLM and human liver microsomes (HLM). ATP inhibited dmd.aspetjournals.org 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 at ASPET Journals on September 29, 2021 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 Downloaded from mechanisms to explain the ‘latency’ of UGT. dmd.aspetjournals.org at ASPET Journals on September 29, 2021
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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 Downloaded from 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 dmd.aspetjournals.org co-substrate, UDP-glucuronic acid (UDP-GlcUA), to a substrate (Mackenzie et al,
2005). This enzyme is localized in the endoplasmic reticulum (ER) and at ASPET Journals on September 29, 2021 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 Downloaded from 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 dmd.aspetjournals.org 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 at ASPET Journals on September 29, 2021
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 Downloaded from 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 dmd.aspetjournals.org dehydrogenase and hexose-6-phosphate dehydrogenase, in the lumen of the
ER (Hewitt et al, 2005). Furthermore, molecular chaperones, such as at ASPET Journals on September 29, 2021 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 Downloaded from
ATP content in the ER with and without alamethicin/detergent treatment.
Through these experiments, we investigated the hypothesis that a change in the dmd.aspetjournals.org 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 at ASPET Journals on September 29, 2021 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 Downloaded from were obtained from Wako Pure Chemicals, Co. Ltd. (Osaka, Japan).
4-Methylumbelliferyl-β-D-glucuronide (4-MUG) and polyoxyethylene cetyl dmd.aspetjournals.org alcohol ether (Brij-58) were purchased from Nakalai Tesque (Kyoto, Japan).
ENLITEN® rLuciferase/Luciferin reagent and 5xPassive lysis buffer were at ASPET Journals on September 29, 2021 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 Downloaded from determined by the published methods (Hanioka et al., 2001) with slight modifications (Nishimura et al., 2007). Human UGT1A9 SupersomeTM and dmd.aspetjournals.org human liver microsomes (pooled microsomes from 150 donors) were purchased from BDGentest (Woburn, MA). at ASPET Journals on September 29, 2021
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, Downloaded from
CA).
Prediction of the three-dimentional structure and ligand binding sites of dmd.aspetjournals.org
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 at ASPET Journals on September 29, 2021
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 Downloaded from structural simulation of the other region involving the highly variable C-terminal domain, the structure of this region was tentatively predicted by ab initio dmd.aspetjournals.org 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 at ASPET Journals on September 29, 2021 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 Downloaded from 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 dmd.aspetjournals.org
shows the IC50 values of the compounds examined. Of the substances tested,
ATP exhibited the strongest inhibitory effect in both Brij-58 and at ASPET Journals on September 29, 2021
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 Downloaded from
Brij-58-treated RLM. The inhibitory potential of adenine-nucleotides also depends on the number of phosphate groups [ATP >> ADP > AMP (= almost no dmd.aspetjournals.org 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. at ASPET Journals on September 29, 2021
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 Downloaded from 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 dmd.aspetjournals.org 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 at ASPET Journals on September 29, 2021 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). Downloaded from
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 dmd.aspetjournals.org
AMP itself lacks any inhibitory potential.
Mechanism underlying the UGT latency caused by detergent- and at ASPET Journals on September 29, 2021 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 Downloaded from
(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 dmd.aspetjournals.org
Brij-58-treated than in alamethicin-treated RLM.
Although the ER appears to contain a substantial concentration of adenine at ASPET Journals on September 29, 2021 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 Downloaded from remaining in the microsomes. For example, it is suggested that an ATP concentration sufficient for UGT inhibition still remains in microsomes even after dmd.aspetjournals.org alamethicin treatment. Conversely, it would be reasonable to believe that the release of ATP from microsomes after detergent treatment results in the abolition at ASPET Journals on September 29, 2021 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 Downloaded from 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 dmd.aspetjournals.org 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, at ASPET Journals on September 29, 2021
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 Downloaded from
UGT isoforms involved in 4-MU glucuronidation. In this case, we used alamethicin-treated UGT1A9 Supersome™ which is used widely for research dmd.aspetjournals.org purposes. As shown in Table 5, ATP inhibited UGT1A9-catalyzed 4-MU
glucuronidation with an IC50 lower than that obtained in alamethicin-treated HLM. at ASPET Journals on September 29, 2021
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 Downloaded from 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 dmd.aspetjournals.org on UGT.
at ASPET Journals on September 29, 2021
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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 Downloaded from 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. dmd.aspetjournals.org
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. at ASPET Journals on September 29, 2021
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 Downloaded from isomerase (PDI), UGT1A1 and UGT1A7, which are sensitive to phosphorylation
(Quéméneur et al, 1994; Basu et al., 2005). Furthermore, molecular chaperones, dmd.aspetjournals.org 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., at ASPET Journals on September 29, 2021
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 Downloaded from 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 dmd.aspetjournals.org the evening than in the morning (Supplemental Figure S1). The concentration of nocturnal ATP was close to 30 µM. Also, the extracellular production of at ASPET Journals on September 29, 2021 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 Downloaded from latency of UGT. As mentioned before, in untreated microsomes, the luminal concentration of ATP is around 30 µM (Supplemental Figure S1), which is dmd.aspetjournals.org
comparable with its IC50 value for UGT inhibition. This observation suggests that
UGT function is partially suppressed by ATP under normal physiological at ASPET Journals on September 29, 2021 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 Downloaded from 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, dmd.aspetjournals.org
2011). However, it has not been discovered why these EGFR-protein tyrosine kinase inhibitors suppress UGT activity. In this study, we suggest that Gefitinib at ASPET Journals on September 29, 2021 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 Downloaded from 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 dmd.aspetjournals.org mitochondrial function which varies in response to a change in physiological conditions. However, many more studies will be needed to fully understand how at ASPET Journals on September 29, 2021 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, Downloaded from
2009; Wass et al., 2010)(Supplemental Figure S2).
Further studies are needed to confirm which residues are relevant for dmd.aspetjournals.org the modulation of UGT activity by adenine nucleotides.
at ASPET Journals on September 29, 2021
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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 Downloaded from
Wrote or contributed to the writing of the manuscript: Ishii, An, Yamada
dmd.aspetjournals.org
at ASPET Journals on September 29, 2021
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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]. Downloaded from
Address correspondence to: Yuji Ishii, Ph.D., Graduate School of dmd.aspetjournals.org
Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan, E-mail: [email protected] at ASPET Journals on September 29, 2021
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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. Downloaded from
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 dmd.aspetjournals.org
4-MU (10 to 1000 µM). The effect of ATP (C, G) and NADP+ (D, H) in
alamethicin-treated microsomes was also determined. The at ASPET Journals on September 29, 2021
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 Downloaded from
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). dmd.aspetjournals.org
Microsomes were treated with Brij-58 (0.25 mg/mg protein) or
alamethicin (0.05 mg/mg protein). 4-MU and UDP-GlcUA concentrations at ASPET Journals on September 29, 2021 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 Downloaded from
were fixed at 100 µM and 2 mM, respectively. Significantly different from
the activity in the absence of inhibitory compounds (**, p<0.01; ***, dmd.aspetjournals.org
p<0.001).
at ASPET Journals on September 29, 2021 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 Downloaded from
30 µM. Each plot represents the average of triplicate assays.
dmd.aspetjournals.org
at ASPET Journals on September 29, 2021
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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] Downloaded from ADP 769±251 1258±54 [1.6]
Adenine <1000 1890±89 [-] dmd.aspetjournals.org
AMP >1000 >2000 [-]
Adenosine >1000 >2000 [-] at ASPET Journals on September 29, 2021 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.
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 29, 2021
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Table 2. Ki values of ATP and NADP+ in terms of 4-MU glucuronidation activity in RLM. Ki (μM) Compounds 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 Downloaded from
dmd.aspetjournals.org at ASPET Journals on September 29, 2021
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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
Km Vmax Vmax/Km Activator (μM) (nmol/min/mg protein) Downloaded from 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 dmd.aspetjournals.org
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 at ASPET Journals on September 29, 2021
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.
- 47 - DMD Fast Forward. Published on July 30, 2012 as DOI: 10.1124/dmd.112.046862 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #46862
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] Downloaded from 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. dmd.aspetjournals.org
at ASPET Journals on September 29, 2021
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DMD #46862
Table 5. IC50 of adenine nucleotides for 4-MU glucuronidation catalyzed by pooled HLM and UGT1A9. HLM UGT1A9
compounds IC50 (µM) Ratio IC50 (µM)
Brij-58 alamethicin (Brij-58=1.0) alamethicin
ATP 33.8±5.2 935±162 27.7 491±22 Downloaded from
ADP >1000 >2000 - >1000
AMP >1000 >2000 - >1000
Microsomes (20 μg) were pretreated with Brij-58 (0.25 mg/mg protein) or dmd.aspetjournals.org 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. at ASPET Journals on September 29, 2021
- 49 - DMD Fast Forward. Published on July 30, 2012 as DOI: 10.1124/dmd.112.046862 This article has not been copyedited and formatted. The final version may differ from this version.
DMD #46862
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 Downloaded from 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. dmd.aspetjournals.org
at ASPET Journals on September 29, 2021
- 50 - DMD Fast Forward. Published on July 30, 2012 as DOI: 10.1124/dmd.112.046862 A ATP (Brij-58)This article has not been copyedited and formatted. The finalE version ATP may (Brij-58) differ from this version.
B NADP+ (Brij-58) F NADP+ (Brij-58) Downloaded from dmd.aspetjournals.org
C ATP (alamethicin) G ATP (alamethicin) at ASPET Journals on September 29, 2021
D NADP+ (alamethicin) H NADP+ (alamethicin)
Figure 1 A C 120 120
100 100 1000 μM This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 1000 μM
80 80 DMD FastForward.PublishedonJuly30,2012asDOI:10.1124/dmd.112.046862 60 60 250 μM 40 40 μ (% control) of
(% control) of 250 M Relative activity Relative activity 20 20 0 μM 0 μM 0 0 0 200 400 600 800 1000 0 500 1000 1500 2000 ATP (μM) ATP (μM) B D 120 120
100 100
80 80 μ 60 60 1000 M 1000 μM 40 40 250 μM (% control) of (% control) of Relative activity μ Relative activity 20 250 M 20 0 μM 0 μM 0 0 0 500 1000 1500 2000 0 500 1000 1500 2000 2500 NADP+ (μM) NADP+ (μM)
Figure 2
Downloaded from from Downloaded dmd.aspetjournals.org at ASPET Journals on September 29, 2021 29, September on Journals ASPET at DMD Fast Forward. Published on July 30, 2012 as DOI: 10.1124/dmd.112.046862 This article has not been copyedited and formatted. The final version may differ from this version. Brij-58-treated microsomes * * ** ** *** *** *** *** *** *** *** *** Downloaded from dmd.aspetjournals.org
Alamethicin-treated microsomes ** at ASPET Journals on September 29, 2021 *** *** *** *** *** *** *** *** ***
Figure 3 DMD Fast Forward. Published on July 30, 2012 as DOI: 10.1124/dmd.112.046862 This article has not been copyedited and formatted. The final version may differ from this version.
14 Brij-58-trated microsomes 12
10
8 6 ***
4-MUG formed 4 *** Downloaded from
(nmol/min/mg protein) 2
0 dmd.aspetjournals.org
control IC50/2 IC50
5 Alamethicin-trated microsomes
at ASPET Journals on September 29, 2021 4 **
3 *** 2 4-MUG formed 1 (nmol/min/mg protein)
0
control IC50/2 IC50
Figure 4 DMD Fast Forward. Published on July 30, 2012 as DOI: 10.1124/dmd.112.046862 This120 article has not been copyedited and formatted.Brij-58-treated The final version may differ fromHLM this version. 100 * ty l) vi o 80 * ti tr ac n ** co e f 60 iv o at el (% 40 R 20 0 0 2.5 25 250
Gefitinib (μM) Downloaded from 120 Alamethicin-treated HLM
100 dmd.aspetjournals.org ty l) vi o 80 ti tr ** ac n co ve f 60 ti o a at ASPET Journals on September 29, 2021 el (% 40 R 20 0 0 2.5 25 250 Gefitinib (μM) 120 Alamethicin-treated UGT1A9 100 * ty l) vi o 80 ti tr * ac n co e f 60 iv o at el (% 40 R ** 20 0 0 2.5 25 250 Gefitinib (μM) Figure 5 DMD Fast Forward. Published on July 30, 2012 as DOI: 10.1124/dmd.112.046862 This article has not been copyedited and formatted. The final version may differ from this version.
100 Gefitinib 80 Gefitinib + ATP 500 μM 60
40 (% control) of
Relative activity 20
0
0 100 200 300 Downloaded from Gefitinib (μM) 100 Gefitinib
80 Gefitinib + dmd.aspetjournals.org AMP 1000 μM 60
40 at ASPET Journals on September 29, 2021 (% control) of
Relative activity 20
0 0 100 200 300 Gefitinib (μM)
Figure 6 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
Drug Metabolism and Disposition
Fig. S1 Circadian variation in the ATP concentration in rat liver microsomes. The liver was removed from rats at 9 a.m. or 9. p.m. Then, microsomes were prepared, and the resuspended microsomes were frozen immediately in liquid nitrogen and stored at -80˚C prior to assay. The ATP content was determined by chemiluminescence produced by firefly-luciferase. In this experiment, rat liver microsomes were diluted to give a protein concentration of 2 to 5 mg/mL. Then, a 1/100 volume of 100%(w/v) trichloroacetic acid was added to the microsomes. The mixture was kept on ice for 30 min, and then centrifuged at 15,000 rpm (4˚C) for 15 min. The resulting supernatant was diluted with 1xPassive Lysis Buffer to measure the ATP content. The concentration was calculated based on the information that the intravesicular space of liver microsomes is 2.34~3.22 µL per mg protein (Marcolongo et al., 1996). The values are the means ± S.E. of three rats. * Significant difference between pairs (p < 0.05).
Reference: Marcolongo P, Fulceri R, Giunti R, Burchell A and Benedetti A (1996) Permeability of liver microsomal membranes to glucose. Biochem Biophys Res Commun 219: 916-922.
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
Drug Metabolism and Disposition
Fig. S2 Prediction of ADP binding sites in UGT1A9. UGT1A9 (GenBank: AAG30418.1) was subjected to protein structure prediction on the web: a case study using the Phyre server (http://www.sbg.bio.ic.ac.uk/phyre2/html/; Kelly and Sternberg, 2009) as described in Materials and Methods. Six templates based on Structural Classification of Proteins and ASTRAL release 1.75A (March 2012) were selected to model UGT1A9 based on heuristics to maximize the confidence, percentage identity and alignment coverage. Six templates used were as follows: 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). 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 (about 65 amino acid residues), the structure of this region was tentatively predicted by ab initio calculation. The final model of UGT1A9 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. A, modeling image of the whole UGT1A9 molecule with ligands ADP (yellow); B, enlarged image with ligands (yellow) and candidate contact sites (blue); C, enlarged image with ligands (yellow) and candidate contact sites (annotated, blue). The following sites are candidate contact sites: 11 SER, 12 HIS, 145 ARG, 229 ARG, 280 GLY, 281 SER, 282 MET, 283 VAL, 285 GLU, 308 ARG, 325 LYS, 326 TRP, 327 LEU, 329 GLN, 344 HIS, 348 HIS, 349 GLY.
References: Brazier-Hicks M, Offen WA, Gershater MC, Revett TJ, Lim EK, Bowles DJ, Davies GJ, and Edwards R (2007) Characterization and engineering of the bifunctional N- and O-glucosyltransferase involved in xenobiotic metabolism in plants. Proc Natl Acad Sci U S A 104: 20238-20243. Kelley LA and Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nature Protoc 4: 363-371. Li L, Modolo LV, Escamilla-Trevino LL, Achnine L, Dixon RA, and Wang X (2007) Crystal structure of Medicago truncatula UGT85H2--insights into the structural basis of a multifunctional (iso)flavonoid glycosyltransferase. J Mol Biol 370: 951-963. Modolo LV, Li L, Pan H, Blount JW, Dixon RA, and Wang X (2009) Crystal structures of glycosyltransferase UGT78G1 reveal the molecular basis for glycosylation and deglycosylation of (iso)flavonoids. J Mol Biol 392: 1292-1302. Mulichak AM, Lu W, Losey HC, Walsh CT, and Garavito RM (2004) Crystal structure of vancosaminyltransferase GtfD from the vancomycin biosynthetic pathway: interactions with acceptor and nucleotide ligands. Biochemistry 43: 5170-5180. Offen W, Martinez-Fleites C, Yang M, Kiat-Lim E, Davis BG, Tarling CA, Ford CM, Bowles DJ, and Davies GJ (2006) Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification. EMBO J 25: 1396-1405. Shao H, He X, Achnine L, Blount JW, Dixon RA, and Wang X (2005) Crystal structures of a multifunctional triterpene/flavonoid glycosyltransferase from Medicago truncatula. Plant Cell 17: 3141-3154. Wass MN, Kelley LA, and Sternberg MJ (2010) 3DLigandSite: predicting ligand-binding sites using similar structures. Nucleic Acids Res 38: W469-473.
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
Drug Metabolism and Disposition
Table S1 Kinetic parameters of 4-MU glucuronidation catalyzed by RLM in the absence or presence of detergent. ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Vmax Km (4-MU or UDP-GlcUA) Activator (nmol/min/mg) (µM) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Kinetics by varying 4-MU a None 5.8 ± 0.3 65.1 ± 10.7 Brij-58 100.8 ± 6.7 105.6 ± 22.2
Kinetics by varying UDP-GlcUA b None 17.9 ± 0.6 1478 ± 139 Brij-58 125.5 ± 4.9 1069 ± 129 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ a4-MU glucuronide formation was estimated at seven concentrations of 4-MU (10 to 800 µM). The concentration of UDP-GlcUA was set at 2 mM in this assay. b4-MU glucuronide formation was estimated at seven concentrations of UDP-GlcUA (0.1 to 8 mM). The concentration of 4-MU was set at 800 µM in this assay. Each value represents the optimized value ± S.E.