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.