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Development and prevalidation of predictive models for catechol drug conjugation and their evaluation for rational drug design

Risultati finali

Rat hepatocytes have been cultured as primary cultures and incubated in the presence of various catechols. The hepatocytes have the advantage to express the whole set of drug metabolising enzymes, including the UDP-glucuronosyltransferases, sulfotransferases and catechol-O-methyltransferases that are involved in the metabolism of these substances. Hepatocytes allow to appreciate the relative importance of these three biotransformation pathways, and to predict the in vivo conjugate profile by phase II reactions in rat. Hepatocytes have been isolated from rat liver by the collagenase perfusion method. They were cultured in medium composed of 75% MEM and 25% Medium 199 in presence of fetal calf serum. Selected catechol drugs were added to the culture medium at the concentration of 50 and 500 microM and incubated for 2 and 20 hours. After extraction from the medium, the metabolites were analysed at the University of Helsinki for detection and identification by liquid chromatography coupled to mass spectrometry. This technology allowed us to determine the concomitant formation of glucuronide, sulphate and methyl conjugates of catechols. These results were in qualitative agreement with those observed in rat urine excretion, in vivo, upon administration of the compounds, and with the prediction based on human and rat enzyme data.
The 1-hydroxypyrene UGT method provides a novel tool for quantifying extremely low UGT activities in the human liver as well as in other tissue specimens. This in vitro glucuronidation reaction is preferentially catalyzed by the human recombinant UGT1A6 and UGT1A9 isoforms. On the other hand, the human recombinant UGT1A9 form was capable of glucuronidating the greatest number (n=35) out of the 49 different model catechol compounds that were screened within this EC project, whereas the human UGT1A6 form was catalytically active toward 16 out of the 49 compounds tested (Ethell et al.). It is thus quite evident that the '1-hydroxypyrene UGT' measurement may serve as a sensitive and selective 'biomarker assay' for the screening of human UGT activity with special relevance to isoforms active toward catechol compounds. A nonradioactive biomarker assay for detecting UGT1A9 and UGT1A6 activity in human tissues was developed, and new means were discovered for in vitro screening of individual differences in catechol-glucuronidating activities in human liver or other tissues. This fluorometric HPLC method is based on quantitation of 1-hydroxypyrene-ί-D-glucuronide formed from 1-hydroxypyrene. For isoform-selective enzyme analysis, the UGT activity was measured at the Km concentrations of UGT1A9 (Km = 1mircometers) and UGT1A6 (Km = 45 mircometres) to distinguish between these two activities. The validity of the latter approach is based on (i) the great enough difference in affinity of these UGTs toward 1-hydroxypyrene and (ii) the high sensitivity of the analytical method (quantity limit around 3 pg) allowing activity testing under reaction conditions primarily suited for the detection of UGT1A9 alone, or the combined activity of UGT1A9+UGT1A6. According to the results of the entire EC project, the glucuronidation of 36 out of 49 structurally variant model catechol compounds was catalyzed by human recombinant UGT1A9 and/or UGT1A6. Entacapone and tolcapone, the well-known new COMT-inhibitor catechol drugs, are glucuronidated mainly by the UGT1A9 form. This finding suggests that our 'biomarker' assay for detecting UGT1A9 may become of a true exploitable value, because the sensitivity of this method is manifold greater than that of the direct methods available today for entacapone or tolcapone glucuronidation.
The expression of two human UDP-glucuronosyltransferase isoenzymes in Semliki Forest virus expression system provides the means of producing large quantities of recombinant enzyme for enzyme kinetic determinations. Protocols and methods for the expression of human UDP-glucuronosyltransferase cDNAs using the Semliki Forest virus expression system were developed. Infection of Chinese hamster lung fibroblast V79 cells were with recombinant SFV-UGT viruses resulted in efficient protein expression. The expression of UGT1A6 and 1A9 in the SFV-infected cells was approximately two fold higher than in a stable V79 cell line. The measurement of kinetic parameters using p-nitrophenol and nitrocatechol, entacapone, as substrates showed that the overall kinetic properties of enzymes produced by the two systems were similar. SFV-UGT viruses also efficiently infected other mammalian cells, such as baby hamster kidney (BHK), Chinese hamster ovary (CHO) and human lung (WI-26 VA4) cells leading to high production of recombinant enzymes
The expression of seven sulfotransferase enzymes in E. coli provides the means of producing large quantitites of recombinant enzyme for enzyme kinetic determinations and purification for the production of antibodies. Protocols and methods for the expression of sulfotransferase cDNAs in E. coli were developed. The procedure uses the pET system of expression vectors to provide an inducible, high output expression system. Significant levels of catalytically active enzyme can be readily produced (up to 30mg pure protein may be obtained per litre of culture medium). Expression levels were quantified using SDS-PAGE and enzyme activity determination using standard substrates for each isoform. The expressed enzyme was used to assess the substrate specificities of individual sulfotransferases towards endogenous and xenobiotic catechols. The individual enzymes could be readily purified from E. coli cell free extract using a combination of ammonium sulphate precipitation, anion exchange chromatography and affinity chromatography. Purified enzymes were then used for detailed kinetic determinations of carefully selecte catechols from which QSAR models were built and validated using the X-ray crystal structure of sulfotransferases SULT1A3 and mEST
A new unified assay for the determination of UDP-glucuronosyltransferase (UGT) activities was developed, using radio-labelled UDPGA and High-Performance Liquid Chromatography. The resolution of [C-14]uridine diphosphate glucuronic acid from radiolabeled glucuronides formed by incorporation of this radiolabel can now be achieved by a sensitive and rapid gradient HPLC method which utilizes a radioactivity endpoint as a universal detection method. One important application of this method is the determination of kinetic parameters for cloned and expressed UGT isoforms with greater speed and precision than can be afforded by TLC methodology. Moreover, assays with C-14-labelled substrates indicate that gradient HPLC can easily resolve the substrate from the glucuronide products and present an alternative to the time-consuming optimisation of conditions for organic phase extraction assays.
We have genetically engineered V79 cells (Chinese hamster lung fibroblasts) by cDNA encoding the rat liver UDP-glucuronosyltransferase (UGT) isoforms, UGT1A6 and UGT2B1. These proteins are involved in the glucuronidation of toxic substances, such as phenols (carcinogens, pesticides) and carboxyl drugs (nonsteroidal anti-inflammatory drugs). Upon transfection of the cells, and selection of the clones by antibiotics, high production of catalytically active proteins was achieved. The expression was stable over time, thus allowing a continuous supply of recombinant enzymes. These two metabolic competent cells, which express each a distinct UGT isoform have been used in this study in order to determine if they catalysed the glucuronidation of catechols. They can also be potentially used for other purposes, such as bioavailability, drug metabolism and toxicity of drugs and chemicals, as well as drug-drug interactions. Additionally, heterologous expression of UGTs provides an opportunity to study the structure and the function of these membrane proteins. V79 cells were co-transfected with a eukaryotic expression vector carrying the cDNA encoding the rat liver UGT1A6 or UGT2B1 and a plasmid encoding the Geneticin resistance gene. Upon transfection, the cells were incubated in a medium containing Geneticin in order to select the recombinant clones. The expression of the recombinant enzymes was monitored by measurement of the activity with 1-naphthol or ketoprofen, as substrates and by determining the amount of protein by Western blot with specific antibodies. The most producing and stable clones were selected and stored in liquid nitrogen. Membrane enriched fractions were prepared from cellular homogenate by differential ultracentrifugations. These microsomes were used to investigate the in vitro glucuronidation of catechols by the recombinant UGT1A6 and UGT2B1.
The expression of five human UDP-glucuronosyltransferase isoenzymes in V79 cells provides the means of producing large quantities of recombinant enzyme for enzyme kinetic determinations and for the characterisation of antibodies. Protocols and methods for the expression of human UDP-glucuronosyltransferase cDNAs in V79 Chinese hamster lung fibroblast cells were developed. The procedure uses the pCI and pCDNA series of expression vectors to provide a high-output stable expression system. Significant levels of catalytically active enzyme can be readily produced. Expression levels were assessed using immunoblotting and enzyme activity determination using standard substrates for each isoform. The expressed enzyme was used to assess the substrate specificities of individual UDP-glucuronosyltransferases towards endogenous and xenobiotic catechols. The individual expressed enzymes were then used for detailed kinetic determinations of carefully selected catechols.
By using the method developed it is possible to determine the rate of methylation catalyzed by the enzyme catechol O-methyltransferase practically for any catechol-type substrate. A new chromatographic catechol O-methyltransferase (COMT) assay based on S-adenosyl-L-[methyl-14C]methionine and on-line radioactivity detection was developed. With minor modifications in the mobile phase composition the methylation velocities for 30 structurally diverse compounds including simple catechols, neurotransmitters, catecholestrogens and catecholic drugs could be measured using human and rat recombinant soluble COMT. The radiochemical method was validated using 3,4-dihydroxybenzoic acid as a model substrate and was shown that accurate and reproducible methylation velocity values could be achieved for both the catecholic hydroxyls. The method proved to be suited for determining the enzyme kinetic parameters.