Characterization of a new class of inhibitors of the recombinant human liver UDP-glucuronosyltransferase, UGT1*6.

The inhibitory effect of a series of novel structurally related compounds on the human UDP-glucuronosyltransferase UGT1*6 stably expressed in a V79 cell line was investigated. The inhibitors contain a lipophilic N-acyl phenylaminoalcohol residue and a uridine moiety connected by a spacer varying for each compound. The effects of these compounds on the glucuronidation reaction measured with 4-methylumbelliferone as substrate were determined. The best inhibitor of the series, D-DPMSU, had an IC50 of 39 microM in the assay conditions. Low Ki values were found toward both UDP-glucuronic acid and 4-methylumbelliferone (17 and 21 microM, respectively). The inhibition was competitive toward both substrates. A similar strong and competitive inhibitory effect was observed with two other inhibitors, DHPASU and DHPASiU. Another compound, D-DPASiU, showed a pure competitive inhibition towards UDP-glucuronic acid, but a non-competitive inhibition towards the acceptor substrate. These data and the optimization of the structures of the inhibitors by molecular modeling suggest that D-DPMSU and DHPASiU compounds may be transition state analog inhibitors of the recombinant UGT1*6 enzyme.

Characterization of UDP-glucuronic acid transport in rat liver microsomal vesicles with photoaffinity analogs.

The endoplasmic reticulum (ER) of rat liver contains several well characterized UDP-glucuronosyltransferases (UGTs), membrane-bound proteins of 50-54 kDa, and also less well identified UDP-glucosyltransferases, with nucleotide binding sites located on the lumenal surface. There is evidence that the substrates for these enzymes, UDP-glucuronic acid (UDP-GlcUA) and UDP-glucose (UDP-Glc), biosynthesized in the cytosol, are transported into the lumen of the ER via unknown mechanisms, the characteristics of which are poorly defined. A new approach for the study of the transport process has been devised using two active-site directed photoaffinity analogs, [beta-32P]5-azido-UDP-GlcUA and [beta-32P]5-azido-UDP-Glc. Photoincorporation of these probes into the lumenally oriented UGTs of intact rat liver microsomal vesicles was used as an indicator of transport. In intact vesicles, [32P]5N3UDP-GlcUA was efficiently incorporated into UGTs in a time, temperature and concentration dependent manner. In contrast, [32P]5N3UDP-Glc apparently was not transported effectively; maximal photolabeling of the 50-54 kDa proteins by this probe was dependent on detergent disruption of the vesicles. Vesicular uptake of and subsequent photolabeling of the 50-54 kDa proteins by [32P]5N3UDP-GlcUA were inhibited by UDP-GlcUA and 5N3UDP-GlcUA while UDP-Glc, 5N3UDP-Glc, UDP-xylose and UDP-N-acetylglucosamine were less inhibitory, suggesting a high degree of specificity for the uptake/photolabeling process. The anionic transport inhibitors DIDS and SITS inhibited [32P]5N3UDP-GlcUA photoincorporation into UGTs in intact vesicles, but also inhibited photolabeling of these and other enzymes in detergent disrupted vesicles. These data suggest the presence in rat liver microsomal vesicles of a specific, carrier-mediated transport process for UDP-GlcUA which is distinct from the mechanism of UDP-Glc transport.

Photoaffinity labeling for evaluation of uridinyl analogs as specific inhibitors of rat liver microsomal UDP-glucuronosyltransferases.

The UDP-glucuronosyltransferases (UGT) involved in glucuronidation of endogenous and exogenous toxic compounds transfer the glucuronic acid residue from UDP-glucuronic acid (UDP-GlcUA), to various acceptor groups. A series of compounds that contain N-acyl phenylaminoalcohol derivatives linked to uridine or isopropylideneuridine were tested as UGT inhibitors. The potency of these inhibitors was determined by studying their effect on the photoaffinity labeling of rat liver microsomal UGTs by two photoaffinity probes, [beta-32P]5-azido-UDP-glucuronic acid (5N3UDP-GlcUA) and [beta-32P]5-azido-UDP-glucose (5N3UDP-Glc) and on the enzymatic formation of the two glucuronide conjugates (3-O- and carboxyl-specific) of lithocholic acid. All but one of the compounds tested proved to have an inhibitory effect on UGTs, both in the photoaffinity labeling system and in the enzymatic glucuronidation assay. In the photoaffinity labeling system, the inhibitors containing the isopropylidene moiety were less effective than their unprotected derivatives; however, the protected forms were, with one exception, more potent inhibitors of enzymatic activity. The photoaffinity labeling of UGTs with [beta-32P]5N3UDP-Glc was more susceptible to inhibition by all derivatives than that with [beta-32P]5N3UDP-GlcUA. The effect of one inhibitor, PP50B, on the two enzymatic activities involved in LA glucuronidation was extensively tested. A double-reciprocal plot suggested a competitive inhibition for UDP-GlcUA with an apparent Ki of 35 microM for LA 3-O-glucuronide formation and 94 microM for the carboxyl-linked glucuronide of the same substrate.

Determination of the human liver UDP-glucuronosyltransferase 2B4 domains involved in the binding of UDP-glucuronic acid using photoaffinity labeling of fusion proteins.

The interactions between UDP-glucuronic acid and two human liver UDP-glucuronosyltransferase 2B4 peptides (14-150 and 299-446) purified from E. coli as Staphylococcus aureus protein A fusion proteins have been investigated. Photoaffinity labeling with azidonucleotides ([beta-32P]5N3UDP-Glucuronic acid and [beta-32P]5N3UDP-Glucose) and competition experiments with UDP-glucuronic acid and structurally related compounds emphasized the presence of a specific UDP binding site between amino acids 299 and 446. Moreover, competition experiments strongly suggested an interaction between the amino terminal part of the protein and glucuronic acid. It would involve an electrostatic bond in the binding of the cosubstrate via the carboxyl group of UDP-glucuronic acid and a positively charged amino acid of the N-terminal domain of the enzyme.

Purification and characterization of a catalytically active human liver UDP-glucuronosyltransferase expressed as a fusion protein in E. coli.

The purification and the characterization of functional human liver UDP-glucuronosyltransferase 2B4 produced as a Staphylococcus aureus protein A fusion protein in E. coli are described. The purified fusion protein was able to catalyze the glucuronidation of hyodeoxycholic acid, the major substrate described for this isoform to date. The effects of the amount and the nature of the phospholipids upon reconstitution into phospholipid micelles were investigated. Apparent determined Km values for hyodeoxycholic acid and UDP-glucuronic acid were 0.55 and 0.43 mM, respectively. Moreover, photoaffinity labelling of the fusion protein with a photoactivatable analog of UDP-glucuronic acid strongly suggested that this recombinant protein exhibited similar binding properties as the microsomal protein, which emphasizes its use for further structural analyses.