Regioselective sulfonation of dopamine by SULT1A3 in vitro provides a molecular explanation for the preponderance of dopamine-3-O-sulfate in human blood circulation.

SULT1A3 is an enzyme that catalyzes the sulfonation of many endogenous and exogenous phenols and catechols. The most important endogenous substrate is dopamine (DA), which is often used as a probe substrate for SULT1A3. We developed a new method for analyzing the SULT1A3 reaction products by high-performance liquid chromatography (HPLC) with electrochemical detection. The sulfonate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), DA and the two dopamine sulfates, DA-3-O-sulfate and DA-4-O-sulfate, can be separated within 3 min. This enables quantitation of the sulfates without radioactive PAPS or the precipitation of unreacted PAPS. Both sulfates were synthesized as reference substances and characterized by (1)H and (13)C nuclear magnetic resonance (NMR), mass spectrometry (MS) and tandem mass spectrometry (MS/MS). The purity of the dopamine sulfates was estimated by HPLC using a diode array detector. We determined the enzyme kinetic parameters for formation of DA-3-O-sulfate and DA-4-O-sulfate using purified recombinant human SULT1A3. The reactions followed Michaelis-Menten kinetics up to 50 microM DA concentration, and strong substrate inhibition was observed at higher concentrations. The apparent K(m) values for sulfonation at both hydroxy groups were similar (2.21+/-0.764 and 2.59+/-1.06 microM for DA-4-O-sulfate and DA-3-O-sulfate, respectively), but the V(max) was approximately six times higher for the formation of the 3-O-sulfate (344+/-139 nmol/min/mg protein) than the 4-O-sulfate (45.4+/-16.5 nmol/min/mg protein). These results are in accordance with the observation that DA-3-O-sulfate is more abundant in human blood than DA-4-O-sulfate and that in the crystal structure of SULT1A3 with dopamine bound to the active site, the 3-hydroxy group is aligned to form hydrogen bonds with catalytic residues of the enzyme.

A modification of the Hammett equation for predicting ionisation constants of p-vinyl phenols.

Currently there are several compounds used as drugs or studied as new chemical entities, which have an electron withdrawing group connected to a vinylic double bond in a phenolic or catecholic core structure. These compounds share a common feature--current computational methods utilizing the Hammett type equation for the prediction of ionisation constants fail to give accurate prediction of pK(a)'s for compounds containing the vinylic moiety. The hypothesis was that the effect of electron-withdrawing substituents on the pK(a) of p-vinyl phenols is due to the delocalized electronic structure of these compounds. Thus, this effect should be additive for multiple substituents attached to the vinylic double bond and quantifiable by LFER-based methods. The aim of this study was to produce an improved equation with a reduced tendency to underestimate the effect of the double bond on the ionisation of the phenolic hydroxyl. To this end a set of 19 para-substituted vinyl phenols was used. The ionisation constants were measured potentiometrically, and a training set of 10 compounds was selected to build a regression model (r2 = 0.987 and S.E. = 0.09). The average error with an external test set of six compounds was 0.19 for our model and 1.27 for the ACD-labs 7.0. Thus, we have been able to significantly improve the existing model for prediction of the ionisation constants of substituted p-vinyl phenols.

Kinetic characterization of the 1A subfamily of recombinant human UDP-glucuronosyltransferases.

The initial glucuronidation rates were determined for eight recombinant human UDP-glucuronosyltransferases (UGTs) of the 1A subfamily, and the bisubstrate kinetics and inhibition patterns were analyzed. At low substrate concentrations, the reactions followed general ternary complex kinetics, whereas at higher concentrations of both substrates, the reactions were mostly characterized by ternary complex kinetics with substrate inhibition. The glucuronidation of entacapone by UGT1A9 was inhibited by 1-naphthol in a competitive fashion, with respect to entacapone, and an uncompetitive fashion, with respect to UDP-glucuronic acid (UDPGA). Its inhibition by UDP, on the other hand, was noncompetitive with respect to entacapone and competitive with respect to UDPGA. These inhibition patterns are compatible with a compulsory ordered bi bi mechanism in which UDPGA is the first-binding substrate. Despite the identical primary structure of the C-terminal halves of the UGT1A isoforms, there were marked differences in the respective K(m) values for UDPGA, ranging from 52 microM for UGT1A6 to 1256 microM for UGT1A8. Relative specificity constants were calculated for the eight UGT1A isoforms with 1-hydroxypyrene, 4-nitrophenol, scopoletin, 4-methylumbelliferone, and entacapone as aglycone substrates. The results demonstrated that seven of the UGT1A isoforms are capable of conjugating phenolic substrates with similar highest k(cat) values, and UGT1A4 has a lower relative turnover rate. The highest specificity constants were obtained for 1-hydroxypyrene, even with UGT1A6, which has been regarded as a specific isoform for small planar phenols. A k(cat) value of 1.9 s(-1) was calculated for the glucuronidation of scopoletin by purified UGT1A9.

Assessment of catechol induction and glucuronidation in rat liver microsomes.

Catechols are substances with a 1,2-dihydroxybenzene group from natural or synthetic origin. The aim of this study was to determine whether catechols (4-methylcatechol, 4-nitrocatechol, 2,3-dihydroxynaphthalene) and the antiparkinsonian drugs, entacapone and tolcapone, at doses 150 to 300 mg/kg/day, for 3 days, are able to enhance their own glucuronidation. The induction potency of catechols on rat liver UDP-glucuronosyltransferases (UGTs) was compared with that of a standard polychlorinated biphenyl (PCB) inducer, Aroclor 1254. The glucuronidation rate of these catechols was enhanced up to 15-fold in the liver microsomes of PCB-treated rats, whereas treatment with catechols had little effect. Entacapone, tolcapone, 4-methylcatechol, catechol, 2,3-dihydroxynaphthalene, and 4-nitrocatechol were glucuronidated in control microsomes at rates ranging from 0.12 for entacapone to 22.0 nmol/min/mg for 4-nitrocatechol. Using 1-naphthol, entacapone, and 1-hydroxypyrene as substrates, a 5-, 8-, and 16-fold induction was detected in the PCB rats, respectively, whereas the catechol-induced activities were 1.1- to 1.5-fold only. Entacapone was glucuronidated more efficiently by PCB microsomes than by control microsomes (Vmax/Km, 0.0125 and 0.0016 ml/min/mg protein, respectively). Similar kinetic results were obtained for 1-hydroxypyrene. The Eadie-Hofstee plots suggested the contribution of multiple UGTs for the glucuronidation of 1-hydroxypyrene (Km1, Km2, Km3 = 0.8, 9.7, and 63 microM, and Vmax1, Vmax2, Vmax3 = 11, 24, and 55 nmol/min/mg, respectively), whereas only one UGT could be implicated in the glucuronidation of entacapone (Km = 130 microM, Vmax = 1.6 nmol/min/mg). In conclusion, catechols are poor inducers of their own glucuronidation supported by several UGT isoforms. Their administration is unlikely to affect the glucuronidation of other drugs administered concomitantly.

CoMFA modeling of human catechol O-methyltransferase enzyme kinetics.

Three-dimensional QSAR models with different charge calculation methods (MOPAC-AM1-ESP, MOPAC-AM1-Coulson and Gasteiger-Hückel) were developed for predicting all three enzyme kinetic parameters Km, Vmax and Vmax/Km for catecholic substrates of human soluble catechol O-methyltransferase (S-COMT). The empirical parameters of 45 substrates were correlated to the steric and electronic molecular fields of the substrates utilizing Comparative Molecular Field Analysis (CoMFA). Alignment rules for CoMFA were developed based on the catalytic mechanism and crystal structure of S-COMT, and the analysis was optimized using an all-space search technique. Leave-one-out and leave-n-out cross-validation (with 5 and 10 cross-validation groups) was carried out, and all developed models proved to be statistically significant with q2 values up to 0.84. The models based on MOPAC charge calculations predicted the empirical values clearly better than the Gasteiger-Hückel method. The derived CoMFA coefficient contour maps of steric and electrostatic interactions correlated clearly with the S-COMT crystallographic structures.

CoMFA modeling of enzyme kinetics: K(m) values for sulfation of diverse phenolic substrates by human catecholamine sulfotransferase SULT1A3.

Three-dimensional QSAR models were developed for predicting kinetic Michaelis constant (K(m)) values for phenolic substrates of human catecholamine sulfating sulfotransferase (SULT1A3). The K(m) values were correlated to the steric and electronic molecular fields of the substrates utilizing Comparative Molecular Field Analysis (CoMFA). The evaluated SULT1A3 substrate data set consisted of 95 different substituted phenols, catechols, catecholamines, steroids, and related structures for which the K(m) values were available. The data set was divided in three different subgroups in the initial analysis: (1). for the first CoMFA model substrates with only one reacting hydroxyl group were selected (n = 51), (2).the second model was build with structurally rigid substrates (n = 59), and (3). finally all substrates of the data set were included in the analysis (n = 95). Substrate molecules were aligned using the aromatic ring and the reacting hydroxyl group as a template. After the initial analysis different substrate alignment rules based on the existing knowledge of the SULT1A3 active site structure were evaluated. After this optimization a final CoMFA model was built including all 95 substrates of the data set. Cross-validated q(2) values (leave-one-out and leave-n-out) and coefficient contour maps were calculated for all derived CoMFA models. All four CoMFA models were statistically significant with q(2) values up to 0.624. These predictive QSAR models will provide us information about the factors that affect substrate binding at the active site of human catecholamine sulfotransferase SULT1A3.

Prediction of physicochemical properties based on neural network modelling.

The literature describing neural network modelling to predict physicochemical properties of organic compounds from the molecular structure is reviewed from the perspective of pharmaceutical research. The standard three-layer, feed-forward neural network is the technique most frequently used, although the use of other techniques is increasing. Various approaches to describe the molecular structure have been successfully used, including molecular fragments, topological indices, and descriptors calculated by semi-empirical quantum chemical methods. Some physicochemical properties, such as octanol-water partition coefficient, water solubility, boiling point and vapour pressure, have been modelled by several research groups over the years using different approaches and structurally diverse large training sets. The prediction accuracy of most models seems to be rather close to the performance of the experimental measurements, when the accuracy is assessed with a test set from the working database. Results with independent test sets have been less satisfactory. Implications of this problem are discussed.

Conjugation of catechols by recombinant human sulfotransferases, UDP-glucuronosyltransferases, and soluble catechol O-methyltransferase: structure-conjugation relationships and predictive models.

Conjugation of a structurally diverse set of 53 catechol compounds was studied in vitro using six recombinant human sulfotransferases (SULTs), five UDP-glucuronosyltransferases (UGT) and the soluble form of catechol O-methyltransferase (S-COMT) as catalyst. The catechol set comprised endogenous compounds, such as catecholamines and catecholestrogens, drugs, natural plant constituents, and other catechols with diverse substituent properties and substitution patterns. Most of the catechols studied were substrates of S-COMT and four SULT isoforms (1A1, 1A2, 1A3, and 1B1), but the rates of conjugation varied considerably, depending on the substrate structure and the enzyme form. SULT1E1 sulfated fewer catechols. Only low activities were observed for SULT1C2. UGT1A9 glucuronidated catechols representing various structural classes, and almost half of the studied compounds were glucuronidated at a high rate. The other UGT enzymes (1A1, 1A6, 2B7, and 2B15) showed narrower substrate specificity for catechols, but each glucuronidated some catechols at a high rate. Dependence of specificity and rate of conjugation on the molecular structure of the substrate was characterized by structure-activity relationship analysis and quantitative structure-activity relationship modeling. Twelve structural descriptors were used to characterize lipophilicity/polar interaction properties, steric properties, and electronic effects of the substituents modifying the catechol structure. PLS models explaining more than 80% and predicting more than 70% of the variance in conjugation activity were derived for the representative enzyme forms SULT1A3, UGT1A9, and S-COMT. Several structural factors governing the conjugation of catechol hormones, metabolites, and drugs were identified. The results have significant implications for predicting the metabolic fate of catechols.

Expression and characterization of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than other UGTs and was purified as an active dimeric enzyme.

Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of alpha-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged UGT1A9. Membranes from the co-infected cells, or a mixture of membranes from separately infected cells, were subjected to detergent extraction and IMAC, and the resulting fractions were analyzed for the presence of each type of UGT1A9 using tag-specific antibodies. In the case of separate infection, the HA-tagged UGT1A9 did not bind to the column. When co-infected with His-tagged UGT1A9, however, part of the HA-tagged enzyme was bound to the column and was eluted by imidazole concentration gradient together with the His-tagged UGT1A9, suggesting the formation of stable dimers that contain one His-tagged and one HA-tagged UGT1A9 monomers.

Comparison of electrospray, atmospheric pressure chemical ionization, and atmospheric pressure photoionization in the identification of apomorphine, dobutamine, and entacapone phase II metabolites in biological samples.

The applicability of different ionization techniques, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and a novel atmospheric pressure photoionization (APPI), were tested for the identification of the phase II metabolites of apomorphine, dobutamine, and entacapone in rat urine and in vitro incubation mixtures (rat hepatocytes and human liver microsomes). ESI proved to be the most suitable ionization method; it enabled detection of 22 conjugates, whereas APCI and APPI showed only 12 and 14 conjugates, respectively. Methyl conjugates were detected with all ionization methods. Glucuronide conjugates were ionized most efficiently with ESI. Only some of the glucuronides detected with ESI were detected with APCI and APPI. Sulfate conjugates were detected only with ESI. MS/MS experiments showed that the site of glucuronidation or sulfation could not be determined, since the primary cleavage was a loss of the conjugate group (glucuronic acid or SO3), and no site-characteristic product ions were formed. However, it may be possible to determine the site of methylation, since methylated products are more stable than glucuronides or sulfates. Furthermore, the loss of CH3 is not necessarily the primary cleavage, and site characteristic products may be formed. Identification and comparison of conjugates formed from the current model drugs were successfully analyzed in different biological specimens of common interest to biomedical research. A fairly good relation was obtained between the data from in vivo and in vitro models of drug metabolism.

N-Glucuronidation of some 4-arylalkyl-1H-imidazoles by rat, dog, and human liver microsomes.

N-Glucuronidation in vitro of six 4-arylalkyl-1H-imidazoles (both enantiomers of medetomidine, detomidine, atipamezole, and two other closely related compounds) by rat, dog, and human liver microsomes and by four expressed human UDP-glucuronosyltransferase isoenzymes was studied. Human liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles with high activity, with apparent V(max) values ranging from 0.59 to 1.89 nmol/min/mg of protein. In comparison, apparent V(max) values for two model compounds forming the N-glucuronides 4-aminobiphenyl and amitriptyline were 5.07 and 0.56 nmol/min/mg of protein, respectively. Atipamezole showed an exceptionally low apparent K(m) value of 4.0 microM and a high specificity constant (V(max)/K(m)) of 256 compared with 4-aminobiphenyl (K(m), 265 microM; V(max)/K(m), 19) and amitriptyline (K(m), 728 microM; V(max)/K(m), 0.8). N-Glucuronidation of medetomidine was highly enantioselective in human liver microsomes; levomedetomidine exhibited a 60-fold V(max)/K(m) value compared with dexmedetomidine. Furthermore, two isomeric imidazole N-glucuronides were formed from dexmedetomidine, but only one was formed from levomedetomidine. Dog liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles at a low rate and affinity, with apparent V(max) values ranging from 0.29 to 0.73 nmol/min/mg of protein and apparent K(m) values from 279 to 1640 microM. Rat liver microsomes glucuronidated these compounds at a barely detectable rate. Four expressed human UDP-glucuronosyltransferase isoenzymes (UGT1A3, UGT1A4, UGT1A6, and UGT1A9) were studied for 4-arylalkyl-1H-imidazole-conjugating activity. Only UGT1A4 glucuronidated these compounds at an activity of about 5% of that measured for 4-aminobiphenyl. The observed activity of UGT1A4 does not explain the high efficiency of glucuronidation of 4-arylalkyl-1H-imidazoles in human liver microsomes.

Characterization of catechol glucuronidation in rat liver.

Catechols are a class of substances from natural or synthetic origin that contain a 1,2-dihydroxybenzene group. We have characterized the glucuronidation by rat liver microsomes and by the rat liver recombinant UDP-glucuronosyltransferase isoforms UGT1A6 and UGT2B1 of a series of 42 structurally diverse catechols, including neurotransmitters, polyphenols, drugs, and catechol estrogens. Small catechols (4-nitrocatechol, 2,3-dihydroxybenzaldehyde, 4-methylcatechol, and tetrachlorocatechol), tyrphostine A23, and octylgallate were glucuronidated at the highest rate by rat liver microsomes and the recombinant enzymes. By contrast, polyphenols from green tea (catechin and related compounds), 3,5-dinitrocatechol, the catechol-O-methyltransferase inhibitor drugs (entacapone, nitecapone, and tolcapone), the carboxyl catechols (gallic acid and dihydroxybenzoic acid derivatives), and the neurotransmitters and dopaminergic drugs, except dobutamine, were glucuronidated at low rate. Glucuronidation of most catechols was increased upon treatment of rats by 3-methylcholanthrene (3-MC) or Aroclor 1254. No induction was observed after administration of phenobarbital and clofibrate or treatment with catechols. Partial least-squares modeling was carried out to explain the variations of glucuronidation activity by liver microsomes of nontreated and 3-MC-treated rats. The model developed explained 82% and predicted 61% of the variations of glucuronidation activities. Among the 17 electronic and substructure parameters used that characterize the catechols, the hydrophobicity/molar volume ratio of catechols showed a strong positive correlation with the glucuronidation rate. The effect of the pK(a) of the catechol group was modeled to be nonlinear, the optimal pK(a) value for glucuronidation being between 8 and 9. Hydrogen bonding and steric effects also were important to account for to predict the glucuronidation rates.