Influence of mutations associated with Gilbert and Crigler-Najjar type II syndromes on the glucuronidation kinetics of bilirubin and other UDP-glucuronosyltransferase 1A substrates

OBJECTIVES: UGT1A1 coding region mutations, including UGT1A1*6 (G71R), UGT1A1*7 (Y486D), UGT1A1*27 (P229Q) and UGT1A1*62 (F83L), have been linked to Gilbert syndrome in Asian populations, whereas homozygosity for UGT1A1*7 is associated with the Crigler-Najjar syndrome type II. This work compared the effects of (a) the individual UGT1A1 mutations on the glucuronidation kinetics bilirubin, beta-estradiol, 4-methylumbelliferone (4MU) and 1-naphthol (1NP), and (b) the Y486 mutation, which occurs in the conserved carboxyl terminal domain of UGT1A enzymes, on 4MU, 1NP and naproxen glucuronidation by UGT1A3, UGT1A6 and UGT1A10. METHODS: Mutant UGT1A cDNAs were generated by site-directed mutagenesis and the encoded proteins were expressed in HEK293 cells. The glucuronidation kinetics of each substrate with each enzyme were characterized using specific high-performance liquid chromatography (HPLC) methods. RESULTS: Compared with wild-type UGT1A1, in-vitro clearances for bilirubin, beta-estradiol, 4MU and 1NP glucuronidation by UGT1A1*6 and UGT1A1*27 were reduced by 34-74%, most commonly as a result of a reduction in Vmax. However, the magnitude of the decrease in the in-vitro clearances varied from substrate to substrate with each mutant. The glucuronidation activities of UGT1A1*7 and UGT1A1*62 were reduced by >95%. Introduction of the Y486D mutation essentially abolished UGT1A6 and UGT1A10 activities, and resulted in 60-90% reductions in UGT1A3 in-vitro clearances. CONCLUSIONS: The glucuronidation of all UGT1A1 substrates is likely to be impaired in subjects carrying the UGT1A1*6 and UGT1A1*62 alleles, although the reduction in metabolic clearance might vary with the substrate. The Y486D mutation appears to greatly reduce most, but not all, UGT1A activities.     

Glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan (CPT-11), by human UGT1A1 variants, G71R, P229Q, and Y486D.

7-Ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of antitumor agent irinotecan (CPT-11), is conjugated and detoxified to SN-38-glucuronide by UDP-glucuronosyltransferase (UGT) 1A1. Genetic polymorphisms in UGT1A1 are thought to contribute to severe diarrhea and/or leukopenia caused by CPT-11. In this regard, it has been reported that polymorphisms in the promoter region could affect the CPT-11 pharmacokinetics and interindividual variation of toxicity. However, little information is available on the influence of UGT1A1 polymorphisms in the coding region on the SN-38 glucuronidation activity. In the present study, wild-type (WT) and three variant (G71R, P229Q, and Y486D) cDNAs of human UGT1A1s were transiently expressed in COS-1 cells, and the kinetic parameters of these UGT1A1s were determined for SN-38 glucuronidation. A partially reduced UGT1A1 protein expression was observed in COS-1 cells for G71R and Y486D. WT UGT1A1 catalyzed SN-38 glucuronidation with an apparent K(m) value of 11.5 microM, whereas those of G71R, P229Q, and Y486D were 14.0, 18.0, and 63.5 microM, respectively. The SN-38 glucuronidation efficiency ratio (V(max)/K(m)) normalized for the level of expression was 1.4, 0.66 (47% of WT), 0.73 (52%), and 0.07 (5%) microl/min/mg of protein for WT, G71R, P229Q, and Y486D, respectively. Thus, the SN-38 glucuronidation activity of Y486D was drastically reduced, whereas the reduction in the G71R and P229Q activities was fractional. The decreased SN-38 glucuronidation efficiency ratio of G71R and P229Q could be critical in combination with other polymorphisms in the UGT1A1 gene.     

Key amino acid residues responsible for the differences in substrate specificity of human UDP-glucuronosyltransferase (UGT)1A9 and UGT1A8

Human UDP-glucuronosyltransferase (UGT)1A9 is one of the major isoforms in liver and extrahepatic tissues, catalyzing the glucuronidation of a variety of drugs, dietary constituents, steroids, fatty acids, and bile acids. UGT1A9 shows high amino acid homology with UGT1A7, UGT1A8, and UGT1A10 with overlapping substrate specificity. However, the affinities for substrates are different among them. Amino acid alignment analysis revealed that 14 amino acids, Cys3, Arg42, Lys91, Ala92, Tyr106, Gly111, Tyr113, Asp115, Asn152, Leu173, Leu219, His221, Arg222, and Glu241, are unique to UGT1A9 compared with UGT1A7, UGT1A8, and UGT1A10. In this study, we constructed expression systems in human embryonic kidney 293 cells for seven mutants (Mut) UGT1A9, Mut 1 (R42Q), Mut 2 (K91M, A92D), Mut 3 (Y106F, G111S, D115G), Mut 4 (N152A), Mut 5 (L173A), Mut 6 (L219F, H221Q, R222Y), and Mut 7 (E241A), in which the amino acids were substituted to those of UGT1A8. Using these mutants, the effects of the amino acid changes on the activities of 4-methylumbelliferone (4-MU), p-nitrophenol (p-NP), and 3-hydroxydesloratadine glucuronidations were investigated. For 4-MU and p-NP O-glucuronidations, Mut 1 and Mut 4 exhibited higher K(m) values and Mut 3 and Mut 4 exhibited higher V(max) values compared with wild-type UGT1A9. It is interesting to note that only Mut 4 was active toward 3-hydroxydesloratadine O-glucuronidation that is specific for UGT1A8. The findings reveal that the residues Arg42 and Asn152 may have a large contribution to the difference in the substrate specificity with that of UGT1A8, although all of the unique amino acids of UGT1A9 would be collectively involved in the catalytic property.     

Morphine glucuronide-to-morphine plasma ratios are unaffected by the UGT2B7 H268Y and UGT1A1*28 polymorphisms in cancer patients on chronic morphine therapy

OBJECTIVE: UDP-glucuronosyltransferase (UGT) 2B7 is the major UGT isoform responsible for the 3- and 6-glucuronidation of morphine in humans. Studies in rats have indicated that UGT1A1 may also contribute to the formation of morphine 3-glucuronide (M3G). Our objective was to investigate whether the UGT2B7 H268Y and UGT1A1*28 polymorphisms contribute to the variability in morphine glucuronide-to-morphine plasma ratios among cancer patients undergoing analgesic therapy with morphine. METHODS: Seventy patients with normal hepatic and renal function using slow-release morphine to relieve cancer pain were included. UGT2B7 genotyping was performed using restriction enzyme analysis of polymerase chain reaction (PCR)-amplified DNA fragments. Wild-type and variant alleles of the UGT1A1 gene were identified using sizing of PCR-amplified fragments. Morphine 6-glucuronide (M6G)/morphine, M3G/morphine, and M3G/M6G plasma ratios were compared between genotypes. RESULTS: The M3G/morphine, M6G/morphine, and M3G/M6G plasma ratios varied 16-, 42-, and sevenfold, respectively, among individuals. No statistically significant differences in plasma ratios were found between individuals possessing UGT2B7 H/H ( n=20), H/Y ( n=30), or Y/Y ( n=20) genotypes. Five patients were homozygous for the UGT1A1 TA(7) allele, which is associated with reduced UGT1A1 gene expression. However, the mean M3G/M6G and M3G/morphine plasma ratios in TA(7) homozygous subjects did not differ significantly from those of heterozygous or homozygous wild-type (TA(6)) individuals. CONCLUSION: The UGT2B7 H268Y polymorphism cannot account for the considerable variation in glucuronide-to-morphine ratios in cancer patients. Moreover, the contribution of UGT1A1 to the formation of M3G appears to be of minor biological significance, at least in a UGT2B7 background.     

Effect of D256N and Y483D on propofol glucuronidation by human uridine 5'-diphosphate glucuronosyltransferase (UGT1A9)

Uridine 5'-diphosphate glucuronosyltransferases (UGTs) are part of a major elimination pathway for endobiotics and xenobiotics. UGT1A9 is a UGT that catalyses the conjugation of endogenous oestrogenic and thyroid hormones, acetaminophen, SN-38 (an active metabolite of irinotecan) and phenols. UGT1A9 is the only isoform that catalyses the glucuronidation of propofol (2,6-diisopropylphenol) in the liver. In the present study, we analysed polymorphisms of UGT1A9 in 100 healthy adult Japanese volunteers. A transversion of 766G > A resulting in the amino acid substitution of D256N was detected in exon 1. The allele frequency of D256N is 0.005. We investigated the effects of D256N and Y483D, which is located on the common exon of UGT1, on propofol glucuronidation by an in vitro expression study. The K(m) of wild-type, D256N and Y483D for propofol glucuronidation were 111.2, 43.6 and 64.5 microM, respectively. The V(max) of D256N and Y483D were 8.1% and 28.8%, and the efficiencies (V(max)/K(m)) were 19.1% and 57.1% of the wild-type, respectively. For mycophenolic acid, 1-naphthol and naringenin, the D256N variant lowered glucuronidation activity considerably, compared to Y483D. The V(max) value of D256N variant for mycophenolic acid was only 9.5% of the wild-type. This study shows the importance of D256N in differences between individuals concerning adverse effects of drugs that are catalysed primarily by UGT1A9. Carriers of D256N may be at risk of suffering adverse effects of propofol and other substrates that are primarily metabolized by UGT1A9.     

Influence of genetic variants in UGT1A1 and UGT1A9 on the in vivo glucuronidation of SN-38

The uridine diphosphate glucuronosyltransferase (UGT) 1A1 and 1A9 isoforms are involved in the phase II biotransformation of the irinotecan metabolite, SN-38. Recently, several variants in the UGT1A1 and UGT1A9 genes have been described with altered functionality in vitro. The aim of this study was to evaluate the functional consequence of the UGT1A1(TA)(7)TAA (UGT1A1(*)28), UGT1A9 766G>A (D256N; UGT1A9(*)5), and UGT1A9 98T>C (M33T; UGT1A9(*)3) variants in Caucasian patients treated with irinotecan. Pharmacokinetic studies were performed after the first course of irinotecan in 47 males and 47 females. The mean (SD) area under the curves (AUCs) of irinotecan and SN-38 were 20,348 +/- 6466 ng x h/mL and 629 +/- 370 ng x h/mL, respectively, which is in line with earlier findings. For UGT1A9(*)5,novariant alleles were observed, whereas for UGT1A9(*)3, 1 patient with the variant allele was found (allele frequency, 0.633%). The distribution of the UGT1A1(*)28 variant showed 44 wild-type patients (Wt), 37 heterozygotes (Het), and 5 homozygotes (Var). The median AUC ratio of SN-38G to SN-38 was significantly reduced in carriers of the variant UGT1A1(*)28 allele (7.00 [Wt] vs. 6.26 [Het] vs. 2.51 [Var]; p =.022). It is concluded that UGT1A9 functional variants are rare in Caucasians and likely to be clinically insignificant in irinotecan regimens. Screening for the UGT1A1(*)28 polymorphism may identify patients with altered SN-38 pharmacokinetics.     

Common human UGT1A polymorphisms and the altered metabolism of irinotecan active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38)

7-Ethyl-10-hydroxycamptothecin (SN-38) is the pharmacologically active metabolite of irinotecan, in addition to being responsible for severe toxicity. Glucuronidation is the main metabolic pathway of SN-38 and has been shown to protect against irinotecan-induced gastrointestinal toxicity. The purpose of this study was to determine whether common polymorphic UDP-glucuronosyltransferase (UGT) affects SN-38 glucuronidation. First, kinetic characterization of SN-38-glucuronide (SN-38-G) formation was assessed for all known human UGT1A and UGT2B overexpressed in human embryonic kidney 293 cells. To assess the relative activity of UGT isoenzymes for SN-38, rates of formation of SN-38-G were monitored by liquid chromatography/mass spectrometry analysis and normalized by level of UGT cellular expression. Determination of intrinsic clearances predicts that hepatic UGT1A1 and UGT1A9 and the extrahepatic UGT1A7 are major components in SN-38-G formation, whereas a minor role is suggested for UGT1A6, UGT1A8, and UGT1A10. In support of the involvement of UGT1A9, a strong coefficient of correlation was observed in the glucuronidation of SN-38 and a substrate, mainly glucuronidate, by UGT1A9 (flavopiridol) by human liver microsomes (coefficient of correlation, 0.905; p = 0.002). In vitro functional experiments revealed a negative impact of the UGT1A1 allelic variants. Residual activities of 49, 7, 8, and 11% were observed for UGT1A1*6 (G(71)R), UGT1A1*27 (P(229)Q), UGT1A1*35 (L(233)R), and UGT1A1*7 (Y(486)D), respectively. Common variants of UGT1A7, UGT1A7*3 (N(129)K;R(131)K;W(208)R), and UGT1A7*4 (W(208)R), displayed residual activities of 41 and 28% compared with the UGT1A7*1 allele. Taken together, these data provide the evidence that molecular determinants of irinotecan response may include the UGT1A polymorphisms studied herein and common genetic variants of the hepatic UGT1A9 isoenzyme yet to be described.     

The main role of UGT1A9 in the hepatic metabolism of mycophenolic acid and the effects of naturally occurring variants.

Mycophenolic acid (MPA), a standard immunosuppressive drug, is characterized by unexplained highly variable pharmacokinetics in transplant recipients. The primary metabolic pathway of MPA is glucuronidation; however, literature reports are inconsistent and the contribution of all human UDP-glucuronosyltransferases (UGTs) has never been systematically assessed. Sixteen heterologously expressed UGTs were studied for MPA 7-O-glucuronidation and compared with liver, kidney, and intestine microsomes. For direct measurement of MPA and mycophenolic acid 7-O-glucuronide (MPAG), we used a liquid chromatography-mass spectrometry protocol. Metabolic studies demonstrated that the glucuronidation of MPA in humans occurs mainly in the liver at variable rates, as well as in the kidney and the intestine. Kinetic data normalized for UGT expression levels point to 1A8 and 1A9 as the main UGTs, as well as to low-activity enzymes, 1A1, 1A7, and 1A10. According to their expression profiles, 1A8 and 1A9 are proposed, respectively, as the main extra-hepatic and the only hepatic enzymes involved. The effects of naturally occurring 1A8 ((*)2 and (*)3) and 1A9 ((*)2 and (*)3) variants on MPA 7-O-glucuronidation were studied and data suggest that the C(277)Y of 1A8 and the M(33)T of 1A9 may influence MPAG levels. A number of low-activity alleles were also identified in 1A1 [(*)6 (G(71)R), (*)7 (Y(486)D), (*)27 (P(229)Q), and (*)35 (L(233)R)], 1A7 [(*)3 (K(129)K(131)R(208)), (*)4 (R(208)), (*)5 (S(115)), (*)8 (K(129)K(131)D(139)R(208)), and (*)9 (S(115)K(129)K(131))], and 1A10 [(*)2 (E(139)K)]. Our study provides preliminary evidences that genetic factors, especially in the hepatic UGT1A9, may contribute to the variability of MPA pharmacokinetics observed in transplant patients.     

5-Nitro-2-pyridyl-1-thioglucosides: application in synthesis of analogues of glycosyltransferases natural substrates

5-Nitro-2-pyridyl-1-thioglucosides were used in synthesis of complex uridine derivatives (13-16) in two different sequences of reactions. In one route, the first step was glycosylation of selectively protected 5-nitro-2-pyridyl-1-thioglucoside 1 with two different glycosyl donors (5 or 6), next, the nitro group in aglycone of obtained disaccharides 7 or 8 was reduced and then obtained products 9 or 10 were condensed with uridine derivatives 3 or 4 using DMT-MM as condensing agent under microwave irradiation. In the second route, condensation and glycosylation reactions were applied in reverse order. As it turned up, a sequence of reactions affected the yield of final glycoconjugates 13-16 and depended on the type of uridine derivatives used.     

Malate dehydrogenase in helminth parasites. Inhibition by benzimidazoles and pyrimidine derivatives

A study was performed of the activities of both cytoplasmic and mitochondrial, malate dehydrogenase (MDH) (E.C.1.1.1.37) in purified extracts of whole specimens of male and female nematodes of four species: T. canis, T. cati, T. leonina and A. suum (and tissues), two trematodes: F. hepatica and D. dendriticum, and four cestodes: M. expansa, M. benedeni, D. caninum and T. hydatigena. The results show that there exist species and sexual differences in the enzyme activities of both enzymes. The relative importance of this energy pathways of these helminth species is discussed. Determinations were made of the in vitro inhibitory activities of four benzimidazoles and six synthesised pyrimidine derivatives on MDH (soluble and mitochondrial) from helminth parasites. Several pyrimidine derivatives (6-amino-5-methyl-5-nitro-uracil, 4-amino-1-methyl-2-methylthio-5-nitro-6-oxo-1,2,3,4-tetrahydropyrimid ine and 4-amino-2-methylthio-5-nitro-6-oxo-1,2,3,4-tetrahidropyrimidine) produced double percent in vitro inhibitions of those shown by the benzimidazoles.     

The human UDP-glucuronosyltransferase: identification of key residues within the nucleotide-sugar binding site

UDP-glucuronosyltransferases (UGTs) play important roles in the metabolism, detoxification,and clearance of many different xenobiotics, including drugs and endogenous compounds. Structural information about these membrane-bound enzymes of the endoplasmic reticulum is limited. We do not know the identity or the location of the key residues for catalysis and binding of the aglycone substrate and the cosubstrate UDP-glucuronic acid (UDPGA). One suggestion was that His371 (UGT1A6 numbering) is the "catalytic base" that deprotonates the phenol group. We have now re-examined this hypothesis by analyzing the activities of the corresponding mutants, 6H371A (in UGT1A6) and the 9H369A (in UGT1A9). The K(m) values of mutant 6H371A for scopoletin and UDPGA were higher by 4- and 11-fold, respectively, than in UGT1A6. The K(d) for the enzyme-UDPGA complex, derived from bisubstrate kinetics, was about 9-fold higher in 6H371A than in UGT1A6, indicating severely impaired cosubstrate binding by the mutant. The effect of mutation on V(max) was large in UGT1A6 but variable in UGT1A9, suggesting that His371 does not play the catalytic role previously hypothesized. In both UGTs, the E379A mutation (UGT1A6 numbering) had an effect similar to that of the H371A mutations. A homology model of the putative UDPGA binding region of UGT1A6 was built using distant homologous protein structures from the "GT1 class." The combined results of activity determinations, kinetic analyses, and modeling strongly suggest that His371 and Glu379 are directly involved in UDPGA binding but are not the general acid or general base.     

Human UDP-glucuronosyltransferase, UGT1A8, glucuronidates dihydrotestosterone to a monoglucuronide and further to a structurally novel diglucuronide.

We identified human UDP-glucuronosyltransferase (UGT) isoforms responsible for producing dihydrotestosterone (DHT) diglucuronide, a novel glucuronide in which the second glucuronosyl moiety is attached at the C2' position of the first glucuronosyl moiety, leading to diglucuronosyl conjugation of a single hydroxyl group of DHT at the C17 position. Incubation of the DHT monoglucuronide with 12 cDNA-expressed recombinant human UGT isoforms and uridine 5'-diphosphoglucuronic acid resulted in a low but measurable DHT diglucuronidation activity primarily with UGT1A8, a gastrointestinal UGT, and to a lesser extent with UGT1A1 and UGT1A9. In contrast, the activity of DHT monoglucuronidation was high and was found in UGT2B17, UGT2B15, UGT1A8, and UGT1A4 in descending order. Among the 12 UGT isoforms tested, only UGT1A8 was capable of producing DHT diglucuronide from DHT. The kinetics of DHT diglucuronidation by microsomes from human liver and intestine fitted the Michaelis-Menten model, and the V(max)/K(m) value for the intestinal microsomes was approximately 4 times greater than that for the liver microsomes.     

Opioids bind to the amino acids 84 to 118 of UDP-glucuronosyltransferase UGT2B7

The UDP-glucuronosyltransferase UGT2B7 is an important human UGT isoform that catalyzes the conjugation of many endogenous and exogenous compounds, among them opioids, resulting in the formation of D-glucuronides. The binding site of the aglycone is located in the N-terminal half of the protein. Using NMR analysis, we demonstrate that the opioid binding site in UGT2B7 is within the 84 to 118 N-terminal amino acids. Three maltose binding protein-UGT2B7 fusion proteins, 2B7F3 and 2B7F4 incorporating the amino acids 24 to 118 and 24 to 96 of UGT2B7, respectively, and 2B7F5 incorporating amino acids 84 to 118 of UGT2B7 were expressed in Escherichia coli and purified by affinity chromatography. NMR analysis showed that morphine was bound to the fusion protein 2B7F3 with a K(D) value similar to the K(D) values obtained for the previously produced fusion proteins, which included amino acids 24 to 180. Morphine did not bind to 2B7F4, but it did bind to 2B7F5. Both NMR 1-D spectra and NOESY experiments indicated that the 2B7F5 protein was mediating magnetization transfer within the morphine. These results allowed us to predict and model a binding site within the amino acids 96 to 101 of UGT2B7. A mutant fusion protein 2B7F3 with the substitution D99A was produced, and the NMR spectroscopy analysis of the protein supported the model. A marked reduction of morphine binding was observed when the charged aspartate was substituted with alanine.     

Troglitazone glucuronidation in human liver and intestine microsomes: high catalytic activity of UGT1A8 and UGT1A10.

Troglitazone glucuronidation in human liver and intestine microsomes and recombinant UDP-glucuronosyltransferases (UGTs) were thoroughly characterized. All recombinant UGT isoforms in baculovirus-infected insect cells (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) exhibited troglitazone glucuronosyltransferase activity. Especially UGT1A8 and UGT1A10, which are expressed in extrahepatic tissues such as stomach, intestine, and colon, showed high catalytic activity, followed by UGT1A1 and UGT1A9. The kinetics of the troglitazone glucuronidation in the recombinant UGT1A10 and UGT1A1 exhibited an atypical pattern of substrate inhibition when the substrate concentration was over 200 micro M. With a Michaelis-Menten equation at 6 to 200 micro M troglitazone, the K(m) value was 11.1 +/- 5.8 micro M and the V(max) value was 33.6 +/- 3.7 pmol/min/mg protein in recombinant UGT1A10. In recombinant UGT1A1, the K(m) value was 58.3 +/- 29.2 micro M and the V(max) value was 12.3 +/- 2.5 pmol/min/mg protein. The kinetics of the troglitazone glucuronidation in human liver and jejunum microsomes also exhibited an atypical pattern. The K(m) value was 13.5 +/- 2.0 micro M and the V(max) value was 34.8 +/- 1.2 pmol/min/mg for troglitazone glucuronidation in human liver microsomes, and the K(m) value was 8.1 +/- 0.3 micro M and the V(max) was 700.9 +/- 4.3 pmol/min/mg protein in human jejunum microsomes. When the intrinsic clearance was estimated with the in vitro kinetic parameter, microsomal protein content, and weight of tissue, troglitazone glucuronidation in human intestine was 3-fold higher than that in human livers. Interindividual differences in the troglitazone glucuronosyltransferase activity in liver microsomes from 13 humans were at most 2.2-fold. The troglitazone glucuronosyltransferase activity was significantly (r = 0.579, p < 0.05) correlated with the beta-estradiol 3-glucuronosyltransferase activity, which is mainly catalyzed by UGT1A1. The troglitazone glucuronosyltransferase activity in pooled human liver microsomes was strongly inhibited by bilirubin (IC(50) = 1.9 micro M), a typical substrate of UGT1A1. These results suggested that the troglitazone glucuronidation in human liver would be mainly catalyzed by UGT1A1. Interindividual differences in the troglitazone glucuronosyltransferase activity in S-9 samples from five human intestines was 8.2-fold. The troglitazone glucuronosyltransferase activity in human jejunum microsomes was strongly inhibited by emodin (IC(50) = 15.6 micro M), a typical substrate of UGT1A8 and UGT1A10, rather than by bilirubin (IC(50) = 154.0 micro M). Therefore, it is suggested that the troglitazone glucuronidation in human intestine might be mainly catalyzed by UGT1A8 and UGT1A10.     

Characterizing the effect of UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A9 genetic polymorphisms on enantioselective glucuronidation of flurbiprofen

Flurbiprofen (FPF), available commercially as a racemic mixture, is a propionic acid derivative of non-steroidal anti-inflammatory drugs (NSAIDs) with known stereoselective glucuronidation. The major enzyme catalyzing this conjugation reaction is UDP-glucuronosyltransferase (UGT) 2B7, with minor contributions by UGT1A9. This study examines the role of the genetic variants of UGT2B7 and 1A9 enzymes involved in the formation of acyl glucuronides (FPFGs). Variants caused by three single nucleotide polymorphisms (SNPs) (A71S, 211G>T; H268Y, 802C>T; and D398N, 1192G>A) in UGT2B7 and three SNPs (C3Y, 8G>A; M33T, 98T>C; D256N, 766G>A) in UGT1A9 showed activity changes toward different substrates. However the functional impacts of these SNPs on chiral substrates were not examined. Upon stable expression in Bac-to-Bac system, UGT2B7*71S (A⁷¹S), UGT2B7*2 (H²⁶⁸Y) and UGT2B7*5 (D³⁹⁸N) were all associated with a decrease in the formation of FPFGs. Compared with UGT2B7*1 (wild-type), UGT2B7*71S exhibited a >2-fold lower intrinsic clearance mainly by altered capacities (V_max). Furthermore, a >14-fold decreased intrinsic clearance of the *1 protein was produced by UGT2B7*2 and UGT2B7*5. However, no significantly stereoselective difference for the formation of (R)- and (S)-FPFG was found among these UGT2B7 allozymes. UGT1A9*2 (C³Y) exhibited a higher V_max (3.2-fold), K_m (2.1-fold) and intrinsic clearance (1.6-fold) toward (S)-FPF than UGT1A9*1 (wild-type). UGT1A9*3 (M³³T) almost lost the catalytic activity to FPF. A significantly stereoselective difference on the glucuronidation of rac-FPF was seen between the two variants compared with the wild type of UGT1A9.     

Amino acid residue ILE211 is essential for the enzymatic activity of human UDP-glucuronosyltransferase 1A10 (UGT1A10).

Conjugation of exogenous and endogenous compounds by uridine diphosphoglucuronosyltransferases (UGTs) is a pathway catalyzing the transfer of a glucuronic acid molecule from UDP glucuronic acid to lipophilic aglycones, which become more polar and more easily excretable in the bile or urine. UGTs are divided into two major families, UGT1 and UGT2. The isoform UGT1A10, along with UGT1A7 and UGT1A8, is expressed exclusively in extrahepatic tissues, notably in the gastrointestinal tract. Here, we report the isolation of a mutant clone of the human UGT1A10, at position 211 of the protein, where a threonine residue replaces an isoleucine residue (allele Thr211). Because the isoleucine is conserved among many UGT1A isoforms, we proceeded to the analysis of the activity of the wild-type UGT1A10 (T211I) and compared it with that of the variant enzyme (I211T(*)). In vitro assays with microsomal extracts from stably expressing human embryonic kidney 293 (HEK293) cells showed that the mutant enzyme lost all detectable activity toward major substrates, which demonstrate that the residue isoleucine at position 211 is essential for UGT1A10 activity. Mutant UGT1A10 (I211T(*)) also lost all detectable activity toward mycophenolic acid. Genomic DNA from 103 unrelated individuals was sequenced for this mutation, and two heterozygous genotypes were detected for this mutation (frequency: 2 per 100 individuals). Because UGT1A10 appears to be expressed in all gastrointestinal tissues and is active toward a wide range of substrates, lack of activity of this isoform may have an impact on individual glucuronidation efficiency.     

The interactions between the N-terminal and C-terminal domains of the human UDP-glucuronosyltransferases are partly isoform-specific, and may involve both monomers

The pathological mutation Y486D was previously shown to reduce the activities of the UDP-glucuronosyltransferases (UGTs) 1A1 and 1A6 by about 88% and 99%, respectively. Surprisingly, the corresponding mutation in UGT1A9 (Y483D) doubled the Vmax of scopoletin glucuronidation, whereas the entacapone glucuronidation rate was decreased by about 50%. Due to the primary structure identity of the C-terminal half of all the human UGTs of the 1A subfamily, the sharp differences between them in the effect of a mutation deep inside the C-terminal half suggested that there are isoform-specific interactions between the variable N- and the conserved C-terminal halves. In dimeric enzymes, like the UGTs, such interactions might either occur within the same polypeptide, or between opposite monomers. The latter implies functional monomer-monomer interactions, and this was investigated using hetero-dimeric UGTs. Insect cells were co-infected with mixtures containing different combinations of recombinant baculoviruses encoding either UGT1A4 or 1A9Sol. The UGT1A4 was selected because it glucuronidates neither entacapone nor scopoletin at significant rates. The active enzyme in these hetero-dimers was 1A9Sol, a truncation mutant of UGT1A9 that exhibited a very low ratio of entacapone to scopoletin glucuronidation rates. Interestingly, the ratio of entacapone to scopoletin glucuronidation rates in the co-infected cells was dependent on, and markedly increased with, the probability that 1A9Sol forms hetero-dimers with UGT1A4. In addition, the apparent Km for entacapone in the hetero-dimers was much lower than in 1A9Sol, and resembled the corresponding value in full-length UGT1A9. The results, thus, revealed important monomer-monomer interactions within the UGTs.     

An active and water-soluble truncation mutant of the human UDP-glucuronosyltransferase 1A9

The UDP-glucuronosyltransferases (UGTs) are integral membrane proteins, and previous attempts to generate a water-soluble UGT by removing the single trans-membrane helix yielded inactive and membrane-bound proteins. We have now replaced the 45 C-terminal amino acids of the human UGT1A9, including its trans-membrane helix, with a fusion peptide ending with six His residues. Detergent-free extraction of insect cells expressing this mutant, UGT1A9Sol, released scopoletin glucuronidation activity into the supernatant, and subsequent ultracentrifugation did not sediment that activity. UGT1A9Sol was purified by immobilized metal affinity chromatography (IMAC) in the absence of detergents throughout the entire process. The IMAC purification increased somewhat the apparent K(m) of UGT1A9 toward scopoletin and rendered the enzyme sensitive to freezing. The activity of UGT1A9Sol in the cell extract was partly inhibited by Triton X-100, irrespective of the presence or absence of phospholipids. UGT1A9Sol exhibited a relatively high rate of scopoletin glucuronidation, whereas its activity toward 1-naphthol, entacapone, umbelliferone, and 4-nitrophenol was much lower. The kinetics and substrate specificity of UGT1A9Sol resembled the detergent-suspended full-length UGT1A9 rather than the membrane-bound UGT1A9. The apparent K(m) value of UGT1A9Sol for scopoletin was similar to that of the full-length UGT1A9 in the presence of detergent, but much higher than the respective value in the membrane-bound enzyme. The results suggest that either the detergent binding to the trans-membrane helix within the full-length UGT1A9, or the removal of this helix by gene manipulation, affect the interaction of the enzyme with its aglycone substrate in a similar manner.     

UDP-glucuronosyltransferase 1A4 polymorphisms in a Japanese population and kinetics of clozapine glucuronidation.

The UDP-glucuronosyltransferase (UGT) family plays a major role in the excretion of endobiotics and xenobiotics and their metabolites. Human UGT1A4 catalyzes the glucuronidation of primary, secondary, and tertiary amines, sapogenins, androgens, and progestins. We directly sequenced polymerase chain reaction-amplified fragments of the UGT1A4 gene from 100 healthy adult Japanese volunteers and calculated their mutation frequency. We identified four single nucleotide polymorphisms (SNPs): three in exon 1 (142T > G: L48V, 448T > C: L150L, 804G > A: P268P), and one in intron 1 (867 + 43C > T). We found three types of alleles with distinct SNP combinations that coded for different amino acid sequences: L48V-L150L-P268P-867 + 43C > T (frequency, 0.155), L48V (0.01), and P268P (0.01) (wild-type frequency was 0.825). The L48V mutant gave twice the efficiency (V(max)/K(m)) for the antipsychotic drug clozapine as the wild-type. Efficiencies of L48V for trans-androsterone, imipramine, and cyproheptadine were increased, but the efficiency for tigogenin was reduced. L48V therefore increased or decreased the glucuronidation activity, depending upon the substrates. This study shows the importance of identifying patients with the L48V polymorphism when calculating dosage, and when considering the potential adverse effects of drugs that are substrates of UGT1A4.     

Single-step identification of all length polymorphisms in the UGT1A1 gene promoter

AIM: To provide a sensitive genetic screening method for rapid identification of all known length polymorphisms in the promoter region of the uridine 5'-diphosphoglucose glucuronosyltransferase (UGT) 1A1 gene comprising (TA)5, (TA)7 and (TA)8 repeats as opposed to the non-mutated (TA)6 allele. METHODS: The UGT1A1 promoter genotype was assessed in 115 subjects by means of a newly developed pyrosequencing method. PCR-generated DNA templates of heterozygous (TA)5 and (TA)7 carriers were cloned into a TOPO TA vector and verified by sequencing. In addition, a (TA)8 segment was produced by cloning to demonstrate the ability of the method to detect this mutation. RESULTS: All length polymorphisms of the UGT1A1 promoter described in the literature were clearly identified. Fifteen subjects had Gilbert's syndrome with elevated serum bilirubin associated with a homozygous (TA)7TAA/(TA)7TAA genotype. Two subjects with the rare genotypes (TA)5TAA/(TA)6TAA and (TA)5TAA/(TA)7TAA were found, where only the latter one displayed elevated serum bilirubin levels. Allelic frequencies were 0.9%, 66.1% and 33% for the (TA)5TAA, (TA)6TAA and (TA)7TAA allele, respectively. CONCLUSION: Our method enables reliable genetic single-step screening for all known length polymorphisms in the UGT1A1 gene promoter that cause Gilbert's syndrome. This facilitates pharmacogenetic-guided dosing of drugs with known toxicity metabolized by UGT1A1.