Inhibition of UDP-glucuronosyltransferase activity by possible transition-state analogues in rat-liver microsomes (original) (raw)
Related papers
Biochimica et Biophysica Acta (BBA) - General Subjects, 1995
The inhibitory effect of a series of novel structurally related compounds on the human UDP-glucuronosyltransferase UGTl * 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 IC,, of 39 PM in the assay conditions. Low Ki values were found toward both UDP-glucuronic acid and 4-methylumbelliferone (17 and 21 PM, 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, o-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 UGTl* 6 enzyme.
Novel inhibitors and substrates of bilirubin: UDP-glucuronosyltransferase Arylalkylcarboxylic acids
European Journal of Biochemistry, 1989
The in vitro inhibitory potency of 20 structurally related alkanoic and arylalkanoic acids has been investigated on rat liver UDP-glucuronosyltransferase. These compounds were tested on the microsomal and purified enzyme, and a cloned cDNA expressed in COS 7 cell cultures. Among all the acids tested, 7,7,7-triphenylheptanoic acid was the most powerful inhibitor of bilirubin : UDP-glucuronosyltransferase with a lower effect on 1-naphtol, androsterone and testosterone glucuronidation. The inhibition was competitive towards the microsomal and purified bilirubin : UDP-glucuronosyltransferases with K!pp values of 12.0 pM and 1.6 pM, respectively. Twenty analogues were examined, and the results showed that their inhibitory potency on bilirubin : UDP-glucuronosyltransferase activity was a function of at least three structural features (a) the presence of a hydrophobic triphenyl moiety; (b) the length of the aliphatic chain and (c) the presence of a carboxylic group. These inhibitors were also tested as possible substrates of UDP-glucuronosyltransferases. The strongest inhibitors were poor substrates of rat liver microsomal UDP-glucuronosyltransferases. However, 7,7,7-triphenylheptanoic acid was actively glucuronidated by purified bilirubin : UDP-glucuronosyltransferase, in contrast to its analogues with decreasing alkyl chain length. In addition, glucuronidation of this molecule was enhanced by clofibrate treatment but could not be detected in Gunn rats, which are deficient in bi1irubin:UDPglucuronosyltransferase, further indicating that the glucuronidation of this compound was catalysed by bilirubin : UDP-glucuronosyltransferdse. The results suggest that 7,7,7-triphenylheptanoic acid may be a useful structural probe to investigate the molecular basis of glucuronidation of bilirubin and carboxylic acids. Conjugation to glucuronic acid enhances the solubility of a large variety of endogenous and exogenous molecules, which are therefore easily eliminated in bile or urine. However, recent reports have documented that several therapeutic agents with a carboxylic group are possibly responsible for adverse reactions via their acylglucuronide metabolites [l]. Bilirubin IXa, the endogenous final product of heme breakdown, also forms unstable glucuronides, which may become covalently bound to albumin in some pathological conditions [2]. Glucuronide formation is catalysed by UDP-glucuronosyltransferase (UDPGT), a multigene family of enzymes including at least three subfamilies. Functional heterogeneity has been defined in earlier studies (see [3-51 for review). Enzyme purification and expression of the cDNAs corresponding to a single enzyme in mammalian cell cultures allowed a partial characterization of the substrate specificity of the rat and human UDPGTs involved in the conjugation of hydroxylated molecules (steroids and phenols) [6-101.
Journal of Biochemical and Molecular Toxicology, 1998
A series of potent and competitive inhibitors of UDP-glucuronosyltransferase derived from 7,7,7-triphenylheptanoic acid has been synthesized in order to probe the active site of the isozyme involved in the glucuronidation of the endogenous toxic compound, bilirubin IX␣. Like triphenylalkylcarboxylic acids, triphenyl alcohols were found to be very effective competitive inhibitors of the reaction (K i 12 to 180 lM). Superimposition of the best inhibitors with bilirubin by computer modeling showed a marked spatial similarity, which accounts for the observed competitive-type inhibition. The bulky triphenylmethyl moiety of the inhibitor superimposed well on the part of the bilirubin molecule containing three of the four pyrrole rings. In agreement, substitution of the triphenylmethyl moiety by planar structures such as fluorenyl or indenyl rings completely suppressed the inhibition. In addition, the weak inhibition exerted by the shortest carboxylic acids could be related to the higher acidity of these molecules. The inhibition potency depended on the acidity of the molecules; the more acidic, the less inhibitory, suggesting that the presence of a negative charge on the inhibitor molecule prevents bilirubin glucuronidation. Based on these results, a reaction mechanism for bilirubin glucuronidation is postulated.
Natural and synthetic inhibitors of UDP-glucuronosyltransferase
Pharmacology & Therapeutics, 2001
Glucuronidation is a major detoxification pathway in vertebrates. The reaction is catalyzed by a family of UDP-glucuronosyltransferases (UGTs) and involves conjugation of many endobiotic and xenobiotic substances with glucuronic acid, forming inactive water-soluble glucuronides. UGT prevents the accumulation of potentially toxic compounds and/or their subsequent bioactivation to more toxic intermediates, although biologically active glucuronides are also known. Impairment of UGTs may have important toxicological consequences. Substances found to inhibit or down-regulate UGT activity include endogenous compounds, a wide range of clinically used drugs, environmental contaminants, and natural toxic substances present in the diet. The development of selective, active-site-directed UGT inhibitors greatly enables the study of various UGT isoenzymes. A promising approach offers the design of transition-state analogs of the glucuronidation reaction. D
Drug Metabolism and Disposition, 2005
Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGTs). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and human liver microsomes in a concentration-dependent manner (fu inc 0.20-0.59). When corrected for nonspecific binding, K m values for TFP glucuronidation were similar for both UGT1A4 (4.1 M) and human liver microsomes (6.1 ؎ 1.2 M) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin, alone, was selective; significant inhibition was observed only for UGT1A4 (IC 50 1.5 M). Using phenylbutazone and quinine as "models," inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further, because the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7, with respective K i values of 335 ؎ 128 M and 186 M. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive nonselective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone are nonselective UGT inhibitors.
Drug Metabolism and Disposition, 2005
Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGT). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine and sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7 and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and to human liver microsomes in a concentration-dependent manner (fu inc 0.20-0.59). When corrected for non-specific binding, K m values for TFP glucuronidation were similar for both UGT1A4 (4.1 µM) and human liver microsomes (6.1 ± 1.2 µM) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin alone was selective; significant inhibition was observed only for UGT1A4 (IC 50 1.5 µM). Using phenylbutazone and quinine as 'models', inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further since the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7 with respective K i values of 335 ± 128 µM and 186 µM. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive non-selective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine and sulfinpyrazone are non-selective UGT inhibitors.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1994
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, [fl-3Zp]5-azido-UDP-glucuronic acid (5N3UDP-GIcUA) and [fl-32p]5-azido-UDP-glucose (5NaUDP-Gic) and on the enzymatic formation of the two glucuronide conjugates (3-0-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 [/3-32p]5N3UDP-Glc was more susceptible to inhibition by all derivatives than that with [fl-32p]5NaUDP-GIcUA. 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-GIcUA with an apparent K i of 35/xM for LA 3-O-glucuronide formation and 94/xM for the carboxyl-linked glucuronide of the same substrate.