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GLYCOSIDATION OF AN ENDOTHELIN ET_A RECEPTOR ANTAGONIST AND DICLOFENAC IN HUMAN LIVER MICROSOMES: AGLYCONE-DEPENDENT UDP-SUGAR SELECTIVITY

Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania

(Received June 7, 2005; accepted August 29, 2005)

	Abstract

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Following the finding that UGT2B7 catalyzes the transfer of the glycosyl group from both UDP-glucuronic acid (UDP-GlcA) and UDP-glucose (UDP-Glc) to an endothelin ET_A receptor antagonist, Compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b] pyridine 6-carboxylic acid], to form an acyl glucuronide and a glucoside (Tang et al., 2003), two additional nucleotide sugars [UDP-galactose (UDP-gal) and UDP-N-acetyl glucosamine (UDP-GlcNAc)] were examined as cosubstrates in human liver microsomes. It was found that UDP-gal, but not UDP-GlcNAc, also served as a sugar donor primarily through catalysis by UGT2B7, although at a significantly reduced catalytic rate. These three UDP-sugars showed pH-dependent kinetics and appeared to compete with each other, with IC₅₀ values parallel to their respective apparent K_m values. In contrast, only UDP-GlcA served as the sugar donor for the conjugation of diclofenac, a known UGT2B7 substrate, with an apparent K_m for UDP-GlcA of 96 ± 17 µM. UDP-Glc and UDP-gal, two futile sugar donors for diclofenac, were found to competitively inhibit the glucuronidation of this aglycone. Different from the case with Compound A, UDP-Glc and UDP-gal displayed K_i values of 2054 ± 108 µM and 1277 ± 149 µM, >10-fold greater than the K_m for UDP-GlcA, indicating that these two nucleotide sugars were also capable of binding to the enzyme but with a lower affinity. The findings of this study indicate that the selectivity of UGT2B7 toward UDP-sugars is aglycone-dependent. With Compound A as the acceptor substrate, human UGT2B7 becomes more accommodative in the transfer of the glycosyl group from UDP-sugars beyond UDP-GlcA. The mechanism may involve enzyme conformational changes associated with Compound A binding to the enzyme.

Human UDP-glycosyltransferases involved in drug metabolism are membrane-bound enzymes located in the endoplasmic reticulum with their catalytic sites facing the lumen. They highly prefer UDP-GlcA as the sugar donor as opposed to other UDP-sugars and, therefore, are named UDP-glucuronsyltransferases (UGTs). Interestingly, the preference for UDP-sugar donors of wild-type UGTs has been found not absolute. There are a considerable number of cases where the glycosyl group in the conjugate could also be derived from other UDP-sugars, especially UDP-Glc (Tang et al., 1979, 2003; Arima, 1990; Senafi et al., 1994; Chmela et al., 2001; Shipkova et al., 2001; Mackenzie et al., 2003). Even more intriguing is the reversed sugar donor specificity found by Toide et al. (2004) showing that human UGT2B isoforms utilize only UDP-Glc, rather than UDP-GlcA, as a cofactor to conjugate an aldose reductase inhibitor. Although it is possible that the tridimensional structure of the sugar donor binding site is not fully conserved among UGT isoforms, it appears that sugar donor selectivity may be aglycone-dependent. For instance, only with bilirubin can UGT1A1 use UDP-GlcA, UDP-xylose, and UDP-Glc as sugar donors to form respective glycosides (Senafi et al., 1994). It is very fascinating that some aglycones are able to influence the sugar donor selectivity of certain UGTs.

We have observed that human UGT2B7 is capable of utilizing both UDP-GlcA and UDP-Glc to conjugate an endothelin ET_A receptor antagonist, Compound A (Fig. 1), with a comparable catalytic potential (V_max/K_{m (Compound A)}) for both glucuronidation and glucosidation (Tang et al., 2003). In addition, these two sugar donors competitively inhibit their counterpart conjugations with the inhibition constant (K_i) close to their K_m values. In expansion of these findings, we examined two additional UDP-sugars, UDP-gal and UDP-N-acetyl-glucosamine (UDP-GlcNAc), as cosubstrates in human liver microsomes. The study also was extended to diclofenac, a known UGT2B7 substrate (King et al., 2001), to examine the impact of aglycone substrate on the selectivity of the enzyme toward UDP-sugars. We demonstrated that in addition to UDP-GlcA and UDP-Glc, UDP-gal also served as a sugar donor for the formation of a galactoside of Compound A in human liver microsomes, and the conjugation was again primarily mediated via UGT2B7. In contrast, diclofenac underwent only glucuronidation in human liver microsomes.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Structures of Compound A, Compound B, UDP-GlcA, UDP-Glc, UDP-gal, and UDP-GlcNAc.

	Materials and Methods

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Chemicals and Reagents. Compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b] pyridine 6-carboxylic acid] and its analog Compound B [(5S,6R,7R)-5-(1,3-benzodioxol-5-yl)-7-[2-(3-hydroxy-2-methylpropyl)-4-(methyloxy)phenyl]-2-[(1-methylethyl)amino]-6,7-dihydro-5H-cyclopenta[b]pyridine-6-carboxylic acid], used as an internal standard (Fig. 1), were synthesized by Banyu Pharmaceutical Co. (Ibaraki, Japan). UDP-GlcA, UDP-Glc, UDP-gal, UDP-GlcNAc, ³H-UDP-gal (galactose-6-³H), alamethicin, diclofenac, and phenytoin were obtained from Sigma-Aldrich (St. Louis, MO). Solvents used for liquid chromatography were of analytical or HPLC grade. Pooled male human (n = 10) liver microsomal preparations were purchased from Xenotech LLC (Kansas City, KS). Recombinant human UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and 2B15 (Supersomes) were obtained from BD Gentest (Woburn, MA) and baculosomes of UGT1A7 and 1A10 from PanVera Corp. (Madison, WI).

Incubation Conditions. The incubations for identification of Compound A and diclofenac glycosides by LC-MS were carried out in the presence of respective acceptor substrates (25 µM), four respective tested UDP-sugars (2 mM), human liver microsomes (1 mg/ml), MgCl₂ (10 mM), and alamethicin in the phosphate buffer (0.1 M, pH 6.0). After the desired length of incubation (30 and 60 min for Compound A with UDP-GlcA and UDP-Glc, and 120 min for the remaining incubations), an equal volume of acetonitrile was added to terminate the reactions. After brief vortexing and centrifugation (10 min at 3800 rpm), the supernatant was subjected to LC-MS assay.

For the quantitation of Compound A conjugate formation, incubations were carried out at 37°C in a Fisher shaking water bath, using 1.1-ml polypropylene disposable deep well tubes purchased from Matrix Technologies Corp. (Hudson, NH). The incubation mixture (final volume of 250 µl) consisted of the following: 0.1 M potassium phosphate buffer (pH 7.4 or 6.0), 10 mM MgCl₂, UDP-sugars (5 mM or varied concentrations), liver microsomes (0.05 mg protein/ml) or expressed UGTs (0.1 mg protein/ml), and Compound A (25 µM or varied concentrations from 100-fold concentrated stock solution in water/acetonitrile, 50:50). The concentration of alamethicin, when applied, was 100 µg/mg protein. The reaction was started by the addition of the respective UDP-sugars and terminated with acetonitrile (200 µl) after a 15-min (for glucuronidation and glucosidation) or 30-min (for galactosidation and N-acetyl-glucosamidation) incubation. The internal standard (Compound B) solution (50 µl at 2.5 µM in water/acetonitrile, 50:50) was added to the samples. After brief vortexing and centrifugation (10 min at 3800 rpm), the supernatant was transferred to 96-well microtiter plates for LC-MS assay.

As for diclofenac glucuronidation, the incubation mixture (final volume of 250 µl in 100 mM phosphate buffer at pH 6.0) contained diclofenac at 60 µM (3-fold of its reported K_m; King et al., 2001) or varied concentrations, 10 mM MgCl₂, human liver microsomes (0.1 mg protein/ml), UDP-sugars (5 mM or varied concentrations), and alamethicin (100 µg/mg protein). The incubation was carried out for 15 min and terminated with acetonitrile (200 µl). The internal standard (phenytoin) solution (100 µl at 5.0 µM in water/acetonitrile, 50:50) was added to the samples.

Determination of UDP-Sugar IC₅₀ or K_i Values. The values of IC₅₀, rather than K_i, were determined for glycosidation of Compound A due to the involvement of multiple UDP-sugars. All incubations contained Compound A (25 µM), liver microsomes (0.05 mg/ml), and alamethicin (100 µg/mg microsomal protein) in a total volume of 250 µl. Concentrations of UDP-sugars as cosubstrates were close to their K_m values in human liver microsomes determined at pH 6.0 and 7.4. As inhibitors, their concentrations spanned from 0.01 to 5000 µM. The experimental design is detailed in Table 1.

The K_i value was determined for UDP-Glc and UDP-gal as inhibitors toward diclofenac glucuronidation in human liver microsomes. The incubation mixture consisted of diclofenac (60 µM), liver microsomes (0.1 mg/ml), and alamethicin (100 µg/mg microsomal protein) in a total volume of 250 µl. Concentrations of UDP-GlcA and UDP-Glc/UDP-gal ranged from 0.1 to 5 mM.

Identification of Glycosides of Compound A and Diclofenac. The chromatographic separation of Compound A and its respective glycosides was performed on a reverse phase C18 column (Synergi MAX-RP, 2.0 x 150 mm, 4 µm; Phenomenex, Torrance, CA) using a Rheos 4000 binary pump (LEAP Technologies, Carrboro, NC) with a flow rate of 200 µl/min. The same mobile phases and gradient described previously (Tang et al., 2003) were applied. The separation of diclofenac and its resultant glycosides was achieved with the same system except for a different gradient (the initial mobile phase consisted of 5% of solvent B, which was linearly increased to 45% over 20 min, then to 80% in another 2 min, and held for an additional 3 min). Mass spectrometric analysis was performed on an LCQ ion trap mass spectrometer equipped with an electrospray ionization source (Thermo Electron Corporation, Waltham, MA), as described previously (Tang et al., 2003), but electrospray ionization was operated in positive and negative modes for Compound A and diclofenac, respectively. Mass spectral data were analyzed using the manufacturer's software package (XCaliber 1.2).

Quantitation of Glycosidic Conjugates of Compound A and Diclofenac. The purification and quantitation of glucuronide and glucoside conjugates of Compound A have been described previously (Tang et al., 2003), but radiochromatographic separation and scintillation counting were utilized to quantify the galactoside due to its low yield. Compound A (50 µM) was incubated with human liver microsomes (0.5 mg/ml) supplemented with UDP-gal (2 mM) at pH 6.0 overnight. UDP-gal was mixed with its tritium-labeled analog to obtain a specific radioactivity of 0.0132 µCi/nmol. The biosynthesis was terminated by the addition of acetonitrile. After centrifugation (5 min at 3800 rpm), the supernatant was subjected to brief evaporation under a nitrogen stream to remove the organic solvent. The resultant samples were allowed to pass through a pretreated OASIS HLB extraction cartridge (1 ml, Waters, Milford, MA). The cartridge was washed with water and then with 20% aqueous methanol to remove the remaining UDP-gal. The eluant from the final wash with methanol was collected, evaporated to dryness, and reconstituted with a given volume of 50% acetonitrile. The radioactivity in a 0.05-ml aliquot of the reconstituted solution was measured by liquid scintillation counting, and the purity of ³H-galactoside of Compound A in the solution was assessed by radiochromatography. Values obtained from both methods were used to determine the concentration of the galactoside in the solution, which was then used for the generation of calibration curves.

The separation of Compound A, its conjugates, and internal standard was accomplished on a Betasil C18 column (2.1 x 50 mm, 5 µm; Thermo Electron Corporation). Solvent A consisted of 0.02% aqueous acetic acid, pH adjusted to 4.5 with NH₄OH, and acetonitrile (90:10), and solvent B consisted of acetonitrile and water (90:10). The mobile phase was delivered at a flow rate of 0.5 ml/min with a linear increase of solvent B from 15% to 85% over 1 min and held for another 1 min. Equilibration was allowed for an additional 1.5 min, giving a total chromatographic run time of 3.5 min. Under these conditions, the glucuronide, glucoside, and galactoside conjugates eluted at 1.3, 1.7, and 1.7 min, respectively. The separation of diclofenac, its glucuronide, and internal standard (phenytoin) was achieved with the same conditions except that solvent B was increased to 75% rather than 85%.

A tandem mass experiment was performed on an MDS Sciex (Concord, ON, Canada) model API 3000 triple quadrupole mass spectrometer interfaced to the column eluant via an MDS Sciex turbospray probe operating at 350°C. Operating conditions for Compound A (and its glucuronide and glucoside conjugates), diclofenac, and their respective internal standards were optimized by infusion along with the liquid chromatography flow (200 µl/min, solvent A/B = 50/50). Selected reaction monitoring experiments in the positive (for Compound A and its conjugates) or negative (for diclofenac and its conjugates) ionization mode were performed using a dwell time of 200 ms per transition to detect ion pairs at m/z 533/339 (Compound A), 709/533 (Compound A glucuronide), 695/533 (Compound A glucoside and galactoside), 519/339 (Compound B), 294/259 (diclofenac), 470/294 (diclofenac glucuronide), 456/294 (diclofenac glucoside), and 251/208 (phenytoin). The lower limit of quantitation for Compound A glucuronide, glucoside, and galactoside in this study was 4 nM. The assay was linear over the range of 4 to 1600 nM for each conjugate. However, only the peak area ratio of diclofenac glucuronide to its internal standard was used for the measurement of this conjugate due to the lack of the authentic standard.

Data Analysis. Estimates of apparent K_m and V_max were obtained by fitting the untransformed data to Michaelis-Menten kinetics (eq.1) using KaleidaGraph (Abelbeck/Synergy, Reading, PA).

(1)

where v is the rate of reaction, V_max is the maximum velocity, K_m is the Michaelis constant, and S is the substrate concentration.

The IC₅₀ values were determined by fitting the data to the Dose Response Logistic model (eq. 2) with the same software.

(2)

where %v_remaining is the remaining reaction rate in the presence of an inhibitor, and I is the inhibitor concentration.

The K_i value of UDP-Glc toward diclofenac glucuronidation was determined by the model of competitive inhibition described in eq. 3.

(3)

where K_s is apparent K_m for UDP-GlcA in the presence of UDP-Glc, and I is the concentration of UDP-Glc.

	Results

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Glycosidation of Compound A and Diclofenac in Human Liver Microsomes. In addition to the previously characterized acyl glucuronide and glucoside of Compound A formed in human liver microsomes in the presence of UDP-GlcA and UDP-Glc (Tang et al., 2003), a metabolite with a protonated molecule (MH⁺) at m/z 695 (identical to that of Compound A glucoside) was detected when supplemented with UDP-gal. Upon tandem mass spectrometry fragmentation, this new conjugate gave rise to a dominant fragment at m/z 533, the ion of protonated Compound A. Based on the supplemented sugar donor (UDP-gal) and corresponding mass change, this conjugate can be assumed to be a galactoside of Compound A, whereas the site of the glycosyl group on the aglycone remains to be determined. However, no product was observed when UDP-GlcNAc was used as a sugar donor. The formation of glycosides of Compound A in the presence of their respective sugar donors is illustrated in Fig. 2A. On the contrary, diclofenac only accepted the glucuronic acid from UDP-GlcA to form a glucuronide. No conjugation was detected when the incubation mixture was supplemented with UDP-Glc or UDP-gal. The extracted ion current chromatogram for diclofenac glucuronide ([M – H]^– at m/z 470) revealed additional minor peaks that appeared to increase with longer incubation (data not shown). They may be derived from acyl migration of the acyl glucuronide of diclofenac, a reaction reported by other investigators (Grillo et al., 2003). When incubation was performed at pH 6.0, the formation of these migrated conjugates was negligible in fresh samples (Fig. 2B). Therefore, incubations for the quantitation of diclofenac glucuronide were carried out in a buffer at pH 6.0.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Base peak chromatograms of human liver microsomal incubates with Compound A (A) and diclofenac (B) supplemented with UDP-GlcA, UDP-Glc, and UDP-gal, respectively. Incubations were carried out at pH 6.0.

Meanwhile, the rate of glycosidation as a function of UDP-sugar concentration (0.025–5 mM) was measured at a fixed concentration of Compound A (25 µM) in a buffer at pH 6.0 and 7.4. Kinetic analysis of glucuronidation, glucosidation, and galactosidation indicated that they all obeyed Michaelis-Menten kinetics. Consistent with the results described previously (Tang et al., 2003), glucuronidation and glucosidation displayed comparable apparent K_m values for UDP-GlcA (K_{m (UDP-GlcA),} 761 ± 42 µM) and UDP-Glc (K_{m (UDP-Glc),} 540 ± 20 µM) when reactions were carried out at pH 7.4. In fact, the apparent K_m value for UDP-gal (K_{m (UDP-Glc),} 1250 ± 190 µM) also was close to that for UDP-GlcA. However, the sugar donor-associated kinetics of glucuronidation and glucosidation were sensitive to pH. As summarized in Table 2, the K_{m (UDP-GlcA)} and K_{m (UDP-Glc)} obtained at pH 6.0 was 525-fold lower than that obtained at pH 7.4, whereas the V_max remained comparable. In contrast, galactosidation responded to pH changes by altering its V_max (2-fold lower at pH 7.4) instead of its K_{m (UDP-gal)}.

Kinetic Properties of Diclofenac Glucuronidation. The rate of glucuronidation of diclofenac as a function of diclofenac concentration (5–100 µM) was measured at a fixed concentration of UDP-GlcA (2 mM) in a buffer at pH 6.0. The reaction appeared to follow Michaelis-Menten kinetics with an average K_m for diclofenac (K_{m (diclofenac)}), from three determinations of 20.6 ± 3.3 µM, comparable to the value reported by other investigators (King et al., 2001). Similarly, the glucuronidation of diclofenac as a function of UDP-GlcA (0.25–5 mM) was measured at a fixed concentration of diclofenac (60 µM) in a buffer at pH 6.0. The result was indicative of typical Michaelis-Menten kinetics with an apparent K_m value for UDP-GlcA (K_{m (UDP-GlcA)}) of 96 ± 17 µM.

UGT2B7 Catalyzed Both Glucosidation and Galactosidation of Compound A in Human Liver Microsomes. The screening for Compound A galactosidation activity in 10 commercially available microsomes expressing specific human UGT isoforms revealed that UGT2B7 showed the highest activity [2.60 pmol/(min · mg protein)] for this conjugation (Fig. 3). The activity of other UGT isoforms was only at the basal level. This result was substantiated by another finding that the rate of galactosidation correlated well with that of glucosidation in human liver microsomal preparations from 16 subjects (Fig. 4, r² = 0.95, p < 0.01). Since UGT2B7 is also primarily responsible for the glucosidation of Compound A in human liver microsomes (Tang et al., 2003), it is conceivable that both glucosidation and galactosidation of Compound A in human liver microsomes were catalyzed primarily by UGT2B7.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Formation of Compound A galactoside by stably expressed UGT isoforms. Incubations were carried out at pH 6.0. Data are expressed as mean ± S.D. of triplicates.

View larger version (18K): [in this window] [in a new window]   FIG. 4. Formation of Compound A glucoside versus galactoside in 16 human liver microsomal preparations. Incubations were carried out at pH 6.0.

Although they were not accepted as glycosyl moiety donors to conjugate diclofenac, UDP-Glc and UDP-gal were inhibitory toward diclofenac glucuronidation in human liver microsomes. Their kinetic mode of inhibition was characterized, and their inhibition constant (K_i) values were determined. As shown by the double reciprocal plot in Fig. 5, the presence of various amounts of UDP-Glc and UDP-gal changed only the apparent K_{m (UDP-GlcA)} and did not affect the V_max, suggesting a competitive inhibition of diclofenac glucuronidation by these two nucleotide sugars. The K_i values determined using nonlinear regression were 2054 ± 108 and 1277 ± 149 µM for UDP-Glc and UDP-gal, respectively. These values are >10-fold greater than the K_{m (UDP-GlcA)}.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Double reciprocal plots for the inhibition of diclofenac glucuronidation by UDP-Glc (A) and UDP-gal (B). Incubations were carried out at pH 6.0. Data are expressed as mean ± S.D. of triplicates.

	Discussion

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It is believed that in the process of glucuronidation, the carboxyl group in UDP-GlcA is the functionality that interacts with a crucial residue, arginine, in the active site (Zakim et al., 1983; Senay et al., 1997; Ouzzine et al., 2002) to allow for an optimal orientation/alignment of an acceptor-donor-enzyme complex. The lack of a carboxyl group in UDP-Glc and UDP-gal may deprive such optimization in the case of diclofenac, although these two donors are able to bind to the same binding site for UDP-GlcA, as indicated by their competitive inhibition of diclofenac glucuronidation (Fig. 5). In contrast, with Compound A as an acceptor, the lack of a carboxyl group in UDP-Glc and UDP-gal seems to be compensated for and UGT2B7 becomes more promiscuous. Although more studies are needed to understand the mechanism, one possibility may involve distinct interaction of Compound A and diclofenac with the enzyme.

It is well documented that substrate binding can induce conformational changes in many enzymatic systems (Zhou et al., 2000; Lopez-Corcuera et al., 2001; Davydov et al., 2004; Segura-Pena et al., 2004). Recent advances in this regard have been described for glycosyltransferases with the availability of X-ray crystal structures of the catalytic domain of a number of enzymes (Qasba et al., 2005). Such changes appear to be necessary in aligning two substrates for catalysis and in configuring the active site. An ordered binding has been proposed by some researchers for mammalian UGTs involved in drug metabolism (Potrepka and Spratt, 1972; Sanchez and Tephly, 1975; Koster and Noordhoek, 1983), by which the acceptor and UDP-sugar donor are the first and second binding substrate, respectively. With this mechanism, it is reasonable to expect acceptor substrate-induced conformational changes and to anticipate such changes to be dependent on the chemical and structural properties of a particular substrate. Supporting this hypothesis is the evidence of aglycone-dependent sugar donor kinetics in human and animal liver microsomes. The wide range of apparent K_m values of UDP-GlcA with different aglycones (Lin and Wong, 2002) has suggested that the binding affinity of this sugar donor is dependent on the chemical nature of the acceptor substrate; in other words, the interaction of acceptor substrate with the enzyme. It appears that such interaction not only affects the affinity of enzyme to its preferred UDP-sugar, but in some cases alters the selectivity of sugar donors. Depending on the aglycones, UGTs can be highly selective for UDP-GlcA (most common), accommodative for other sugar donors (Compound A), or highly selective for UDP-Glc rather than UDP-GlcA (an aldose reductase inhibitor; Toide et al., 2004).

Compared with diclofenac, Compound A is bulkier and possesses more hydrogen bond donors and acceptors. Thus, once binding to the enzyme, presumably through additional Van der Waals forces or hydrogen bonds, Compound A may elicit conformational changes that assist in positioning Compound A and UDP-Glc in an orientation/alignment optimal for an in-line displacement reaction mechanism (S_N2), a situation identical to the glucuronidation catalyzed by mammalian UGTs (Radominska-Pandya et al., 1999; Qasba et al., 2005). However, the binding of diclofenac to UGT2B7 may be unable to induce such conformational changes favorable for UDP-Glc. On the other hand, it appears that the lack of the carboxyl group is more readily compensated in UDP-Glc than in UDP-gal, as exemplified in this study by the large difference in the activities of glucosyltransferase and galactosyltransferase with respect to Compound A. This finding indicates the importance of configuration of the C4 hydroxyl group on sugar moiety, because the only difference between UDP-Glc and UDP-gal is the equatorial versus axial hydroxyl group. In addition, UDP-GlcNAc lacks 2-hydroxyl on the sugar portion and it is not accommodated at all. Clearly, chemical and structural properties of sugar donors could constitute a contributing factor to this occurrence.

In summary, we herein report that UGT2B7 is capable of transferring the glycosyl group from an additional sugar nucleotide, UDP-gal, to Compound A, and have demonstrated that the sugar donor selectivity of this enzyme is aglycone-dependent. Although conformational changes induced by aglycone binding to UGT2B7 are postulated to contribute to altered sugar donor selectivity, a full understanding of the mechanism is awaiting the availability of the crystal structure of UGT2B7. However, computational methodologies have been developed to predict acceptor substrate selectivity and to provide a measure of substrate binding (Smith et al., 2003, 2004; Sorich et al., 2002, 2004). Therefore, with this approach, compounds known to have the ability to change sugar donor selectivity could be utilized for further investigation of the properties of the corresponding UGTs.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.005801.

ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; UDP-GlcA, UDP-glucuronic acid; UDP-Glc, UDP-glucose; UDP-gal, UDP-galactose; UDP-GlcNAc, UDP-N-acetyl-glucosamine; K_m, apparent Michaelis constant; K_i, inhibition constant; V_max, maximal velocity.

The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Cuyue Tang, Department of Drug Metabolism, Merck Research Laboratories, Sumneytown Pike, P.O. Box 4, WP75-100, West Point, PA 19486-0004. E-mail: cuyue_tang{at}merck.com

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