Thomas Friedberg

Sequence similarity of mammalian epoxide hydrolases to the bacterial haloalkane dehalogenase and other related proteins. Implication for the potential catalytic mechanism of enzymatic epoxide hydrolysis

Direct comparison of the amino acid sequences of microsomal and soluble epoxide hydrolase superficially indicates that these enzymes are unrelated. Both proteins, however, share significant sequence similarity to a bacterial haloalkane dehalogenase that has earlier been shown to belong to the α/β hydrolase fold family of enzymes. The catalytic mechanism for the dehalogenase has been elucidated in detail [Verschueren et al. (1993) Nature 363, 693‐698] and proceeds via an ester intermediate where the substrate is covalently bound to the enzyme. From these observations we conclude (i) that microsomal and soluble epoxide hydrolase are distantly related enzymes that have evolved from a common ancestral protein together with the haloalkane dehalogenase and a variety of other proteins specified in the present paper, (ii) that these enzymes most likely belong to the α/β hydrolase fold family of enzymes and (iii) that the enzymatic epoxide hydrolysis proceeds via a hydroxy ester intermediate, in contrast to the presently favoured base‐catalyzed direct attack of the epoxide by an activated water.

Endogenous drug transporters in in vitro and in vivo models for the prediction of drug disposition in man.

The epithelial canine and porcine kidney cell lines MDCK, MDCKII and LLC-PK1, respectively are employed to establish recombinant models of drug transport. Endogenous drug carriers in these cells may contribute to the activities of recombinant drug transporters, thus making it difficult to assess their properties. We analysed the expression of endogenous transporters in these cell lines by RT-PCR and by determining drug transporter activities. Concerning drug efflux, multidrug resistance protein 1 (MDR1) and MRP1 mRNAs were found in all lines. MRP2 mRNA was expressed in all cell lines except MDCK. Transepithelial transport of vinblastine and its modulation by a MDR1-specific inhibitor or by the MDR1- and MRP-inhibitor verapamil, indicated that MDCKII cells have, in comparisons to the other cell lines, relatively high levels of functional MDR1 while vinblastine transport in MDCK cells is likely to be mediated more by MRP1. Notably, LLC-PK1 cells displayed little activity attributable to either MDR1 and MRP1, thus making them suitable for the expression of these efflux pumps. Of the drug uptake carriers, OATP-A mRNA was only expressed in MDCK cells. OATP-C mRNA was barely detectable in MDCK cells and absent in MDCKII and LLC-PK1 cells. In agreement with transcriptional profiling, the OATP-mediated uptake of either estradiol-glucuronide or estrone-sulfate was either absent or barely detectable in all cell lines thus implying that they are suitable to establish recombinant models for human OATPs. Transcriptional profiling was also performed on porcine and canine tissues and revealed that MRP1 was expressed in canine but not in human or porcine liver, whereas surprisingly OATP-C was expressed in canine kidney but only in human and porcine liver. The findings presented are relevant to the use of porcine and canine models for drug disposition.

An amperometric biosensor with human CYP3A4 as a novel drug screening tool.

We developed a biosensor based on the redox properties of human CYP3A4 to directly monitor electron transfer to the heme protein. Enzyme films were assembled on gold electrodes by alternate adsorption of a CYP3A4 layer on top of a polycation layer. Direct, reversible electron transfer between the electrode and CYP3A4 was observed with voltammetry under anaerobic conditions. In the presence of oxygen, the oxidation peak of the hemoprotein disappeared, and the reduction peak increased 2- to 3-fold. Addition of CYP3A4 substrates (verapamil, midazolam, quinidine, and progesterone) to the oxygenated solution caused a concentration-dependent increase in the reduction current in cyclic voltammetric and amperometric experiments. Product analyses after electrolysis with the enzyme film showed catalytic activity of the biosensor depending on substrate concentration, its inhibition by ketoconazole, and a minor contribution of H(2)O(2) to the catalytic cycle. These results suggest that electron exchange between the electrode and the immobilized CYP3A4 occurred, and that metabolic activity of the enzyme was maintained. Thus, important requirements for the application of human CYP biosensors in order to identify drugs or drug candidates as substrates or inhibitors to the attached enzyme are fulfilled.

Differential Effects of Fluvoxamine and Other Antidepressants on the Biotransformation of Melatonin

Melatonin, the predominant product of the pineal gland, is involved in the maintenance of diurnal. rhythms. Nocturnal blood concentrations of melatonin have been shown to be enhanced by fluvoxamine, but not by other serotonin reuptake inhibitors. Because fluvoxamine is an inhibitor of several cytochrome P450 (CYP) enzymes, the authors studied the biotransformation of melatonin and the effects of fluvoxamine on the metabolism of melatonin in vitro using human liver microsomes and recombinant human CYP isoenzymes. Melatonin was found to be almost exclusively metabolized by CYP1A2 to 6-hydroxymelatonin and N-acetylserotonin with a minimal contribution of CYP2C19. Both reactions were potently inhibited by fluvoxamine, with a Ki of 0.02 μM for the formation of 6-hydroxymelatonin and 0.05 μM for the formation of N-acetylserotonin. Other than fluvoxamine, fluoxetine, paroxetine, citalopram, imipramine and desipramine were also tested at 2 and 20 μM. Among the other antidepressants, only paroxetine was able to affect the metabolism of melatonin at supratherapeutic concentrations of 20 μM, which did not reach by far the magnitude of the inhibitory potency of fluvoxamine. The authors concluded that fluvoxamine is a potent inhibitor of melatonin degradation. Because this inhibitory action is also found in vivo, fluvoxamine might be used as an enhancer of melatonin, which might offer new therapeutic possibilities of fluvoxamine.

Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes

A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase.

Establishment of functional human cytochrome P450 monooxygenase systems in Escherichia coli.

Cytochromes P450 (CYP) have been expressed in a variety of systems such as mammalian cells, yeast, and bacteria. The bacterial system is technically the least demanding and provides large amounts of catalytically active P450s for metabolic and structural studies relating to preclinical drug development. This chapter provides a detailed technical description of the processes that allow the coexpression of various CYP isoforms together with CYP reductase in Escherichia coli and gives some examples of the results that can be achieved for the expression of human P450s.

MERITS AND LIMITATIONS OF RECOMBINANT MODELS FOR THE STUDY OF HUMAN P450-MEDIATED DRUG METABOLISM AND TOXICITY: AN INTRALABORATORY COMPARISON

A wide variety of pharmacological and toxicological properties of drugs are determined by cytochrome P450-mediated metabolism. Characterization of these pathways and of the P450 isoenzymes involved constitutes an essential part of drug development. Similarly, because P450s are catalyzing the toxication and detoxication of environmental pollutants, an understanding of these reactions facilitates risk assessment in environmental toxicology. Recently, a variety of recombinant expression systems has been employed to study the role of human P450s in these reactions. These include insect, bacterial, yeast, and mammalian models. As these were developed and characterized by different laboratories, evaluation of their merits and limitations is inherently difficult. To resolve this problem, we have established and characterized the latter three systems and present the key results here. In general, the catalytic properties of P450 isozymes in the various models were rather similar. However, taking technical considerations into account as well as the high level of functional expression of P450s achieved in bacteria make this system ideally suited for drug metabolism research, including the generation of milligram quantities of metabolites for structural determinations. For toxicological studies, however, expression of P450s in mammalian cells was most appropriate. This is exemplified here by studies into the role of human P450s in the activation and inactivation of chemotherapeutic drugs.

Regio- and stereoselective regulation of monooxygenase activities by isoenzyme-selective phosphorylation of cytochrome P450

The phosphorylation of the two major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in hepatocytes by the action of the membrane permeating cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP. Under these conditions the dealkylation of 7-pentoxyresorufin, a selective substrate of cytochrome P450IIB1 and P450IIB2 was markedly reduced. 16 beta-Hydroxylation of testosterone which is catalyzed specifically only by cytochrome P450IIB1 and IIB2 was strongly reduced; for 16 alpha-hydroxylation which is also catalyzed by cytochrome P450IIB1 and IIB2 but additionally by 3 further cytochrome P450 isoenzymes, this reduction was less pronounced; for the oxidation of the 17 beta-hydroxyl group which besides cytochromes P450IIB1 and IIB2 is additionally catalyzed not only by other cytochromes P450 but also by 17 beta-hydroxysteroid dehydrogenase there was a clear tendency of reduction which, however, no longer reached statistical significance. Hydroxylation at other positions of testosterone which are catalyzed by other cytochrome P450 isoenzymes were not significantly changed. Hence isoenzyme-selective phosphorylation of cytochrome P450 leads to a corresponding isoenzyme-selective modulation of monooxygenase activity which holds promise to be especially important as a fast regulation of the control of genotoxic metabolites.

Perfluorodecanoic acid decreases the enzyme activity and the amount of glutathione S-transferases proteins and mRNAs in vivo

The effects of the anti-wetting agent perfluoro-n-decanoic acid (PFDA) on various glutathione S-transferase (GST) enzyme activities were studied in vitro and in vivo. In addition the effects of PFDA treatment on the amount of some glutathione S-transferase subunits and their corresponding translatable mRNA were studied in vivo. PFDA like some other peroxisome proliferators was a non-competitive inhibitor of several GST enzyme activities in vitro. In vivo PFDA reduced the enzyme activity towards substrates which are indicative for the Ya, Yb1 and Yb2 subunits of GSTs to a larger extent than the enzyme activity towards the substrate indicative for the Yc subunit. Whereas the reduction of GST enzyme activities by other peroxisome proliferators was shown to be caused by an inhibition of the relevant enzymes in vivo, PFDA was found to decrease the GST enzyme activities at least in part by lowering the amount of the various GST subunits in vivo due to a lowered concentration of translatable mRNA coding for these enzymes. In addition PFDA abolished the inducibility of GST mRNAs by phenobarbital. Thus PFDA might be an interesting tool for mechanistic studies of the control of GST expression in the liver.The effects of the anti-wetting agent perfluoro-n-decanoic acid (PFDA) on various glutathione S-transferase (GST) enzyme activities were studied in vitro and in vivo. In addition the effects of PFDA treatment on the amount of some glutathione S-transferase subunits and their corresponding translatable mRNA were studied in vivo. PFDA like some other peroxisome proliferators was a non-competitive inhibitor of several GST enzyme activities in vitro. In vivo PFDA reduced the enzyme activity towards substrates which are indicative for the Ya, Yb1 and Yb2 subunits of GSTs to a larger extent than the enzyme activity towards the substrate indicative for the Yc subunit. Whereas the reduction of GST enzyme activities by other peroxisome proliferators was shown to be caused by an inhibition of the relevant enzymes in vivo, PFDA was found to decrease the GST enzyme activities at least in part by lowering the amount of the various GST subunits in vivo due to a lowered concentration of translatable mRNA coding for these enzymes. In addition PFDA abolished the inducibility of GST mRNAs by phenobarbital. Thus PFDA might be an interesting tool for mechanistic studies of the control of GST expression in the liver.

Selective detection of mRNA forms encoding the major phenobarbital inducible cytochromes P450 and other members of the P450IIB family by the RNAse A protection assay

The identification of P450 mRNAs in a tissue poses the problem that members of the same P450 gene family share a high sequence homology. Studies based on oligomer probes rely on a probe covering only a few base pairs. In contrast in our study on the expression of the P450IIB gene family we used in vitro-generated antisense transcripts, covering several hundred base pairs, of the hypervariable and constant regions of the P450IIB1 and P450IIB2 cDNA, in the RNAse A protection assay of mRNA isolated from various tissues. RNAse A concentrations were adjusted to a level where this enzyme still yielded distinct fragments for a defined P450IIB1 antisense/P450IIB2 sense heteroduplex, which contained 24 scattered mismatches within a stretch of 285 nucleotides. In contrast nuclease S1 was not useful for the detection of mismatches within this heteroduplex. With this highly sensitive RNAse A protection assay we were able to distinguish between the expression of P450IIB1 and the expression of P450IIB2 in several organs. Our results strongly support earlier studies on the tissue specific expression of these enzymes, which had used oligomer probes (Omiecinski, C. J., 1986, Nucleic Acids Res. 14, 1525-1539). Moreover we detected the constitutive hepatic expression of a P450IIB gene which was distinct from P450IIB1 and IIB2. In addition we identified a P450IIB mRNA which was expressed at high levels in the preputial gland but not in the liver or any other organ tested.