UDP-Glucuronosyltransferase inhibitors

EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, 1998, Vol. 23, No.4, pp. 453-456

UDP-Glucuronosyltransferase inhibitors E. GOLOVINSKY, Z. NAYDENOVA, K. GRANCHAROV Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria

Keywords: Glucuronidation, UDP-glucuronosyltransferases, detoxification pathway

INTRODUCTION

DESIGN OF UGT INHIBITORS

Glucuronidation is a major detoxification pathway in vertebrates. The reaction is catalyzed by a family of closely related enzymes, the UDP-glucuronosyltransferases (UGT, E.C. 2.4.1.17) (1- 4) and involves the transfer of glucuronic acid from UDP-glucuronic acid (UDPGA) to countless structurally unrelated substances possessing hydroxyl-, amino- , carboxyl- or sulfhydryl groups, converting them to water-soluble ~-(D)-glucu­ ronides.

Over the past 10-12 years, several groups of compounds that specifically inhibit UGTs in vitro have been synthesized. Triphenylacetic acid and the chemically related arylcarboxylic acids have been shown to be potent inhibitors of rat and human liver microsomal bilirubin UGTs (17,18) with the most powerful inhibitor being 7,7,7-triphenylheptanoic acid. We found new effective, specific UGT inhibitors among a wide range of differently substituted pyrimidine derivatives which could be considered as analogs of uracil or the uridine moiety of UDPGA (19). 5- Nitrouracil and its isomer, 4,6-dihydroxy-5-nitropyrimidine, were found to be most potent and selective inhibitors of 4-nitrophenol (4-NP) glucuronidation, and without any effect on phenolphthalein (PPh) conjugation, known to be associated with different UGT isoforms present in the rat liver (20). The inhibition by 5-nitrouracil was found to be mixed type toward 4-NP and atypically competitive toward UDPGA (apparent Ki 0.11 and 0.2 mM, respectively). Significant inhibition of 4-NP-UGT activity also produced three benzylaminosubstituted analogs. Of the orotic acid derivatives synthesized in our laboratory, N"-orotyl-p-chlorophenylalanine N'N'-N"-bis(2-chloroethyl)hydrazide, a novel pyrimidine analog, caused a marked suppression of both 4-NP-UGT and PPh-UGT activity. As a whole, the pyrimidine bases were more inhibitory against 4-NP glucuronidation. This suggests differences in the binding sites of the UGT isoforms that convert 4-NP and PPh, respectively.

Of all the organs in the body the liver has been established as the most important glucuronidation site. Experiments involving chemical modification of UGTs have shown the possible involvement of a histidine residue and a carboxyl group in the catalytic process (5,6). Previously, the number of UGTs was evidenced by their differential inducibility by 3-methylcholantrene or phenobarbital. Recently, multiple forms of UGTs have been observed via their purification to apparent homogeneity and particularly by cloning, sequencing and expression of the cDNAs in cultured cells (7-12). UGTs play an important cytoprotective role either by preventing the accumulation of potentially toxic xenobiotics or by avoiding their subsequent bioactivation to even more toxic reactive intermediates (12- 14) So a series of toxicological events might be expected as a result of in vivo inhibition of the glucuronidation reaction by different compounds. The development of selective inhibitors for the various UGT isoforms would facilitate the study of the active sites of the various enzyme forms. If such inhibitors were also active in vivo, they could be useful in obtaining a better insight into the role and biological significance of a particular conjugation pathway in the detoxification of a number of drugs and xenobiotics (15,16).

Please send reprint requests to: Z. Naydenova, Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad.G.Bonchev Str. B1.21, 1113 Sofia, Bulgaria

A recent approach towards the synthesis of selective, active site - directed inhibitors has been based on the presumed transition state of the glucuronidation reaction (Fig.1). Noort et al.(21) have designed a series of possible transition-state analogs of the glucuronidation reaction. They contain both a UDP moiety and a lipophilic acceptor substrate moiety. 2-(I-Naphthyl)ethyl-UDP and 2,2,2-(triphenyl)ethyl-UDP were found to be the most effective inhibitors of 4-NP glucuronidation in rat liver microsomes, with a com-

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Eur. J. Drug Metab. Pharmacokinet., 1998, No. 4

COO~ ,f[3JR OH

: 0 ',II OH

OH

HN~I

Inhibitory effects of pesticides on UGT activity in rat liver microsomes.

O~N

0 II OH

HO Fig. 1.

Table I.

OH

Presumed transition state for the glucuronidation reaction. R = substituent on the phenyl ring (21).

petitive inhibitory effect towards UDP-glucuronic acid and 4-NP, which should be the case if these inhibitors function as transition-state analogs. As an extension of this study, a series of chemically related ill, ill, illtriphenylalcohols and the corresponding ill, ill, co-triphenylalkyl-UDP derivatives were synthesized and tested on various UGTs including recombinant rat liver UGTs expressed in V79 cells (22). UDP-linked inhibitors have shown. a high inhibitory potency which increases as a function of the length of the hydrocarbon chain. The above compounds were designed to take advantage of both the high affinity of UGT for the UDP moiety of UDPGA and the specific structural requirements for an acceptor substrate. It was also suggested that both phosphate groups might be required for correct binding. However, structurally related compounds in which the linker between uridine and aglycone was replaced by a diphosphate-like 5-atom spacer have been reported to be strong inhibitors of various UGT isoforms. These compounds contain lipophilic N-acyl phenylaminoalcohol derivatives which are linked to uridine or isopropylideneuridine (23-25). Some of the inhibitors were found to behave as transition-state analogs. In an attempt to develop effective UGT inhibitors as possible transition-state analogs, various protected L-amino acids were linked to 5'-O-isopropylideneuridine, thus introducing a variable spacer between the nucleoside and a lipophilic residue (26,27). The serine derivative of isopropylideneuridine was found to be a most potent inhibitor of both 4-NP and PPh glucuronidation, with 1050 values of 0.45 and 0.22 mM, respectively. Leucine and valine derivatives were also active at a 1 mM concentration, eliciting a similar decrease in 4-NP or PPh conjugation (49-55%).

For details, see Refs. 28 and 29.

Inhibition of UGT by environmental chemicals An important ecological aspect of the present study was to examine the influence of some widely used pesticides as well as their metabolites on different isoforms of this enzyme. A series af pesticides representative of different chemical groups were tested for their inhibitory effect on the glucuronidation of 4-NP and PPh by rat liver microsomes (28,29). The herbicides simazine, propazine, prometryn, ametryn, cyanazine, chlorsulfuron, the insecticides dioxacarb, carbaryl and the fungicide zineb significantly decreased UGT activity (Table I). The carbamate insecticide dioxacarb was found to be the most potent inhibitor; at a 1 mM concentration it suppressed 4-NP-UGT activity completely, and over half the activity associated with PPh conjugation. The same concentration of simazine and carbaryl only affected 4-NP glucuronidation, while prometryn and cyanazine were found to be selective inhibitors of PPh conjugation. Chlorsulfuron, propazine and zineb caused marked inhibition of both 4-NP and PPh conversion. Concentrations of 0.1 mM carbaryl, dioxacarb and zineb were still inhibitory against 4-NPUGT, with zineb exhibiting activity even at 0.01 mM.

New UGT inhibitors are being designed. Recently developed transition-state analogs may be useful in gaining insight into the active site of various UGT forms as well as the pharmaceutical agents which prevent the rapid elimination of a number of drugs

E. Golovinsky et al., UDP-Glucuronosyltransferase inhibitors from the organism. On the other hand, numerous environmental contaminants (pesticides, industrial pollutants and known toxic substances), natural mutagens and carcinogens could affect these detoxifying enzymes,

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with unpredictable toxicological consequences. Studies are now being undertaken to elucidate this aspect of UGT inhibition.

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