Pharmacophore and quantitative structure-activity relationship modeling: complementary approaches for the rationalization and prediction of UDP-glucuronosyltransferase 1A4 substrate selectivity

Pharmacophore, two-dimensional (2D), and three-dimensional (3D) quantitative structure-activity relationship (QSAR) modeling techniques were used to develop and test models capable of rationalizing and predicting human UDP-glucuronosyltransferase 1A4 (UGT1A4) substrate selectivity and binding affinity (as K(m,app)). The dataset included 24 structurally diverse UGT1A4 substrates, with 18 of these comprising the training set and 6 an external prediction set. A common features pharmacophore was generated with the program Catalyst after overlapping the sites of conjugation using a novel, user-defined "glucuronidation" feature. Pharmacophore-based 3D-QSAR (r(2) = 0.88) and molecular-field-based 3D-QSAR (r(2) = 0.73) models were developed using Catalyst and self-organizing molecular field analysis (SOMFA) software, respectively. In addition, a 2D-QSAR (r(2) = 0.80, CV r(2) = 0.73) was generated using partial least-squares (PLS) regression and variable selection using an unsupervised forward selection (UFS) algorithm. Both UGT1A4 pharmacophores included two hydrophobic features and the glucuronidation site. The 2D-QSAR showed the best overall predictivity and highlighted the importance of hydrophobicity (as log P) in substrate-enzyme binding.     

Multiple pharmacophores for the investigation of human UDP-glucuronosyltransferase isoform substrate selectivity

The UDP-glucuronosyltransferase (UGT) enzyme 'superfamily' contributes to the metabolism of a myriad of drugs, nondrug xenobiotic agents, and endogenous compounds. Although the individual UGT isoforms exhibit distinct but overlapping substrate selectivities, structural features of substrates that confer selectivity remain largely unknown. Using methods developed for pharmacophore fingerprinting combined with optimization and pattern recognition techniques, subsets of pharmacophores associated with the substrates and nonsubstrates of 12 human UGT isoforms were selected to generate predictive models of substrate selectivity and to elucidate the chemical and structural features associated with substrates and nonsubstrates. For all 12 UGT isoforms, the pharmacophore model generated showed predictive ability, as determined by a test set comprising 30% of the available data for each isoform. Models for UGT1A6, -1A7, -1A9, and -2B4 displayed the best predictive ability (>75% of test set predicted correctly) and were further analyzed to interpret the pharmacophores selected as important. The individual pharmacophores differed among isoforms but generally represented relatively simple structural and chemical features. For example, an aromatic ring attached to the nucleophilic group was found to increase the likelihood of glucuronidation by UGT1A6, UGT1A7 and UGT1A9. A large hydrophobic region close to the nucleophile and a hydrogen bond acceptor 10 A from the nucleophile were found to be common to most UGT2B4 substrates. The pharmacophores further suggest that the environment immediately adjacent to the nucleophilic site of conjugation is an important determinant of metabolism by a particular UGT.     

Evaluation of inhibitors of intestinal UDP-glucuronosyltransferases 1A8 and 1A10 using raloxifene as a substrate in Caco-2 cells: Studies with four flavonoids of Scutellaria baicalensis

UDP glucuronosyltransferases (UGTs) of the gastrointestinal tract play a crucial role in protection against the toxic effects of xenobiotics in the environment. UGTs such as UGT1A8 and UGT1A10 are predominantly expressed in gastrointestinal tissues. In this study, we examined the phase II metabolism of raloxifene in differentiated Caco-2 monolayers by inducing UGT1A8 and UGT1A10 expression in these cells. The present study evaluated the following four flavonoids of Scutellaria baicalensis as model herbal compounds: scutellarein, salvigenin, baicalein, and wogonin. All test compounds, endpoint substrates, and their metabolites were quantified using liquid chromatography and high-resolution mass spectrometry. The transepithelial electrical resistance values for the individual compounds were comparable regardless of whether they were measured individually. Salvigenin significantly inhibited UGT1A8 and UGT1A10 activities in a concentration-dependent manner. All individual compounds except scutellarein inhibited UGT1A8 and UGT1A10 activity at a concentration of 100 μM. In addition, all individual flavonoids at 100 μM, except wogonin, significantly increased the amount of raloxifene in the basolateral chambers. The positive control, canagliflozin, significantly inhibited both UGT1A8 and UGT1A10 activities. These findings suggest that the Caco-2 assay can be utilized for identifying UGT1A8 and UGT1A10 inhibitors and indicate the potential of salvigenin for enhancing the pharmacological effects of UGT substrate drugs.     

Quaternary ammonium-linked glucuronidation of 1-substituted imidazoles: studies of human UDP-glucuronosyltransferases involved and substrate specificities.

A series of eight 1-substituted imidazoles was investigated as model substrates for glucuronidation at an aromatic tertiary amine of polyaza heterocyclic ring systems. The human UDP-glucuronosyltransferases (UGTs) involved and substrate specificities were investigated. Nine expressed enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) were examined, but only UGT1A4 catalyzed the formation of a quaternary ammonium-linked glucuronide metabolite for six of the substrates. UGT1A3 also catalyzed the glucuronidation of the previously investigated 1-phenylimidazole but none of the newly investigated compounds. No glucuronidation was observed with 1-(4-nitrophenyl)imidazole, the compound with the 4-phenyl substituent with the largest electron withdrawing effect. The incubation conditions for the determination of the kinetic constants for UGT1A4 catalysis of six substrates were optimized and included incubation at pH 7.4 with alamethicin at 10 microg/mg of protein. Latency disrupting agents, including alamethicin and sonication, enhanced glucuronidation 1.25-fold at most. There were 17.5- and 2.2-fold variations in the apparent K(m) (range, 0.18-3.15 mM) and V(max) values (range, 0.16-0.35 nmol/min/mg of protein). Linear correlation analyses between UGT1A4 kinetics and substrate physicochemical parameters showed significant correlation between V(max) and both the partition coefficient (log P, n-octanol/water) and pK(a) and between K(m) and pK(a), thereby indicating that the lipophilicity and the ease of availability of the tertiary amine lone pair of electrons of the substrate are important with respect to enzyme catalysis.     

Analysis of substrate specificities and tissue expression of rat UDP-glucuronosyltransferases UGT1A7 and UGT1A8.

The UGT1 complex codes for a subfamily of homologous "1A7-like" UDP-glucuronosyltransferases (UGTs), including UGT1A7 and UGT1A8. Little information is available regarding either the substrate specificities or regulation of the UGT1A7-like forms from rats. We compared the activities and tissue expression of UGT1A7 and UGT1A8, which exhibit 77% identity in their amino terminal sequence. UGT1A7 shows broad specificity, catalyzing the glucuronidation of 31 of 40 randomly selected substrates (100 muM) at rates >0.1 nmol/mg/min. UGT1A7 substrates included both planar and nonplanar compounds, mono- and polycyclic aromatics, and compounds with bulky side chain ring substitutions. UGT1A8 exhibited a narrower substrate specificity that completely overlapped with UGT1A7. UGT1A8 was most active toward the 1-OH, 4-OH, 5-OH, 6-OH, 7-OH, 10-OH, 11-OH, and 12-OH derivatives of benzo[a]pyrene. Other effective UGT1A8 substrates (>0.1 nmol/mg/min) included 9-OH-benzo[a]pyrene, 1-naphthol, 4-methylumbelliferone, 7-hydroxycoumarin, chrysin, quercetin, 4-nitrophenol, and estriol. In general, substrates preferred by UGT1A8 were polyaromatic planar structures with nonbulky substituents and a superimposable 1-naphtho ring structure. Studies of the tissue expression of the UGT1A7 and 1A8 mRNAs using RNase protection analysis suggested that each is expressed in liver and kidney of control rats. A major difference is the higher expression of UGT1A7 mRNA in intestine. These studies suggest complementary functions of the UGT1A7 and UGT1A8 forms in xenobiotic metabolism. Further studies are necessary to determine whether their relative contributions change as a function of development, hormonal status, or exposure to inducing agents.     

Determination of drug glucuronidation and UDP-glucuronosyltransferase selectivity using a 96-well radiometric assay.

A rapid and sensitive radiometric assay for UDP-glucuronosyltransferase (UGT) is described. UGT substrates are incubated in 96-well plates with microsomes in the presence of [14C]UDP-glucuronic acid, and 14C-labeled glucuronidation products are separated from the unreacted nucleotide sugar by solid-phase extraction using 96-well extraction plates. The assay was validated with 15 structurally diverse UGT substrates containing acidic, phenolic, and hydroxyl reacting groups. Glucuronidation velocities for these compounds were determined using human, rat, and dog liver microsomes, and reaction kinetics were studied with 1-naphthol and 4-methylumbelliferone. Results obtained with the new assay confirmed the previously reported rank order of glucuronidation velocity of several typical UGT substrates and the finding that the glucuronidation of most of these compounds is significantly faster in dog than in human liver microsomes. UGT specificity of five compounds was determined using recombinant human UGTs. The major UGT isoforms identified were UGT1A6, UGT1A7, and UGT1A9 for 4-methylumbelliferone; UGT1A6 and UGT1A8 for 1-naphthol; UGT2B7 for naloxone; UGT1A3 and UGT2B7 for ketoprofen; and UGT1A4 for trifluoperazine. Identical results were obtained with a conventional high-performance liquid chromatography method coupled to mass spectrometric detection. The new assay should prove valuable for rapidly benchmarking recombinant UGTs and microsomal preparations from different species and tissues, identifying high-turnover compounds during drug discovery, and reaction phenotyping studies.     

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.     

Use of cloned and expressed human UDP-glucuronosyltransferases for the assessment of human drug conjugation and identification of potential drug interactions.

Glucuronidation is an important pathway for human drug metabolism. Four cloned and expressed human UDP-glucuronosyltransferases (UGT1A1, UGT1A6, UGT1A9, and UGT2B15) were used to screen a series of three potential drug substrates differing only in position of the phenol moiety. The meta and para phenols, UK-156,037 and UK-157,147, were found to be substrates for UGT1A1 with K(m) values of 256 and 105 microM, respectively. The ortho phenol UK-157,261 was glucuronidated predominantly by UGT1A9 with a K(m) of 45 microM. The latter K(m) compares favorably with the known UGT1A9 substrate propofol (K(m) = 200 microM). In a series of competition experiments, UK-157,261 was shown to inhibit the glucuronidation of propofol by UGT1A9 with a K(i) value of 65 microM. This result indicates that even the most potent of these compounds is extremely unlikely to interact in the clinic with the glucuronidation of propofol. This study shows the utility of the expressed human UDP-glucuronosyltransferases in determining substrate structure-activity relationships and potential drug-drug interactions.     

Differential modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed estradiol-3-glucuronidation by the addition of UGT1A1 substrates and other compounds to human liver microsomes.

Previous results demonstrating homotropic activation of human UDP-glucuronosyltransferase (UGT) 1A1-catalyzed estradiol-3-glucuronidation led us to investigate the effects of 16 compounds on estradiol glucuronidation by human liver microsomes (HLM). In confirmation of previous work using alamethicin-treated HLM pooled from four livers, UGT1A1-catalyzed estradiol-3-glucuronidation demonstrated homotropic activation kinetics (S(50) = 22 microM, Hill coefficient, n = 1.9) whereas estradiol-17-glucuronidation (catalyzed by other UGT enzymes) followed Michaelis-Menten kinetics (K(m) = 7 microM). Modulatory effects of the following compounds were investigated: bilirubin, eight flavonoids, 17alpha-ethynylestradiol (17alpha-EE), estriol, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), anthraflavic acid, retinoic acid, morphine, and ibuprofen. Although the classic UGT1A1 substrate bilirubin was a weak competitive inhibitor of estradiol-3-glucuronidation, the estrogens and anthraflavic acid activated or inhibited estradiol-3-glucuronidation dependent on substrate and effector concentrations. For example, at substrate concentrations of 5 and 10 microM, estradiol-3-glucuronidation activity was stimulated by as much as 80% by low concentrations of 17alpha-EE but was unaltered by flavanone. However, at higher substrate concentrations (25-100 microM) estradiol-3-glucuronidation was inhibited by about 55% by both compounds. Anthraflavic acid and PhIP were also stimulators of estradiol 3-glucuronidation at low substrate concentrations. The most potent inhibitor of estradiol 3-glucuronidation was the flavonoid tangeretin. The UGT2B7 substrates morphine and ibuprofen had no effect on estradiol 3-glucuronidation, whereas retinoic acid was slightly inhibitory. Estradiol-17-glucuronidation was inhibited by 17alpha-EE, estriol, and naringenin but was not activated by any compound. This study demonstrates that the interactions of substrates and inhibitors at the active site of UGT1A1 are complex, yielding both activation and competitive inhibition kinetics.