HIF-prolyl hydroxylases and cardiovascular diseases

Prolyl hydroxylases belong to the family of iron- and 2-oxoglutamate-dependent dioxygenase enzyme. Several distinct prolyl hydroxylases have been identified. The hypoxia-inducible factor (HIF) prolyl hydroxylase termed prolyl hydroxylase domain (PHD) enzymes play an important role in oxygen regulation in the physiological network. There are three isoforms that have been identified: PHD1, PHD2 and PHD3. Deletion of PHD enzymes result in stabilization of HIFs and offers potential treatment options for many ischemic disorders such as peripheral arterial occlusive disease, myocardial infarction, and stroke. All three isoforms are oxygen sensors that regulate the stability of HIFs. The degradation of HIF-1α is regulated by hydroxylation of the 402/504 proline residue by PHDs. Under hypoxic conditions, lack of oxygen causes hydroxylation to cease HIF-1α stabilization and subsequent translocation to the nucleus where it heterodimerizes with the constitutively expressed β subunit. Binding of the HIF-heterodimer to specific DNA sequences, named hypoxia-responsive elements, triggers the transactivation of target genes. PHD regulation of HIF-1α-mediated cardioprotection has resulted in considerable interest in these molecules as potential therapeutic targets in cardiovascular and ischemic diseases. In recent years, attention has been directed towards identifying small molecule inhibitors of PHD. It is postulated that such inhibition might lead to a clinically useful strategy for protecting the myocardium against ischemia and reperfusion injury. Recently, it has been reported that the orally absorbed PHD inhibitor GSK360A can modulate HIF-1α signaling and protect the failing heart following myocardial infarction. Furthermore, PHD1 deletion has been found to have beneficial effects through an increase in tolerance to hypoxia of skeletal muscle by reprogramming basal metabolism. In the mouse liver, such deletion has resulted in protection against ischemia and reperfusion. As a result of these preliminary findings, PHDs is attracting increasing interest as potential therapeutic targets in a wide range of diseases.     

Drug-metabolizing ability of molybdenum hydroxylases

Molybdenum hydroxylases, which include aldehyde oxidase and xanthine oxidoreductase, are involved in the metabolism of some medicines in humans. They exhibit oxidase activity towards various heterocyclic compounds and aldehydes. The liver cytosol of various mammals also exhibits a significant reductase activity toward nitro, sulfoxide, N-oxide and other moieties, catalyzed by aldehyde oxidase. There is considerable variability of aldehyde oxidase activity in liver cytosol of mammals: humans show the highest activity, rats and mice show low activity, and dogs have no detectable activity. On the other hand, xanthine oxidoreductase activity is present widely among species. Interindividual variation of aldehyde oxidase activity is present in humans. Drug-drug interactions associated with aldehyde oxidase and xanthine oxidoreductase are of potential clinical significance. Drug metabolizing ability of molybdenum hydroxylases and the variation of the activity are described in this review.     

Tyrosine and tryptophan hydroxylases as therapeutic targets in human disease

Introduction: The ancient and ubiquitous monoamine signalling molecules serotonin, dopamine, norepinephrine, and epinephrine are involved in multiple physiological functions. The aromatic amino acid hydroxylases tyrosine hydroxylase (TH), tryptophan hydroxylase 1 (TPH1), and tryptophan hydroxylase 2 (TPH2) catalyse the rate-limiting steps in the biosynthesis of these monoamines. Genetic variants of TH, TPH1, and TPH2 genes are associated with neuropsychiatric disorders. The interest in these enzymes as therapeutic targets is increasing as new roles of these monoamines have been discovered, not only in brain function and disease, but also in development, cardiovascular function, energy and bone homeostasis, gastrointestinal motility, hemostasis, and liver function.

Areas covered: Physiological roles of TH, TPH1, and TPH2. Enzyme structures, catalytic and regulatory mechanisms, animal models, and associated diseases. Interactions with inhibitors, pharmacological chaperones, and regulatory proteins relevant for drug development.

Expert opinion: Established inhibitors of these enzymes mainly target their amino acid substrate binding site, while tetrahydrobiopterin analogues, iron chelators, and allosteric ligands are less studied. New insights into monoamine biology and 3D-structural information and new computational/experimental tools have triggered the development of a new generation of more selective inhibitors and pharmacological chaperones. The enzyme complexes with their regulatory 14–3–3 proteins are also emerging as therapeutic targets.     

Cytochrome P450 4A fatty acid omega hydroxylases

The Cytochrome P450 4A subfamily is one of eighteen subfamilies in the CYP4 family and presently consists of twenty individual forms in nine different mammalian species. The major substrates for CYP4A forms are fatty acids, but recent studies have shown other non-fatty acid substrates may be metabolized by specific CYP4A forms. The physiological and metabolic functions of the CYP4A subfamily have not been elucidated, but the ability of CYP4A forms to metabolize medium and long chain length fatty acids at their omega (omega)-carbon atom has generated significant interest because of the possible role that omega-hydroxylated fatty acids may have in cell signalling processes and as an alternative pathway for fatty acid metabolism. A number of different compounds or physiological conditions have been shown to regulate the expression of CYP4A forms in liver and/or kidney. Several CYP4A forms may serve as a marker for the exposure to compounds that are classified as peroxisome proliferators. There is also considerable interest why multiple CYP4A forms exist in different tissues. Recent studies in the rat and human indicate that other CYP4 forms besides CYP4A forms may be responsible for the metabolism of arachidonic acid to its omega-hydroxy product. The focus of this review will be to summarize recent studies that have characterized the substrate specificity of rat, rabbit and human CYP4A forms and discuss the significance of CYP4A-mediated hydroxylation of fatty acids. In addition, dietary effects or novel compounds that have been reported to regulate CYP4A expression in the rat and mouse will be discussed.     

Tetrahydrobiopterin binding to aromatic amino acid hydroxylases. Ligand recognition and specificity

The three aromatic amino acid hydroxylases (phenylalanine, tyrosine, and tryptophan hydroxylase) and nitric oxide synthase (NOS) all utilize (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) as cofactor. The pterin binding site in the three hydroxylases is well conserved and different from the binding site in NOS. The structures of phenylalanine hydroxylase (PAH) and of NOS in complex with BH(4) are still the only crystal structures available for the reduced cofactor-enzyme complexes. We have studied the enzyme-bound and free conformations of BH(4) by NMR spectroscopy and molecular docking into the active site of the three hydroxylases, using endothelial NOS as a comparative probe. We have found that the dihydroxypropyl side chain of BH(4) adopts different conformations depending on which hydroxylase it interacts with. All the bound conformations are different from that of BH(4) free in solution at neutral pH. The different bound conformations appear to result from specific interactions with nonconserved amino acids at the BH(4) binding sites of the hydroxylases, notably the stretch 248-251 (numeration in PAH) and the residue corresponding to Ala322 in PAH, i.e., Ser in TH and Ala in TPH. On the basis of analysis of molecular interaction fields, we discuss the selectivity determinants for each hydroxylase and explain the high-affinity inhibitory effect of 7-tetrahydrobiopterin specifically for PAH.     

Selectivity and affinity determinants for ligand binding to the aromatic amino acid hydroxylases

Hydroxylation of the aromatic amino acids phenylalanine, tyrosine and tryptophan is carried out by a family of non-heme iron and tetrahydrobiopterin (BH4) dependent enzymes, i.e. the aromatic amino acid hydroxylases (AAHs). The reactions catalyzed by these enzymes are important for biomedicine and their mutant forms in humans are associated with phenylketonuria (phenylalanine hydroxylase), Parkinson's disease and DOPA-responsive dystonia (tyrosine hydroxylase), and possibly neuropsychiatric and gastrointestinal disorders (tryptophan hydroxylase 1 and 2). We attempt to rationalize current knowledge about substrate and inhibitor specificity based on the three-dimensional structures of the enzymes and their complexes with substrates, cofactors and inhibitors. In addition, further insights on the selectivity and affinity determinants for ligand binding in the AAHs were obtained from molecular interaction field (MIF) analysis. We applied this computational structural approach to a rational analysis of structural differences at the active sites of the enzymes, a strategy that can help in the design of novel selective ligands for each AAH.     

Tissue and sex differences in the activation of aromatic hydrocarbon hydroxylases in rats

Betamethasone and α-naphthoflavone produced similar activation of biphenyl 2-hydroxylase and benzo[a]pyrene 3-hydroxylase in control male rat liver microsomes. In small intestinal epithelial microsomes, betamethasone had no effect whereas α-naphthoflavone caused a pronounced activation of benzo[a]pyrene hydroxylation and a lesser activation of biphenyl 2-hydroxylation. In lung microsomes, betamethasone had no effect on either enzyme activity whereas α-naphthoflavone had no effect on biphenyl 2-hydroxylase but inhibited benzo[a]pyrene hydroxylase. In kidney cortex microsomes from male rats both compounds caused inhibition or had no effect whereas in kidney cortex microsomes from female rats betamethasone activated whereas α-naphthoflavone had no effect.Activation also occurred in isolated viable hepatocytes from male rats. The response of biphenyl 2-hydroxylase was very similar to that found in male rat liver microsomes but benzo[a]pyrene hydroxylase was more sensitive to activation and less sensitive to inhibition than in microsomes. The findings are interpreted as demonstrating the presence of more than one ‘latent’ aromatic hydrocarbon hydroxylase in rodents.     

Reaction of 1-amino- and 1-chlorophthalazine with mammalian molybdenum hydroxylases in vitro

1-Amino- and 1-chlorophthalazine were tested for possible substrate activity with partially purified rabbit-liver aldehyde oxidase and bovine-milk xanthine oxidase. 1-Chlorophthalazine was a more efficient substrate than the parent compound, phthalazine, with either aldehyde oxidase or xanthine oxidase. The oxidation product of 1-chlorophthalazine was identified as 4-chloro-1-(2H)-phthalazinone on the basis of chromatographic, infra-red and mass-spectral data. 1-Aminophthalazine was oxidized by aldehyde oxidase to 4-amino-1-(2H)-phthalazinone but was a competitive inhibitor of xanthine oxidase. Kinetic studies at different pH values indicated that, in each case, it is the unprotonated form of 1-aminophthalazine that reacts with the molybdenum hydroxylases.     

Genetic and hormonal regulation of steroid hydroxylases and drug metabolizing enzymes in rat liver

Two microsomal steroid hydroxylase activities (cholesterol-7-hydroxylase and progesterone-16-hydroxylase) were measured in the livers of Sprague-Dawley and Wistar rats and compared to three other monooxygenase activities (aryl hydrocarbon hydroxylase, p-nitro-anisole-O-demethylase and aminopyrine-N-demethylase). Cholesterol-7-hydroxylase behaves in a very unique manner. It is the only one of the studied enzymes to be more active in the female than in the male, it is very poorly induced by phenobarbital and methylcholanthrene, but responds quickly to the administration of glucocorticoids. In fact, the cholesterol-7-hydroxylase activity presents a very pronounced circadian rhythm which is under the control of the hypothalamo-adrenal axis. Marked differences are also found in the response of the various enzymatic activities to the administration of inducers as well as in their relative activities in untreated male and female animals.Read at the Symposium Relevance of Chronobiology for Toxicology and Pharmacology held at the 16th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section: Toxicology, March 6, 1975, Mainz     

Prenatal induction of benzo(a)pyrene hydroxylases in mice

1. Benzo(a)pyrene hydroxylase (BPH) activity was measured in homogenates of fetal liver (day 18) or of whole-embryos of mice on day 9, 10 or 12 of gestation after maternal pretreatment with B(a)P on 3 consecutive days. A³H-liberation assay with³H-B(a)P labelled either generally or at the 6-position was used. The values obtained with the embryonic/fetal tissues were compared with those found in maternal liver. 2. Three oral doses of 17.5 mg B(a)P/kg body wt were found to just significantly induce BPH in maternal liver. An induction was observed after pretreatment with 24 mg B(a)P/kg body wt in 9-, 10-or 12-day-old whole-embryos, but the V_max reached was only 10–20% (1% on day 9) of that of adult non-induced liver. The K_m (6-hydroxylation) for all tissues tested were in the same range (600–900 nM). The induction was demonstrable in embryos at tissue levels about one order of magnitude lower than those required for induction in maternal liver. 3. Treatment with 25 mg B(a)P/kg body wt on 3 consecutive days was required to induce BPH in fetal liver on day 18 of gestation. The required B(a)P tissue concentrations were about one half of those necessary for induction in maternal liver. 4. Among a variety of other polycyclic hydrocarbons only chrysene showed an inducing potency similar to that of B(a)P in adult and fetal liver. For all compounds tested there was no correlation found in the inducing potency between adult and fetal liver (e.g. coronene). 5. The doses required to induce BPH in the maternal or fetal liver or in whole embryos of rodents are significantly higher (mg range) than those of usual average human exposure or those taken up by smokers (ng range).Abbreviations AHH aryl hydrocarbon hydroxylase - B(a)P benzo(a)pyrene - BPH benzo(a)pyrene hydroxylases - PAH polycyclic aromatic hydrocarbons     

大鼠肝睾酮羟化酶体外诱导模型的建立

目的建立大鼠原代培养肝细胞微粒体睾酮羟化酶系列同工酶(CYP2A1,CYP2B1/2,CYP2C11,CYP3A1/2)的体外诱导模型。方法应用胶原酶原位灌流法分离大鼠肝细胞进行原代培养;用苯巴比妥钠(PB,1mmol·L-1)、地塞米松(Dex,10μmol·L-1)和β-萘酚黄酮(β-NF,50μmol·L-1)诱导培养肝细胞72 h,提取肝细胞微粒体,进行其肝CYP总量、肝微粒体蛋白含量和睾酮羟化酶比活性的测定。另外采用体内诱导方法,SD大鼠分别给予PB 80mg·kg-1,Dex50mg·kg-1和β-NF80mg·kg-1,ip,每天1次,连续5d,停药24h后制备肝微粒体进行上述指标的测定。结果在体外和体内实验中,PB,Dex和β-NF对肝微粒体蛋白含量、CYP总量和睾酮羟化酶同工酶活性均具有较高的诱导效应。PB对肝CYP总量在体外的诱导效应高于体内,对睾酮不同位置羟化作用的诱导效应体内外无显著性差异;Dex和β-NF对肝CYP总量及睾酮不同位置羟化作用的诱导效应体内外无显著性差异。结论大鼠肝睾酮羟化酶体外诱导模型可替代体内实验,用于药物代谢、新药安全性评价及其他外源性化合物代谢和毒性研究。     

Synthetic analogues of tetrahydrobiopterin with cofactor activity for aromatic amino acid hydroxylases

Tetrahydrobiopterin (THB) analogues with 6-alkoxymethyl substituents, 3a-j, where the substituents were straight- and branched-chain alkyl ranging from methyl to octyl, have been synthesized by the Taylor method from pyrazine ortho amino nitriles by guanidine cyclization, hydrolysis in aqueous NaOH, and catalytic hydrogenation over Pt in trifluoroacetic acid (TFA). The best of these compounds, 3b, is an excellent cofactor for phenylalanine hydroxylase, tyrosine hydroxylase (V = 154% of THB), and tryptophan hydroxylase, does not destablize the binding of substrate (Kmtyr = 23 microM), and is recycled by dihydropteridine reductase (V = 419% of THB). The compounds are being evaluated as cofactor replacements in biopterin-deficiency diseases.     

Selective inactivation of four rat liver microsomal androstenedione hydroxylases by chloramphenicol analogs

The steroid androstenedione has been shown to be a valuable tool for the study of the selective inactivation of cytochrome P-450 isozymes in intact rat liver microsomes. The validity of this approach was investigated using microsomes, purified cytochrome P-450 isozymes, antibodies to particular cytochromes P-450, and the known mechanism-based inactivator chloramphenicol. Enzyme inactivation and antibody inhibition studies show that microsomes from both phenobarbital- and non-phenobarbital-treated rats are needed to accurately monitor the inactivation of the major phenobarbital-inducible cytochrome P-450 isozyme (PB-B) and of the major constitutive androstenedione 16 alpha-hydroxylase (UT-A). Similar experiments indicate that, although isozyme P-450g does catalyze the 6 beta-hydroxylation of androstenedione in a reconstituted system, this cytochrome appears to make only a minimal contribution to microsomal 6 beta-hydroxylase activity, which reflects instead the activity of pregnenolone-16 alpha-carbonitrile-induced isozymes. With these parameters investigated, initial enzyme inactivation studies showed that the antibiotic chloramphenicol caused different rates of NADPH-dependent enzyme inactivation among the four androstenedione hydroxylases monitored (16 beta greater than 6 beta greater than 16 alpha greater than 7 alpha). Based on these data, 12 chloramphenicol analogs were examined, and the results with these compounds show that their selectivity as cytochrome P-450 inactivators is a function of at least three structural features: 1) the number of halogen atoms, 2) the presence of a para-nitro group on the phenyl ring, and 3) substitutions on the ethyl side chain. For example, the compound N-(2-phenethyl)dichloroacetamide was shown to reversibly inhibit but not inactivate the cytochrome(s) P-450 responsible for androstenedione 6 beta-hydroxylase activity, whereas N-(2-p-nitrophenethyl) and N-(1,2-diphenethyl)dichloroacetamide rapidly inactivated the 6 beta-hydroxylase. The ability to monitor the activity of multiple isozymes with a single substrate should allow the development of a systematic approach to the design of selective inactivators of rat liver cytochromes P-450.     

肾脏花生四烯酸CYP羟化酶参与高血压的形成及维持正常血压的稳定

目的:研究肾脏特异性表达CYP4A1与血压变化的关系.方法:将含有开放阅读框的CYP4A1 cDNA克隆到真核表达载体pcDNA3.1,构建4Al/pcDNA3.1和反义4Al/pcDNA3.1重组体,舌下静脉注射转染SD雄性大鼠,经尾动脉测量收缩压.用Western blot及Northern blot分析转染对照pcDNA3.1质粒及正、反义CYP4A1重组体后,CYP4A1在大鼠的脑、心、肺、肝、肾组织表达水平.结果:转染正义、反义CYP4A1两周后,大鼠的血压与对照组相比分别增加1.8kPa±0.3kPa(13.2mmHg±2.5mmHg)和减少了1.7kPa±0.3kPa(13.0mmHg±2.2 mmHg).Western blot及Northern blot结果均显示CYP4A1可选择性地在肾脏表达,而在脑、心、肺、肝几乎无表达,且在给予反义CYP4A1的大鼠肾脏中CYP4Al蛋白表达几乎被完全阻断.结论:在正常的SD大鼠肾脏中,转染正、反义CYP4A1可引起血压升高和降低,说明肾脏花生四烯酸CYP羟化酶参与高血压的形成及维持正常血压的稳定.     

Stereochemical biotransformation of warfarin as a probe of the homogeneity and mechanism of microsomal hydroxylases

The hepatic microsomal metabolism of R and S warfarin by normal Wistar and Sprague-Dawley rats was compared with that of phenobarbital (PB) and 3-methylcholanthrene (3-MC)-pretreated animals. In all the microsomal systems examined, R warfarin was metabolized faster than the S enantiomer. Induction of microsomal mixed-function oxidase activity with PB, and especially 3-MC, caused significant alterations in the normal stereochemical pattern of R and S warfarin hydroxylation which were independent of the method of microsomal preparation and the technique employed in the quantitation of hydroxylated products. PB pretreatment of Sprague-Dawley rats resulted in an increase in all hydroxylated products but to differing extents. Similar results were obtained from Wistar rats except for the processes of 4' and benzylic hydroxylation of (S) warfarin. 3-MC pretreatment resulted in the selective induction of 6- and 8-hydroxylation in both species. These results suggest that liver microsomes from normal animals contain at least two major, A and B, and two minor, C and D, mono-oxygenases which differ in their stereoselectivity (as measured by the rates for the formation of two enantiomeric products), regioselectivity (as measured by the ratio of any two isomeric products from the same substrate), and inducibility. In this model, normal animals have hydroxylase activity enzyme A which is not inducible by PB or 3-MC and which is stereoselective for the R enantiomer of warfarin in 7- and benzylic hydroxylation and for the S enantiomer in 4'-hydroxylation. Microsomes from normal and PB-induced animals contain additional hydroxylase activity, enzyme B, which catalyzes both the 6- and 8-hydroxylation of warfarin and which has low stereoselectivity but is regioselective for 6-hydroxylation. Enzyme B may also be responsible for some 4'-hydroxylation. PB-induced animals have additional mono-oxygenase activity, enzyme C, which displays the opposite stereoselectivity compared to enzyme A for benzylic and less stereoselectivity for 7-hydroxylation. 3-MC-induced animals have greatly enhanced levels of 6- and 8-hydroxylase activity, enzyme D, which is stereoselective for the R enantiomer and regioselective for 8-hydroxylation of R warfarin and 6-hydroxylation of S warfarin.     

1-substituted phthalazines as probes of the substrate-binding site of mammalian molybdenum hydroxylases

The interaction of a series of 1-substituted phthalazine derivatives with partially purified aldehyde oxidase from rabbit, guinea-pig and baboon liver, and with bovine milk xanthine oxidase, has been investigated. Of the 18 compounds examined, rabbit liver aldehyde oxidase metabolized 10, whereas guinea-pig and baboon liver enzyme oxidized 13 and 14, respectively. Where metabolites were characterized, oxidation was shown to occur at position four of the phthalazine ring. Km values ranged from 0.003 to 1.8 mM. In contrast, most compounds were competitive inhibitors of bovine milk xanthine oxidase with Ki values ranging from 0.015 to 1.3 mM; the cationic derivative 2-methylphthalazinium iodide was oxidized to 2-methyl-1-phthalazinone by both aldehyde oxidase and, with a much reduced affinity, by xanthine oxidase. In terms of structure-metabolism relationships, Vmax values were relatively insensitive to the electronic effects of substituents, but a trend for the more lipophilic derivatives to show increased affinities (Km and Vmax/Km) towards aldehyde oxidase could be seen. However, calculations of molecular size revealed a species-dependent cut-off threshold above which compounds were not metabolized. Results suggest that the relative size of the active site for hepatic aldehyde oxidase is in the order baboon greater than guinea-pig greater than rabbit, and that in spatial terms the active site of bovine milk xanthine oxidase is similar to that of baboon liver aldehyde oxidase. Thus, the binding site of rabbit liver aldehyde oxidase, a widely used source of the oxidase, is apparently more restricted than in some other species.     

The effects of organochlorine pesticides as inducers of testosterone and benzo[a]pyrene hydroxylases

p,p'-DDE, phenobarbital, dieldrin heptachlor, chlordane and toxaphene induced rat liver microsomes exhibited increased formation of the 4,5-dihydrodiol, 3,6-quinone, 9- and 3-hydroxymetabolites of benzo[a]pyrene and the latter three compounds also induced an increase in the rate of formation of the 9,10-dihydrodiol metabolite. Lindane was inactive as an inducer of benzo[a]pyrene hydroxylase. With the exception of lindane, all the organochlorine pesticides and PB induced testosterone 16 alpha- and 16 beta-hydroxylases; in contrast lindane induced testosterone 6 alpha-, 7 alpha- and 6 beta-hydroxylases and PB also induced testosterone 15 beta-hydroxylase and androstenedione formation. Using a battery of monooxygenase enzyme assays it was evident that there were significant differences between PB and several organochlorine pesticides as inducers of rat hepatic cytochrome P-450-dependent monooxygenases.