Porphyria-induced hepatic porphyrinogen carboxy-lyase inhibitor and its interaction with the active site(s) of the enzyme (original) (raw)
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Effect of α lipoic acid amide on hexachlorobenzene porphyria
IUBMB Life, 1999
The aim of this work is to study the efti~ct of thioctamide -the commercial form of ~x lipoic acid amide-on the porphyrinogenic action of hexachlorobenzene (HCB). For this purpose, porphyria was induced in rats by chronic HCB treatment, with or without simultaneous tbioctamide administration. Two different groups of rats were used as reference: one treated with vehicle (control) and the other treated with thioctamide (TO). Urine 8 aminolevulic acid, porphobilinogen, and porphyrin excretions were lower in the HCB + TO treated group than in the HCB group, and the same happened with liver uroporphyrin accumulation. On the other hand, the second stage of uroporphyrinogen-decarboxylase activity was significantly higher in the HCB + TO group than in the HCB group, fi aminolevulic acid synthase activity was higher in the HCB group. Hepatic thiobarbituric acid reactive substances were lower in HCB + TO group than in HCB group. Thus, we might suggest that TO would decrease HCB effects by means of its flee radical scavenging ability, and by having a direct effect on uroporphyrinogen-decarboxylase activity.
Journal of Biochemical and Molecular Toxicology, 2005
Uroporphyrinogen decarboxylase is an essential enzyme in all organisms and functions in the heme biosynthetic pathway, catalyzing the decarboxylation of the four acetate groups of uroporphyrinogen to form coproporphyrinogen. This work examines whether the four sequential decarboxylations occur at the same active site, and explores whether hexachlorobenzene-induced porphyria affects the behavior of the enzyme. For this purpose, kinetic competition studies were done with mixtures of uroporphyrinogen III and pentacarboxyporphyrinogen III. With the enzyme from normal rats, a constant velocity was obtained with all the mixtures, indicating that uroporphyrinogen and pentacarboxy-porphyrinogen react at the same active site, i.e. the first and fourth decarboxylations occur at the same site. In contrast, in experiments with enzyme from rats with hexachlorobenzeneinduced porphyria, the total rate for mixtures was always lower than the reference rate; and a curve with a deep minimum was obtained, indicating that the two reactions occur at functionally different sites, but with cross-inhibition. This suggests that the modifications induced in the enzyme by hexachlorobenzene cause the two active sites to become nonequivalent and functionally different. The question is discussed how the hexachlorobenzene treatment may produce this abnormal kinetic behavior, and alternative hypotheses are considered. C 2005 Wiley Periodicals, Inc. J
The Journal of Organic Chemistry, 2010
A series of vinylporphyrinogens were prepared to probe the enzyme coproporphyrinogen oxidase (CPO). Six (2-chloroethyl)porphyrins were synthesized from a common dipyrrylmethane via a,cbiladiene intermediates in excellent yields. Subsequent dehydrohalogenation with DBU in refluxing DMF then gave the required vinylporphyrin methyl esters, including harderoporphyrin-I, harderoporphyrin-III, and isoharderoporphyrin. The corresponding porphyrinogen carboxylic acids were incubated with chicken red cell hemolysates, which contain the enzyme CPO, and the products analyzed. The 17-ethyl analogue of harderoporphyrinogen-III, but not its 13-ethyl isomer, was shown to be an excellent substrate for CPO in accord with a proposed model for the active site of this enzyme. In addition, harderoporphyrinogen-VII, the monovinyl intermediate in the metabolism of coproporphyrinogen-IV, was shown to be an equally good substrate for this enzyme. However, isoharderoporphyrinogen, which lacks the correct ordering of peripheral substituents, was also a substrate for CPO. Furthermore, a nonnatural type I isomer of harderoporphyrinogen was shown to be acted on by CPO, but in this case further metabolism was noted and this afforded an unprecedented trivinyl porphyrinogen product. The corresponding porphyrin methyl ester was isolated and characterized by FAB MS and proton NMR spectroscopy. The results from these studies allow the binding requirements of CPO to be further assessed and provide a series of substrates to investigate this poorly understood enzyme.
Photodynamic and light independent action of 8 to 2 carboxylic free porphyrins on some haem-enzymes
The International Journal of Biochemistry & Cell Biology, 2001
Backgrounds and aims: skin lesions in cutaneous porphyrias appear to be determined by the structural properties of the porphyrins accumulated. To better understand the relationship between the structure and physicochemical properties of porphyrins and their specific effect on protein configuration, the action of a whole range of 8 to 2 carboxylic porphyrins has been studied. Materials and methods: d-aminolevulinic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D) partially purified from bovine liver, were exposed to 10 mM uroporphyrin (Uro), phyriaporphyrin (Phyria), hexaporphyrin (Hexa), pentaporphyrin (Penta), coproporphyrin (Copro) or protoporphyrin (Proto), either in the dark or under UV light. All experiments were performed in the enzyme solutions after removing the porphyrins. Results: under both illuminating conditions, all porphyrins inactivated the enzymes (20-70% under control values), indicating photodynamic action mediated by oxidative reactions and conformational changes due to direct binding of porphyrins to the protein. Total thiol content in ALA-D was not significantly changed by most porphyrins under UV light, while all porphyrins increase total sulfhydryl groups in PBG-D (23-52% over the control values) indicating changes in the redox status of SH residues. Free amino groups were reduced by all porphyrins in ALA-D (23-56% under controls), instead they were enhanced in PBG-D (23-51% over controls), suggesting protein fragmentation. The formation of molecular aggregates would be the consequence of cross-links between oxidation products, while fragmentation can be attributed to either rupture of disulphur bridges and/or enhancement of free amino groups on the protein enzyme. Conclusions: the effect of the porphyrins on enzyme activity, total SH groups and free amino groups content, was different for ALA-D and PBG-D, even under the same illuminating conditions. On the basis of these results, no correlation between enzyme alterations and the physicochemical properties of porphyrins could be established.
Acta physiologica latino americana
Soybean callus 5-aminolaevulinate synthetase (ALA-S) has been covalently attached to Sepharose 4B. The optimal conditions for binding have been determined. The water-insoluble ALA-S retained 40% of the activity of the original soluble preparation, the coupling yield was also high. Sepharose--ALA-S could be stored at 4°C for periods up to 40 days with only 25% loss of activity and it could be repeatedly used with little alteration of its enzymic activity, pH optima of the free and bound enzyme were the same.
PK 11195 aggravates 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced hepatic porphyria in rats
Hepatology, 1996
of the heme biosynthetic pathway. Porphyrias are classified There is evidence to suggest that peripheral-type beninto two main categories: hepatic or erythropoietic, dezodiazepine receptors (PBR) are involved in porphyrin pending on whether the excessive porphyrin production is in transport during erythroid differentiation, and it is posthe liver or in the bone marrow, respectively. 3,4 sible that these receptors have an important role in Some chemical agents and drugs can produce biochemical heme biosynthesis. We examined the biochemical and disorders in rats that resemble different forms of human heultrastructural alterations in rat liver following experipatic porphyria. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine mentally induced acute hepatic porphyria, as well as the (DDC) is a fast-acting synthetic compound that is frequently effects of the administration of a selective PBR ligand, used to produce experimental hepatic porphyria in fasting PK 11195. The most severe pathological conditions were rats. 5 The fasting state of the rats is a necessary factor to found in rats that received a combined treatment of the induce the porphyrinogenic effect of DDC, and is termed the porphyrinogenic agent 3,5-diethoxycarbonyl-1,4-dihy-''glucose effect.'' 6 DDC causes the accumulation of a green drocollidine (DDC) and PK 11195. Transmission electron porphyrin-like pigment, N-methylprotoporphyrin IX, which microscopy showed a correlation between the ultrais a potent inhibitor of the enzyme ferrochelatase, which constructural pathology of the liver, the total porphyrin levverts protoporphyrin IX into heme in the heme biosynthetic els in urine and liver, and the porphobilinogen levels in pathway. 7,8 DDC causes greatly increased levels of hepatic urine. Hepatocytes in this acute porphyria showed the protoporphyrin IX and increased levels of urinary excretion of development of large secondary lysosomes containing porphobilinogen (PBG) and d-aminolevulinic acid in a pattern crystalline aggregates of protoporphyrin. Bile canaliculi similar to that of an acute attack of human variegate porwere grossly enlarged, contained aggregates of protophyria. 5 Variegate porphyria is an acute porphyria in which porphyrin crystals, and showed the presence of bile protoporphyrinogen oxidase activity is lowered, and in which thrombi. In addition, prominent bundles of collagen fiprotoporphyrin IX consequently becomes the major circulatbers (fibrosis) were commonly found in livers of rats that ing porphyrin. 3 had been treated with DDC or DDC and PK 11195. We Benzodiazepines (BZ) have anxiolytic, anticonvulsant, conclude that the administration of PK 11195 to pormuscle-relaxant, and hypnotic properties. These therapeutic phyric rats aggravates porphyrin accumulation and celresponses are mediated by the central BZ receptors, abunlular damage in the liver. Perhaps this evidence suggests dant in the central nervous system, and are coupled with gthat PK 11195 blocks the binding of protoporphyrin IX aminobutyric acid receptors and the chloride ion channel. 9 to PBR, thus elevating the content of protoporphyrin IX In addition, BZs bind to peripheral BZ receptors (PBR), which in liver. (HEPATOLOGY 1996;24:697-701.) have been identified in various peripheral tissues as well as in glial brain cells. 10-13 PBR are located mainly in the outer Porphyrins, except for protoporphyrin IX, an intermediate mitochondrial membrane, 14 although they have been found in heme biosynthesis, are by-products that have escaped the in red blood cells, which lack mitochondria. 15 The BZ ligand pathway of heme biosynthesis through irreversible oxidation Ro 5-4864, as well as the isoquinoline carboxamide derivative of the corresponding porphyrinogens. 1,2 The porphyrias are PK 11195, demonstrates a high affinity for PBR. 16 a group of inherited or acquired diseases resulting from over-Displacement experiments of [ 3 H]PK 11195 binding, from production, accumulation, and excretion of excessive porphy-PBR located on various peripheral tissues, with porphyrins rins, and their precursors are secondary to enzymatic defects and hemin have been performed. 17 It was found that pure in the heme biosynthetic pathway. At least seven different hemin and protoporphyrin IX competitively inhibit mitochonporphyrias exist, a result of specific defects in the enzymes drial BZ binding with inhibition constants of 41 and 15 nmol/ L, respectively. Therefore, it was concluded that dicarboxylic acid porphyrins appear to be endogenous ligands for PBR. Recently, it has been found that PBR appear to be involved Abbreviations: DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; PBG, porphobilinogen; in porphyrin transport during erythroid differentiation. 18 BZ, benzodiazepines; PBR, peripheral-type benzodiazepine receptors; DMSO, dimethyl sulf-Consequently, PBR may play an important role in the eryoxide. throid-specific induction of heme biosynthesis. 18
A Porphodimethene Chemical Inhibitor of Uroporphyrinogen Decarboxylase
PLoS ONE, 2014
Uroporphyrinogen decarboxylase (UROD) catalyzes the conversion of uroporphyrinogen to coproporphyrinogen during heme biosynthesis. This enzyme was recently identified as a potential anticancer target; its inhibition leads to an increase in reactive oxygen species, likely mediated by the Fenton reaction, thereby decreasing cancer cell viability and working in cooperation with radiation and/or cisplatin. Because there is no known chemical UROD inhibitor suitable for use in translational studies, we aimed to design, synthesize, and characterize such a compound. Initial in silico-based design and docking analyses identified a potential porphyrin analogue that was subsequently synthesized. This species, a porphodimethene (named PI-16), was found to inhibit UROD in an enzymatic assay (IC 50 = 9.9 mM), but did not affect porphobilinogen deaminase (at 62.5 mM), thereby exhibiting specificity. In cellular assays, PI-16 reduced the viability of FaDu and ME-180 cancer cells with half maximal effective concentrations of 22.7 mM and 26.9 mM, respectively, and only minimally affected normal oral epithelial (NOE) cells. PI-16 also combined effectively with radiation and cisplatin, with potent synergy being observed in the case of cisplatin in FaDu cells (Chou-Talalay combination index ,1). This work presents the first known synthetic UROD inhibitor, and sets the foundation for the design, synthesis, and characterization of higher affinity and more effective UROD inhibitors.
Mechanism of hexachlorobenzene-induced porphyria in rats. Effect of phenobarbitone pretreatment
Biochemical Journal, 1984
The effect of a pretreatment with phenobarbitone (PB) on the porphyrinogenic action exerted by hexachlorobenzene (HCB) was examined in female rats. Kinetic studies of enzyme function after HCB poisoning showed that porphyrinogen carboxy-lyase was the only enzyme of haem biosynthesis that markedly lowered its activity. Both stages of uroporphyrinogen (UPG) III decarboxylation were decreased. This enzyme, together with UPG I synthase (increased levels) were the first enzymes altered. Subsequently, an increase in delta-aminolaevulinate (AmLev) synthase and ferrochelatase was detected; AmLev dehydratase was the last to increase. On long-term exposure, PB alone did not modify the basal values of haem intermediates; only the content of cytochrome P-450 increased. All the enzyme activities studied showed no significant changes, except ferrochelatase, which increased. With both drugs the metabolic impairment promoted by HCB was accelerated and enhanced by prior PB treatment leading to the o...