Stefania Hanau

Purinoceptor function in the immune system

Immune cells express plasma membrane receptors for extracellular adenosine and purine and pyrimidine nucleotides. Overwhelming evidence suggests that these receptors have a pivotal role in the modulation of several immune cell responses, and may therefore be very important in the overall economy of the immune network. Adenosine can be either an activatory or an inhibitory mediator. Nucleotides have a more complex effect, depending not only on the P2 receptor subtype activated but also on doses applied and the length of stimulation. Both metabotropic (G‐protein‐linked) and ionotropic (intrinsic ion channels) P2 receptors are present in immune cells and their expression is modulated by inflammatory cytokines and differentiation agents. The physiological meaning of the P2 receptors is still obscure; however, converging evidence from different laboratories suggest that they may be involved in a wide variety of responses such proliferation, differentiation, cell death, and cytokine release. Drug Dev. Res. 39:319–329, 1996.

6-Phosphogluconate dehydrogenase: the mechanism of action investigated by a comparison of the enzyme from different species.

The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei. The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate. Kinetic comparison showed that an increase in the rate of NADP+ reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate. 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators. The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate. The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed.

In vitro effects of estrogen on tgb and c-myc gene expression in normal and neoplastic human thyroids

The authors investigated the effects of 17 beta-estradiol (E) and thyroid-stimulating hormone (TSH) on tgb (coding for thyroglobulin), c-myc RNA levels, and [3H]thymidine (thy) incorporation in suspension cultures of normal, adenomatous and carcinomatous human thyroid follicles. The cultured follicles showed decreased tgb RNA and enhanced c-myc RNA levels. In the culture of normal and adenomatous samples E caused a significant increase of [3H]thy incorporation and tgb RNA levels, with no effect on c-myc RNA levels. No effect of E was observed in the carcinomatous thyroid culture. TSH induced a significant increase of [3H]thy incorporation and c-myc expression only in adenoma cultures and a significant increase of tgb RNA levels in both normal and adenomatous samples. TSH had no effect on the carcinoma. The results show that E, like TSH, stimulates in vitro the expression of the tgb gene in differentiated cells, without stimulating the expression of the c-myc proto-oncogene, suggesting a possible action of E on normal thyroid function and perhaps growth, even if not associated with increased c-myc expression.

6-Phosphogluconate Dehydrogenase: A Target for Drugs in African Trypanosomes

New drugs are urgently required for Human African Trypanosomiasis (sleeping sickness), a disease which has re-emerged as a major health threat in Sub-Saharan Africa. The third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase, has been shown to be a good target for drugs. The enzyme is essential to the trypanosomes that causes sleeping sickness and structural differences when compared to its mammalian counterpart allow for selective inhibition. Three series of inhibitors have been designed, these include phosphorylated carbohydrate substrate and transition state analogues, non-carbohydrate substrate analogues and also triphenylmethane-based compounds. All have shown selective inhibition of the trypanosomal 6-phosphogluconate dehydrogenase and representatives of each have trypanocidal activity.

Kinetic resolution of vic-diols by Bacillus stearothermophilus diacetyl reductase

Abstract The kinetic resolution of several racemic syn- and anti-1,2-diols by enzymatic oxidation with Bacillus stearothermophilus diacetyl reductase is described. The enantiomerically pure (R,R)- and (R,S)-diols are recovered in almost quantitative yield.

Synthesis and biological evaluation of substrate-based inhibitors of 6-phosphogluconate dehydrogenase as potential drugs against African trypanosomiasis.

The synthesis and biological evaluation of three series of 6-phosphogluconate (6PG) analogues is described. (2R)-2-Methyl-4,5-dideoxy, (2R)-2-methyl-4-deoxy and 2,4-dideoxy analogues of 6PG were tested as inhibitors of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver and also Trypanosoma brucei where the enzyme is a validated drug target. Among the three series of analogues, seven compounds were found to competitively inhibit 6PGDH from T. brucei and sheep liver enzymes at micromolar concentrations. Six inhibitors belong to the (2R)-2-methyl-4-deoxy series (6, 8, 10, 12, 21, 24) and one is a (2R)-2-methyl-4,5-dideoxy analogue (29b). The 2,4-dideoxy analogues of 6PG did not inhibit both enzymes. The trypanocidal effect of the compounds was also evaluated in vitro against T. brucei rhodesiense as well as other related trypanosomatid parasites (i.e., Trypanosoma cruzi and Leishmania donovani).

Differential responsiveness of rat striatal nerve endings to the mitochondrial toxin 3-nitropropionic acid: implications for Huntington's disease.

Rat striatal synaptosomes and slices were used to investigate the responsiveness of different populations of nerve terminals to 3‐nitropropionic acid (3‐NP), a suicide inhibitor of the mitochondrial enzyme succinate dehydrogenase, and to elucidate the ionic mechanisms involved. 3‐NP (0.3–3 mm) stimulated spontaneous γ‐aminobutyric acid (GABA), glutamate and [3H]‐dopamine efflux but left unchanged acetylcholine efflux from synaptosomes. This effect was associated with a >70% inhibition of succinate dehydrogenase, as measured in the whole synaptosomal population. The facilitation was not dependent on extracellular Ca2+ but relied on voltage‐dependent Na+ channel opening, because it was prevented by tetrodotoxin and riluzole. 3‐NP also elevated spontaneous glutamate efflux from slices but in a tetrodotoxin‐insensitive way. To investigate whether energy depletion could change the responsiveness of nerve endings to a depolarizing stimulus, synaptosomes were pretreated with 3‐NP and challenged with pulses of KCl evoking ‘quasi‐physiological’ neurotransmitter release. 3‐NP potentiated the K+‐evoked GABA, glutamate and [3H]‐dopamine release but inhibited the K+‐evoked acetylcholine release. The 3‐NP induced potentiation of GABA release was Ca2+‐dependent and prevented by tetrodotoxin and riluzole whereas the 3‐NP‐induced inhibition of acetylcholine release was tetrodotoxin‐ and riluzole‐insensitive but reversed by glipizide, an ATP‐dependent K+ channel inhibitor. We conclude that the responsiveness of striatal nerve endings to 3‐NP relies on activation of different ionic conductances, and suggest that the selective survival of striatal cholinergic interneurons following chronic 3‐NP treatment (as in models of Huntingtons disease) may rely on the opening of ATP‐dependent K+ channels, which counteracts the fall in membrane potential as a result of mitochondrial impairment.

Sugar derivatives as new 6-phosphogluconate dehydrogenase inhibitors selective for the parasite Trypanosoma brucei

Sugar derivatives mimicking compounds which take part in the catalysed reaction have been assayed as alternative substrates and/or competitive inhibitors of 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Phosphonate analogues have been synthesised and the new compound 5-deoxy-5-phosphono-D-arabinonate shows good selectivity towards the parasite enzyme. A number of 4-carbon and 5-carbon aldonates are strong inhibitors of the parasite enzyme with K(i) values below the substrate K(m) and some acyl derivatives are also potent inhibitors. At least five of the compounds showing a significant selectivity for the parasite enzyme represent leads for trypanocidal drugs against this recently validated target.

Selective inhibition of 6-phosphogluconate dehydrogenase from Trypanosoma brucei

A number of triphenylmethane derivatives have been screened against 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Some of these compounds show good inhibition of the enzymes and also selectivity towards the parasite enzyme. Modelling was undertaken to dock the compounds into the active sites of both enzymes. Using a combination of DOCK 3.5 and FLEXIDOCK a correlation was obtained between docking score and both activity for the enzymes and selectivity. Visualisation of the docked structures of the inhibitors in the active sites of the enzymes yielded a possible explanation of the selectivity for the parasite enzyme.

NADPH activates a decarboxylation reaction catalysed by lamb liver 6-phosphogluconate dehydrogenase

NADP-dependent lamb liver 6-phosphogluconate dehydrogenase catalyses the oxidative decarboxylation of 2-deoxy-6-phosphogluconate, an analogue of the natural substrate. The first products of the reaction are NADPH and 3-keto-2-deoxy-6-phosphogluconate. The NADPH, released from the enzyme, binds to the coenzyme site of the same or the other subunit, activating the decarboxylation reaction in which has not a redox role, since it can be substituted by an analogue devoid of enzymatic redox power. These findings are compared to those obtained with other NADP-dependent decarboxylating dehydrogenases.