Short Communication A Fluorescent Assay Amenable to Measuring Production of b-D-Glucuronides Produced from Recombinant UDP-Glycosyl Transferase Enzymes

b-glucuronidase cleavage of 4-methylumbelliferyl b-D-glucuronide generates the highly fluorescent compound, 4-methylumbelliferone. We show that other b-D-glucuronide compounds act as competitors in this assay. The 4-methylumbelliferyl b-D-glucuronide cleavage assay can easily be adapted to high throughput formats to detect the presence of b-D glucuronides generated using recombinant glycosyl transferase preparations. Thousands of potentially important therapeutic agents are being generated through the use of combinatorial chemistry. Many of the candidate compounds chosen for development fail in clinical trials due to poor pharmacokinetic properties. Obtaining relevant in vitro drug metabolism data at an early stage is considered to help improve the successful selection of candidate compounds. This includes, as a first step, identifying the enzymes responsible for metabolism of a compound and then determining kinetic parameters, which, through appropriate mathematical manipulations, show utility in predicting pharmacokinetic parameters in humans (Obach et al., 1997). UDP glycosyltransferases (UGTs) are a class of enzymes involved in the conjugation of glucuronic acid to a variety of drugs, xenobiotics, and endogenous steroid compounds (Burchell et al., 1995). In humans, at least 15 isoenzymes are encoded by two gene subfamilies (Mackenzie et al., 1997). Glucuronidation is known to be an important detoxification pathway that can lead not only to changes in the pharmacokinetic and pharmacodynamic properties of therapeutic agents, but also to the production of reactive metabolites (Parkinson, 1996). Traditional analytical procedures for measuring glycosyltransferase activities and metabolites, using radiolabeled substrates or cofactors with resolution by thin-layer chromatography or HPLC (Bansal and Gessner, 1980; Ethell et al., 1998), are not suited for high throughput formats. A more rapid, nonradioactive assay that can identify glucuronide formation or presence would be of great utility. Materials and Methods Methods are included in the text and figure legends. b-D-glucuronidase (Part G-7396), a-napthyl b-D-glucuronide, b-trifluoromethylumbelliferyl b-D-glucuronide, b-estradiol 3-(b-D-glucuronide), p-acetominophenyl b-D-glucuronide, 5bandrostane-3a,17a-diol-11-one-17-carboxilic acid-3-(b-D-glucuronide), uridine 59-diphosphoglucuronic acid (UDPGA), and 4-methylumbelliferone b-D-glucuronide were obtained from Sigma Chemical Co. (St. Louis, MO). Tetrahydrocortisone 3-b-D-glucuronide was obtained from Molecular Probes (Eugene, OR). Recombinant control and UGT1A6 membrane preparations were generated at PanVera Corporation and are commercially available from PanVera (Madison, WI). All other reagents were analytical grade or better and purchased from a variety of commercial sources. Results and Discussion We sought to develop an assay that can be used to demonstrate the presence of b-D-glucuronides that might be generated from different classes of UGT enzymes. An assay based on b-glucuronidase activity provides a high throughput screening method to identify structurally different b-D-glucuronides. Cleavage of 4-methylumbelliferyl b-D-glucuronide yielded the highly fluorescent compound 4-methylumbelliferone. Under linear conditions of protein concentration and incubation time, the apparent Km value for cleavage of 4-methylumbelliferyl-b-D-glucuronide was approximately 56 mM (Fig. 1). To show the feasibility of developing a high throughput inhibition assay, we examined the potential of a variety of structurally dissimilar commercially available b-D-glucuronides to act as inhibitors for the cleavage of 4-methylumbelliferyl-b-D-glucuronide. These b-D-glucuronides represent compounds that might potentially be formed by UGT activity, e.g., phenol and steroid glucuronides. Figure 2 shows that all of the b-glucuronides tested inhibited the production of 4-methylumbelliferone by b-glucuronidase. The potential to inhibit 4-methylumbelliferyl-b-D-glucuronide cleavage appeared to be dependent on the chemical nature of the substituted aglycone, with b-estradiol-3-(b-D-glucuronide) and a-naphthyl b-D-glucuronide showing the strongest and weakest inhibition, respectively. UDPGA, an essential cofactor for UGTs, was not an effective competitor over the range tested, probably due to the a configuration of the sugar bond to UDP (Parkinson, 1996). To evaluate whether the fluorescent assay could be used to detect b-glucuronides generated from a biochemical assay, two approaches were taken. First, a-naphthyl b-D-glucuronide was generated in an incubation with recombinant human UGT1A6 and a-naphthol. Fol1 Abbreviations used are: UDPGA, uridine 59-diphosphoglucuronic acid, UGT; UDP-glycosyltransferase. Send reprint requests to: Peter M. Shaw, Bristol-Myers Squibb, New Jersey, P.O. Box 5400, Princeton, NJ 08543-5300. Email: [email protected]; and Olga V. Trubetskoy, PanVera Corporation, 545 Science Dr., Madison, WI 53711. Email: [email protected] 0090-9556/99/2705-0555–557$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 27, No. 5 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 555 at A PE T Jornals on A uust 4, 2017 dm d.aspurnals.org D ow nladed from