Screening of 16 major drug glucuronides for time-dependent inhibition of nine drug-metabolizing CYP enzymes - detailed studies on CYP3A inhibitors.

Time-dependent inhibition of cytochrome P450 (CYP) enzymes has been observed for a few glucuronide metabolites of clinically used drugs. Here, we investigated the inhibitory potential of 16 glucuronide metabolites towards nine major CYP enzymes in vitro. Automated substrate cocktail methods were used to screen time-dependent inhibition of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2J2 and 3A in human liver microsomes. Seven glucuronides (carvedilol ß-D-glucuronide, diclofenac acyl-ß-D-glucuronide, 4-hydroxyduloxetine ß-D-glucuronide, ezetimibe phenoxy-ß-D-glucuronide, raloxifene 4'-glucuronide, repaglinide acyl-ß-D-glucuronide and valproic acid ß-D-glucuronide) caused NADPH- and time-dependent inhibition of at least one of the CYPs investigated, including CYP2A6, CYP2C19 and CYP3A. In more detailed experiments, we focused on the glucuronides of carvedilol and diclofenac, which inhibited CYP3A. Carvedilol ß-D-glucuronide showed weak time-dependent inhibition of CYP3A, but the parent drug carvedilol was found to be a more potent inhibitor of CYP3A, with the half-maximal inhibitor concentration (IC50) decreasing from 7.0 to 1.1 µM after a 30-minute preincubation with NADPH. The maximal inactivation constant (kinact) and the inhibitor concentration causing half of kinact (KI) for CYP3A inactivation by carvedilol were 0.051 1/min and 1.8 µM, respectively. Diclofenac acyl-ß-D-glucuronide caused time-dependent inactivation of CYP3A at high concentrations, with a 4-fold IC50 shift (from 400 to 98 µM after a 30-minute preincubation with NADPH), and KI and kinact values of >2000 µM and >0.16 1/min. In static predictions, carvedilol caused significant (>1.25-fold) increase in the exposure of the CYP3A substrates midazolam and simvastatin. In conclusion, we identified several glucuronide metabolites with CYP inhibitory properties. Based on detailed experiments, the inactivation of CYP3A by carvedilol may cause clinically significant drug-drug interactions.

Solanidine is a sensitive and specific dietary biomarker for CYP2D6 activity.

BACKGROUND: Individual assessment of CYP enzyme activities can be challenging. Recently, the potato alkaloid solanidine was suggested as a biomarker for CYP2D6 activity. Here, we aimed to characterize the sensitivity and specificity of solanidine as a CYP2D6 biomarker among Finnish volunteers with known CYP2D6 genotypes. RESULTS: Using non-targeted metabolomics analysis, we identified 9152 metabolite features in the fasting plasma samples of 356 healthy volunteers. Machine learning models suggested strong association between CYP2D6 genotype-based phenotype classes with a metabolite feature identified as solanidine. Plasma solanidine concentration was 1887% higher in genetically poor CYP2D6 metabolizers (gPM) (n = 9; 95% confidence interval 755%, 4515%; P = 1.88 × 10-11), 74% higher in intermediate CYP2D6 metabolizers (gIM) (n = 89; 27%, 138%; P = 6.40 × 10-4), and 35% lower in ultrarapid CYP2D6 metabolizers (gUM) (n = 20; 64%, - 17%; P = 0.151) than in genetically normal CYP2D6 metabolizers (gNM; n = 196). The solanidine metabolites m/z 444 and 430 to solanidine concentration ratios showed even stronger associations with CYP2D6 phenotypes. Furthermore, the areas under the receiver operating characteristic and precision-recall curves for these metabolic ratios showed equal or better performances for identifying the gPM, gIM, and gUM phenotype groups than the other metabolites, their ratios to solanidine, or solanidine alone. In vitro studies with human recombinant CYP enzymes showed that solanidine was metabolized mainly by CYP2D6, with a minor contribution from CYP3A4/5. In human liver microsomes, the CYP2D6 inhibitor paroxetine nearly completely (95%) inhibited the metabolism of solanidine. In a genome-wide association study, several variants near the CYP2D6 gene associated with plasma solanidine metabolite ratios. CONCLUSIONS: These results are in line with earlier studies and further indicate that solanidine and its metabolites are sensitive and specific biomarkers for measuring CYP2D6 activity. Since potato consumption is common worldwide, this biomarker could be useful for evaluating CYP2D6-mediated drug-drug interactions and to improve prediction of CYP2D6 activity in addition to genotyping.

Ticagrelor Increases Exposure to the Breast Cancer Resistance Protein Substrate Rosuvastatin.

Ticagrelor and rosuvastatin are often used concomitantly after atherothrombotic events. Several cases of rhabdomyolysis during concomitant ticagrelor and rosuvastatin have been reported, suggesting a drug-drug interaction. We showed recently that ticagrelor inhibits breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 1B1, 1B3, and 2B1-mediated rosuvastatin transport in vitro. The aim of this study was to investigate the effects of ticagrelor on rosuvastatin pharmacokinetics in humans. In a randomized, crossover study, 9 healthy volunteers ingested a single dose of 90 mg ticagrelor or placebo, followed by a single 10 mg dose of rosuvastatin 1 hour later. Ticagrelor 90 mg or placebo were additionally administered 12, 24, and 36 hours after their first dose. Ticagrelor increased rosuvastatin area under the plasma concentration-time curve (AUC) and peak plasma concentration 2.6-fold (90% confidence intervals: 1.8-3.8 and 1.7-4.0, P = 0.001 and P = 0.003), and prolonged its half-life from 3.1 to 6.6 hours (P = 0.009). Ticagrelor also decreased the renal clearance of rosuvastatin by 11% (3%-19%, P = 0.032). The N-desmethylrosuvastatin:rosuvastatin AUC0-10h ratio remained unaffected by ticagrelor. Ticagrelor had no effect on the plasma concentrations of the endogenous OATP1B substrates glycodeoxycholate 3-O-glucuronide, glycochenodeoxycholate 3-O-glucuronide, glycodeoxycholate 3-O-sulfate, and glycochenodeoxycholate 3-O-sulfate, or the sodium-taurocholate cotransporting polypeptide substrate taurocholic acid. These data indicate that ticagrelor increases rosuvastatin concentrations more than twofold in humans, probably mainly by inhibiting intestinal BCRP. Because the risk for rosuvastatin-induced myotoxicity increases along with rosuvastatin plasma concentrations, using ticagrelor concomitantly with high doses of rosuvastatin should be avoided.

The effect of hydroxychloroquine on cholesterol metabolism in statin treated patients after myocardial infarction.

Background and aims: To evaluate the effect of hydroxychloroquine (HCQ) on serum and lipoprotein lipids and serum biomarkers of cholesterol synthesis and absorption in myocardial infarction patients with a high-dose statin. Methods: Myocardial infarction patients (n = 59) with a constant statin dose were randomized to receive hydroxychloroquine 300 mg (n = 31) or placebo (n = 28) daily for six months and followed up for one year. Results: Statin reduced total-c (-26 ± 22% in hydroxychloroquine and -28 ± 19% in placebo group, P = 0.931), LDL-c (-38 ± 26% vs. -44 ± 23%, respectively, P = 0.299), and cholesterol synthesis biomarkers zymostenol, desmosterol, and lathosterol ratios from baseline to one year (e.g., serum lathosterol ratio -17 ± 45% vs. -15 ± 41%, respectively, P < 0.001 for both, P = 0.623 between groups). Compensatorily, cholesterol absorption increased during the intervention (e.g., serum campesterol ratio 125 ± 90% vs. 113 ± 72%, respectively, P < 0.001 for both, P = 0.488 between groups). Hydroxychloroquine did not affect cholesterol concentrations or cholesterol absorption. It prevented the statin-induced increase in cholesterol precursor, desmosterol ratio, from six months to one year in the hydroxychloroquine group (P = 0.007 at one year compared to placebo). Conclusions: Combined with a high-dose statin, hydroxychloroquine had no additional effect on serum cholesterol concentration or cholesterol absorption. However, the findings suggest that hydroxychloroquine interferes with lanosterol synthesis, and thereafter, it temporarily interferes with the cholesterol synthesis pathway, best seen in halting the increase of the desmosterol ratio.Trial Registration ClinicalTrials.gov Identifier: NCT02648464.

Introduction of an electrochemical point-of-care assay for quantitative determination of paracetamol in finger-prick capillary whole blood samples.

AIMS: Measuring venous plasma paracetamol concentrations is time- and resource-consuming. We aimed to validate a novel electrochemical point-of-care (POC) assay for rapid paracetamol concentration determinations. METHODS: Twelve healthy volunteers received 1 g oral paracetamol, and its concentrations were analysed 10 times over 12 h for capillary whole blood (POC), venous plasma (high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS)), and dried capillary blood (HPLC-MS/MS). RESULTS: At concentrations >30 µM, POC showed upward biases of 20% (95% limits of agreement [LOA] -22 to 62) and 7% (95% LOA -23 to 38) compared with venous plasma and capillary blood HPLC-MS/MS, respectively. There were no significant differences between mean concentrations for the paracetamol elimination phase. CONCLUSIONS: Upward biases in POC compared with venous plasma HPLC-MS/MS were likely due to higher paracetamol concentrations in capillary blood than in venous plasma and to faulty individual sensors. The novel POC method is a promising tool for paracetamol concentration analysis.

Hydroxychloroquine is Metabolized by Cytochrome P450 2D6, 3A4, and 2C8, and Inhibits Cytochrome P450 2D6, while its Metabolites also Inhibit Cytochrome P450 3A in vitro.

This study aimed to explore the cytochrome P450 (CYP) metabolic and inhibitory profile of hydroxychloroquine (HCQ). Hydroxychloroquine metabolism was studied using human liver microsomes (HLMs) and recombinant CYP enzymes. The inhibitory effects of HCQ and its metabolites on nine CYPs were also determined in HLMs, using an automated substrate cocktail method. Our metabolism data indicated that CYP3A4, CYP2D6, and CYP2C8 are the key enzymes involved in HCQ metabolism. All three CYPs formed the primary metabolites desethylchloroquine (DCQ) and desethylhydroxychloroquine (DHCQ) to various degrees. Although the intrinsic clearance (CLint) value of HCQ depletion by recombinant CYP2D6 was > 10-fold higher than that by CYP3A4 (0.87 versus 0.075 µl/min/pmol), scaling of recombinant CYP CLint to HLM level resulted in almost equal HLM CLint values for CYP2D6 and CYP3A4 (11 and 14 µl/min/mg, respectively). The scaled HLM CLint of CYP2C8 was 5.7 µl/min/mg. Data from HLM experiments with CYP-selective inhibitors also suggested relatively equal roles for CYP2D6 and CYP3A4 in HCQ metabolism, with a smaller contribution by CYP2C8. In CYP inhibition experiments, HCQ, DCQ, DHCQ, and the secondary metabolite didesethylchloroquine were direct CYP2D6 inhibitors, with 50% inhibitory concentration (IC50) values between 18 and 135 µM. HCQ did not inhibit other CYPs. Furthermore, all metabolites were time-dependent CYP3A inhibitors (IC50 shift 2.2-3.4). To conclude, HCQ is metabolized by CYP3A4, CYP2D6, and CYP2C8 in vitro. HCQ and its metabolites are reversible CYP2D6 inhibitors, and HCQ metabolites are time-dependent CYP3A inhibitors. These data can be used to improve physiologically-based pharmacokinetic models and update drug-drug interaction risk estimations for HCQ. SIGNIFICANCE STATEMENT: While CYP2D6, CYP3A4, and CYP2C8 have been shown to mediate chloroquine biotransformation, it appears that the role of CYP enzymes in hydroxychloroquine (HCQ) metabolism has not been studied. In addition, little is known about the CYP inhibitory effects of HCQ. Here, we demonstrate that CYP2D6, CYP3A4, and CYP2C8 are the key enzymes involved in HCQ metabolism. Furthermore, our findings show that HCQ and its metabolites are inhibitors of CYP2D6, which likely explains the previously observed interaction between HCQ and metoprolol.

Systemic hypertonic saline enhances glymphatic spinal cord delivery of lumbar intrathecal morphine.

The blood-brain barrier significantly limits effective drug delivery to central nervous system (CNS) targets. The recently characterized glymphatic system offers a perivascular highway for intrathecally (i.t.) administered drugs to reach deep brain structures. Although periarterial cerebrospinal fluid (CSF) influx and concomitant brain drug delivery can be enhanced by pharmacological or hyperosmotic interventions, their effects on drug delivery to the spinal cord, an important target for many drugs, have not been addressed. Hence, we studied in rats whether enhancement of periarterial flow by systemic hypertonic solution might be utilized to enhance spinal delivery and efficacy of i.t. morphine. We also studied whether the hyperosmolar intervention affects brain or cerebrospinal fluid drug concentrations after systemic administration. Periarterial CSF influx was enhanced by intraperitoneal injection of hypertonic saline (HTS, 5.8%, 20 ml/kg, 40 mOsm/kg). The antinociceptive effects of morphine were characterized, using tail flick, hot plate and paw pressure tests. Drug concentrations in serum, tissue and microdialysis samples were determined by liquid chromatography-tandem mass spectrometry. Compared with isotonic solution, HTS increased concentrations of spinal i.t. administered morphine by 240% at the administration level (T13-L1) at 60 min and increased the antinociceptive effect of morphine in tail flick, hot plate, and paw pressure tests. HTS also independently increased hot plate and paw pressure latencies but had no effect in the tail flick test. HTS transiently increased the penetration of intravenous morphine into the lateral ventricle, but not into the hippocampus. In conclusion, acute systemic hyperosmolality is a promising intervention for enhanced spinal delivery of i.t. administered morphine. The relevance of this intervention should be expanded to other i.t. drugs and brought to clinical trials.

Comparative Hepatic and Intestinal Metabolism and Pharmacodynamics of Statins.

This study aimed to comprehensively investigate the in vitro metabolism of statins. The metabolism of clinically relevant concentrations of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and their metabolites were investigated using human liver microsomes (HLMs), human intestine microsomes (HIMs), liver cytosol, and recombinant cytochrome P450 enzymes. We also determined the inhibitory effects of statin acids on their pharmacological target, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. In HLMs, statin lactones were metabolized to a much higher extent than their acid forms. Atorvastatin lactone and simvastatin (lactone) showed extensive metabolism [intrinsic clearance (CLint) values of 3700 and 7400 µl/min per milligram], whereas the metabolism of the lactones of 2-hydroxyatorvastatin, 4-hydroxyatorvastatin, and pitavastatin was slower (CLint 20-840 µl/min per milligram). The acids had CLint values in the range <0.1-80 µl/min per milligram. In HIMs, only atorvastatin lactone and simvastatin (lactone) exhibited notable metabolism, with CLint values corresponding to 20% of those observed in HLMs. CYP3A4/5 and CYP2C9 were the main statin-metabolizing enzymes. The majority of the acids inhibited HMG-CoA reductase, with 50% inhibitory concentrations of 4-20 nM. The present comparison of the metabolism and pharmacodynamics of the various statins using identical methods provides a strong basis for further application, e.g., comparative systems pharmacology modeling. SIGNIFICANCE STATEMENT: The present comparison of the in vitro metabolic and pharmacodynamic properties of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin and their metabolites using unified methodology provides a strong basis for further application. Together with in vitro drug transporter and clinical data, the present findings are applicable for use in comparative systems pharmacology modeling to predict the pharmacokinetics and pharmacological effects of statins at different dosages.

Relationship of Edoxaban Plasma Concentration and Blood Coagulation in Healthy Volunteers Using Standard Laboratory Tests and Viscoelastic Analysis.

The capability of viscoelastic measurement parameters to screen anticoagulation activity of edoxaban in relation to its plasma concentrations was evaluated in 15 healthy male volunteers. Blood samples were drawn before the oral administration of edoxaban 60 mg and 2, 4, 6, 8, and 24 hours after administration. At each time, standard coagulation tests were performed, blood viscoelastic properties were measured with a thromboelastometry device ROTEM delta analyzer (Instrumentation Laboratory, Werfen, Barcelona, Spain), and edoxaban plasma concentrations were measured. Our primary interest was the possible correlation between edoxaban plasma concentrations and values for ROTEM ExTEM, and FibTEM. We also studied the correlation of edoxaban plasma concentrations with the results of standard coagulation tests. We saw the effect of a single dose of edoxaban most clearly in clotting time (CT) of ROTEM ExTEM and FibTEM. Changes in these parameters correlated significantly with edoxaban plasma concentrations up to 6 hours from the ingestion of the drug. Activated partial thromboplastin time, prothrombin time, and anti-factor Xa were also affected. Peak changes were observed 2 and 4 hours after administration of edoxaban. The changes were mostly reversed after 8 hours. In conclusion, ROTEM CT correlates significantly with edoxaban plasma concentrations and can be used to estimate the effect of edoxaban. ROTEM should be considered as part of the assessment of coagulation, with the big advantage of being readily available on site.

Fluorescence-based high-throughput screening assay for drug interactions with UGT1A6.

The increasing awareness and the rising importance of UDP-glucuronosyltransferases (UGTs) in the pharmacokinetics of drugs have evoked a need to develop more powerful tools for studying the role of UGTs in the metabolism of drug candidates. To this end, we have developed a fluorescent high-throughput screening assay for screening potential inhibitors and/or substrates for recombinant human UGTs-here, for the UGT1A6. The assay is based on the increase in fluorescence intensity when 1-naphthol is glucuronidated. The formation of the highly fluorescent product, 1-naphthylglucuronide, is followed at excitation wavelengths of 295 and 300 nm with fixed emission (335 nm) in real time directly from the reaction mixture. A probe concentration of 5 µM with 2.5 µg of total protein in phosphate buffer at pH 7.4 with 5% dimethyl sulfoxide resulted in optimal linearity and acceptable signal separation (signal-to-base, 3.0) for the probe reaction. The interactions of test compounds with the enzyme are detected as lower rate of 1-naphthylglucuronide formation and thus lower rate of fluorescence increase. The success of the assay was first demonstrated with the known UGT1A6 substrates 4-hydroxyindole and scopoletin (Z' factor ≥0.5) and later with nonsteroidal anti-inflammatory drugs and salicylate derivatives. Diclofenac, 5-methylsalicylic acid, 5-bromosalicylic acid, 5-chlorosalicylic acid, and 5-fluorosalicylic acid decreased the probe glucuronidation rate at 500 µM by >50%. Further, the results gained with the high-throughput screening assay correlated well with the results obtained, in parallel, with the reference high-performance liquid chromatography method.

Glucuronidation of racemic O-desmethyltramadol, the active metabolite of tramadol.

O-Desmethyltramadol, the active metabolite of analgesic tramadol, is metabolised through glucuronidation. The present study was conducted to identify the human UDP-glucuronosyltransferases (UGTs) that catalyse the glucuronidation of O-desmethyltramadol, a racemic mixture of 1R,2R- and 1S,2S-enantiomers. We developed a fast and selective liquid chromatography-mass spectrometry method to separate, analyse and quantify the diastereomeric phenolic O-glucuronides of O-desmethyltramadol. To quantify O-desmethyltramadol glucuronidation, we biosynthesised both phenolic O-glucuronides of O-desmethyltramadol and verified their structure by mass spectrometry and nuclear magnetic resonance spectroscopy. Subsequently, the 16 human UGTs of subfamilies 1A and 2B were screened for O-desmethyltramadol glucuronidation activity. UGTs 1A7-1A10 exhibited a strict stereoselectivity, exclusively glucuroniding the 1R,2R-enantiomer. Similar though not strict enantioselectivity was exhibited by UGT2B15. UGT2B7, on the other hand, glucuronidated both O-desmethyltramadol enantiomers, with slight preference for 1S,2S-O-desmethyltramadol. Enzyme kinetic parameters were determined for the most active UGTs, 1A8 and 2B7. The apparent K(m) or S(50) values were high: 1.2mM±0.23 for 1R,2R-O-desmethyltramadol with UGT1A8 and 1.84±1.2 and 4.6±2.0mM for 1S,2S- and 1R,2R-O-desmethyltramadol enantiomers with UGT2B7, respectively. Glucuronidation analyses of O-desmethyltramadol with human liver microsomes exhibited stereoselectivity, favouring the 1S,2S-O-desmethyltramadol over 1R,2R-O-desmethyltramadol and yielding 62.4 and 24.6pmol/mg/min, respectively. In intestinal microsomes, on the other hand, the two enantiomers were glucuronidated at similar rates, about 6pmol/mg/min. The results shed new light on both tramadol metabolism and the substrate selectivity of the human UGTs.

Glucuronidation of psilocin and 4-hydroxyindole by the human UDP-glucuronosyltransferases.

We have examined the glucuronidation of psilocin, a hallucinogenic indole alkaloid, by the 19 recombinant human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A, and 2B. The glucuronidation of 4-hydroxyindole, a related indole that lacks the N,N-dimethylaminoethyl side chain, was studied as well. UGT1A10 exhibited the highest psilocin glucuronidation activity, whereas the activities of UGTs 1A9, 1A8, 1A7, and 1A6 were significantly lower. On the other hand, UGT1A6 was by far the most active enzyme mediating 4-hydroxyindole glucuronidation, whereas the activities of UGTs 1A7-1A10 toward 4-hydroxyindole resembled their respective psilocin glucuronidation rates. Psilocin glucuronidation by UGT1A10 followed Michaelis-Menten kinetics in which psilocin is a low-affinity high-turnover substrate (K(m) = 3.8 mM; V(max) = 2.5 nmol/min/mg). The kinetics of psilocin glucuronidation by UGT1A9 was more complex and may be best described by biphasic kinetics with both intermediate (K(m1) = 1.0 mM) and very low affinity components. The glucuronidation of 4-hydroxyindole by UGT1A6 exhibited higher affinity (K(m) = 178 microM) and strong substrate inhibition. Experiments with human liver and intestinal microsomes (HLM and HIM, respectively) revealed similar psilocin glucuronidation activity in both samples, but a much higher 4-hydroxyindole glucuronidation rate was found in HLM versus HIM. The expression levels of UGTs 1A6-1A10 in different tissues were studied by quantitative real-time-PCR, and the results, together with the activity assays findings, suggest that whereas psilocin may be subjected to extensive glucuronidation by UGT1A10 in the small intestine, UGT1A9 is likely the main contributor to its glucuronidation once it has been absorbed into the circulation.

UDP-glucuronosyltransferases in conjugation of 5alpha- and 5beta-androstane steroids.

We have examined the glucuronidation of androsterone (5alpha-androstane-3alpha-ol-17-one), etiocholanolone (5beta-androstane-3alpha-ol-17-one), 5alpha-androstane-3alpha-,17beta-diol (5alpha-diol), and 5beta-androstane-3alpha-, 17beta-diol (5beta-diol) by 19 recombinant human UDP-glucuronosyltransferases (UGTs). The results reveal large differences in stereo- and regioselectivity between UGT2B7, UGT2B15, and UGT2B17. UGT2B7 conjugated all four androgens at the 3-OH but not at the 17-OH that is available in both diols. UGT2B7 exhibited a higher glucuronidation rate toward the steroids with a flat backbone, androsterone and 5alpha-diol, compared with etiocholanolone and 5beta-diol, which have a bent backbone. UGT2B17 readily glucuronidated androsterone and, particularly, etiocholanolone at the 3-OH, but in the two diols it exhibited high preference for the 17-OH and low glucuronidation rate at the 3-OH. UGT2B15 did not glucuronidate any of the studied four androgens at the 3-OH, but it did conjugate both diols at the 17-OH, with a clear preference for 5alpha-diol. Of the UGT1A subfamily, only UGT1A4 catalyzed the glucuronidation of androsterone and 5alpha-diol at measurable rates, even if low. UGT2A1 and UGT2A2 glucuronidated most compounds in this study, but mostly at rather low rates. An exception was the glucuronidation of etiocholanolone by UGT2A1 that revealed a very low substrate affinity in combination with very high V(max) value. The results shed new light on the substrate selectivity of individual UGTs in steroid glucuronidation. In addition they bear implications for doping analyses and its dependence of genetic polymorphism because testosterone is a precursor in the biosynthesis of these four androgens, whereas the contribution of UGT2B17 to their glucuronidation varies greatly.

Mutation analysis in UGT1A9 suggests a relationship between substrate and catalytic residues in UDP-glucuronosyltransferases.

UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from UDP-glucuronic acid to endo- and xenobiotics in our body. UGTs belong to the GT1 family of glycosyltransferases and many GT1s use a serine protease-like catalytic mechanism in which an Asp-His pair deprotonates a hydroxyl on the aglycone for nucleophilic attack on the sugar donor. The pair in human UGTs could be H37 and either D143 or D148 (UGT1A9 numbering). However, H37 is not totally conserved, being replaced by either Pro or Leu in UGT1A4 and UGT2B10. We therefore investigated the role of H37, D143 and D148 in UGT1A9 by site-directed mutagenesis, activity and kinetic measurements with several substrates. The results suggest that H37 is not critical in N-glucuronidation, but is so in O-glucuronidation. The V(max) of the H37A mutant was much less affected in N- than O-glucuronidation, while the reverse was true for the Asp mutations, particularly D143A. We suggest that this is due to the opposing properties of O- and N- nucleophiles. O-nucleophiles require the histidine to deprotonate them so that they become effective nucleophiles, while N-nucleophiles develop a formal positive charge during the reaction (RNH(2)(+)-GlcA), and thus require a negatively charged residue to stabilize the transition state.

The UDP-glucuronosyltransferases as oligomeric enzymes.

The UDP-glucuronosyltransferases (UGTs) are integral membrane proteins of the endoplasmic reticulum that play important roles in the defense against potentially hazardous xenobiotics. The UGTs also participate in the metabolism and homeostasis of many endogenous compounds, including bilirubin and steroid hormones. Most human UGTs can glucuronidate several substrates the chemical structures of which may vary significantly. Understanding the structural basis for the complex substrate specificity of the UGTs is a major challenge that is hampered by the lack of sufficient structural information on these enzymes. Nevertheless, there is currently a broad interest in the structure and function of the UGTs and here we have focused on their oligomeric state. The question whether or not the UGTs are oligomeric enzymes, either dimeric or tetrameric, was frequently addressed in the past, as well as in recent studies. The current knowledge of protein-protein interactions among the UGTs is limited, however, primarily due to considerable difficulties in purifying individual recombinant UGTs as fully active and mono-dispersed proteins. Such hurdles in studying the oligomeric state of the UGTs prompted researchers to develop less direct approaches for examining the quaternary structure of the UGTs and its functional significance. In this article we have reviewed, sometimes critically, most of the available studies about the oligomeric state of the UGTs. Concluding that the UGTs are oligomeric enzymes, we discuss hetero-oligomerization among UGTs and its possible implications for the structure, function and substrate specificity of the enzymes.

Eudismic analysis of tricyclic sesquiterpenoid alcohols: lead structures for the design of potent inhibitors of the human UDP-glucuronosyltransferase 2B7.

The epimeric tricyclic sesquiterpenoid alcohols globulol, epiglobulol, cedrol, epicedrol, longifolol, and isolongifolol were investigated in their ability to inhibit the recombinant human UDP-glucuronosyltransferase (UGT) 2B7. The stereoisomers displayed rapidly reversible competitive inhibition, which was substrate-independent. Longifolol and its stereoisomer isolongifolol displayed the lowest competitive inhibition constants (K(ic)) of 23 and 26 nM, respectively. The K(ic) values of cedrol and its epimer epicedrol were 0.15 and 0.21 microM, those of globulol and epiglobulol were 5.4 and 4.0 microM, respectively. The diastereomeric alcohols exhibited nearly identical affinities toward UGT2B7 indicating that the spatial arrangement of the hydroxy group had no influence on the dissociation of the enzyme-terpenoid complex. The high affinities stemmed presumably from mere hydrophobic interactions between the hydrocarbon scaffold of the terpenoid alcohol and the binding site of the enzyme. Glucuronidation assays revealed that there were large differences in the rates at which the epimeric alcohols were conjugated. Therefore, the spatial arrangement of the hydroxy group controlled the rate of the UGT2B7-catalyzed reaction. The introduction of a methyl group into the side chain of isolongifolol and longifolol increased the steric hindrance. As a result, the rate of the UGT2B7-catalyzed reaction was decreased by more than 88%. The findings indicated that the rate of the UGT2B7-catalyzed glucuronidation is significantly controlled by stereochemical and steric factors. Considering the high inhibition levels exerted by the tricyclic sesquiterpenoid alcohols, these compounds might serve as valuable lead structures for the design of potent inhibitors for UGT2B7.

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.

Potent inhibitors of the human UDP-glucuronosyltransferase 2B7 derived from the sesquiterpenoid alcohol longifolol.

The tricyclic sesquiterpenol (+)-longifolol served as a lead structure for the design of inhibitors of the human UDP-glucuronosyltransferase (UGT) 2B7. Twenty-four homochiral and epimeric longifolol derivatives were synthesized and screened for their ability to inhibit the enzyme. The absolute configuration at the stereogenic center C1' was determined by X-ray crystallography and 2D NMR spectroscopy (gHSQC, gNOESY). The phenyl-substituted secondary alcohol 16 b (beta-phenyllongifolol) displayed the highest affinity toward UGT2B7, and its inhibitory dissociation constant was 0.91 nM. The mode of inhibition was rapidly reversible and competitive. The inhibitor was not glucuronidated by UGT2B7 or other hepatic UGTs, presumably as a result of the high steric demand exerted by the phenyl group. Inhibition assays employing 14 other UGT isoforms suggested that inhibitor 16 b was highly selective for UGT2B7.

Isoform-selective inhibition of the human UDP-glucuronosyltransferase 2B7 by isolongifolol derivatives.

A set of 48 derivatives of the tricyclic sesquiterpenol alcohol isolongifolol was synthesized. The set comprised homochiral and diastereomeric alcohols, amines, chlorohydrins, as well as carboxylic acids, phosphonic acids, and their corresponding esters. The absolute configuration of the epimeric compounds was assigned by 2D NMR experiments [gradient heteronuclear single quantum correlation (gHSQC) and gradient nuclear Overhauser enhancement spectroscopy (gNOESY)] in agreement with crystallographic data. The tricyclic derivatives were assessed as inhibitors of the human UDP-glucuronosyltransferase (UGT) 2B7. The phenyl-substituted secondary alcohol 26b was the best inhibitor in this series and its competitive inhibition constant was 18 nM. Compound 26b was not glucuronidated by UGT2B7 and other hepatic UGT enzymes, presumably due to the high steric hindrance exerted by its bulky phenyl substituent. Its inhibitory activity toward 14 other UGT isoforms of subfamily 1A and 2B was determined, and the data indicated that the tricyclic secondary alcohol 26b was highly selective for UGT2B7 (true selectivity >1000).

Interactions with other human UDP-glucuronosyltransferases attenuate the consequences of the Y485D mutation on the activity and substrate affinity of UGT1A6.

OBJECTIVES: To explore the possible role of hetero-oligomerization among the human UDP-glucuronosyltransferases in attenuating the consequences of the pathological Y486D mutation (UGT1A1 numbering) that often causes hyperbilirubinaemia. Owing to exon sharing in the human UGT1A gene, the equivalent mutation is present in all other UGT1As of the affected individuals. It is unknown, however, if this mutation results in clinical conditions, other than impaired bilirubin conjugation by UGT1A1. METHODS: The main experimental approach in this study was to try and form hetero-oligomers of selected UDP-glucuronosyltransferases by coinfecting insect cells with recombinant baculoviruses that encode different human UDP-glucuronosyltransferases and mutants thereof. The infected cells were analysed for both relative expression levels and catalytic activity in each case, the combination of which yielded normalized activity. Kinetic analyses and copurification by affinity chromatography were also performed. RESULTS: Coinfections with UGT1A4 increased the normalized scopoletin glucuronidation of 6YD (the Y485D mutant of UGT1A6) much more than it affected 1YD (the Y486D mutant of UGT1A1). Serotonin glucuronidation analyses revealed that coexpression of 6YD with most other human UDP-glucuronosyltransferases significantly increased the normalized activity of this mutant. Using 1-naphthol as the aglycone substrate, the Km of 6YD for the cosubstrate UDP-glucuronic acid was about 50 times higher than in UGT1A6. Yet, coexpression of 6YD with UGT1A4 lowered the Km for UDP-glucuronic acid to the level of UGT1A6. Coexpression also influenced wild-type UGT1A6 and UGT2B7, increasing the normalized activity of UGT1A6, but decreasing it for UGT2B7. CONCLUSION: Hetero-oligomerization may play an important role in UDP-glucuronosyltransferases activity.