François Noël

Taurine prevents the neurotoxicity of β-amyloid and glutamate receptor agonists: activation of GABA receptors and possible implications for Alzheimer’s disease and other neurological disorders

Alzheimers disease (AD) and several other neurological disorders have been linked to the overactivation of glutamatergic transmission and exci‐totoxicity as a common pathway of neuronal injury. The β‐amyloid peptide (Aβ) is centrally related to the pathogenesis of AD, and previous reports have demon¬strated that the blockade of glutamate receptors pre¬vents Aβ‐induced neuronal death. We show that tau¬rine, a β‐amino acid found at high concentrations in the brain, protects chick retinal neurons in culture against the neurotoxicity of Aβ and glutamate receptor ago¬nists. The protective effect of taurine is not mediated by interaction with glutamate receptors, as demon¬strated by binding studies using radiolabeled glutamate receptor ligands. The neuroprotective action of taurine is blocked by picrotoxin, an antagonist of GABAA receptors. GABA and the GABAA receptor agonists phenobarbital and melatonin also protect neurons against Aβ ‐induced neurotoxicity. These results suggest that activation of GABA receptors decreases neuronal vulnerability to excitotoxic damage and that pharmaco¬logical manipulation of the excitatory and inhibitory neurotransmitter tonus may protect neurons against a variety of insults. GABAergic transmission may repre¬sent a promising target for the treatment of AD and other neurological disorders in which excitotoxicity plays a relevant role.—Louzada, P. R., Lima, A. C. P., Mendonca‐Silva, D. L., Noël, F., de Mello, F. G., Ferreira, S. T. Taurine prevents the neurotoxicity of β‐amyloid and glutamate receptor agonists: activation of GABA receptors and possible implications for Alz¬heimers disease and other neurological disorders.

Heterogeneity of ouabain specific binding sites and (Na+ + K+)-ATPase inhibition in microsomes from rat heart.

Cardiac glycoside binding to microsomes prepared from rat heart ventricles and enriched in (Na+ + K+)-ATPase was measured by a rapid filtration technique. The relation between ouabain binding to microsomes and (Na+ + K+)-ATPase activity has also been examined. Data were statistically analysed by means of two different non linear regression methods. The experimental results were fitted the most closely by a model describing that ouabain specific binding occurred at two classes of independent sites. High affinity sites were characterized by a dissociation constant of 0.21 +/- 0.01 microM and a low capacity (9.4 +/- 1.4 pmoles/enzymatic unit). Low affinity sites were characterized by a dissociation constant equal to 13 +/- 3 microM and a capacity equal to 87 +/- 15 pmoles/enzymatic unit. Similar results were obtained with the more lipophilic glycoside digoxin. It was also observed that dihydroouabain, a ouabain derivative with a saturated lactone ring, competes with 3H-ouabain for the binding to the two classes of sites. Binding to these two classes of sites appeared to be associated with a corresponding inhibition of (Na+ + K+)-ATPase activity.

Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors

The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B–RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC50 values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.

Synthesis and Preliminary Pharmacological Evaluation of New ( ) 1,4-Naphthoquinones Structurally Related to Lapachol

Seven new 1,4-naphthoquinones structurally related to lapachol were synthesized from lawsone and oxygenated arylmercurials. These compounds can also be seen as pterocarpan derivatives where the A-ring was substituted by the 1,4-naphthoquinone nucleus. Pharmacological screening provided evidence of significant biological activities, including effects against proliferation of the MCF-7 human breast cancer cell line, against Herpes Simplex Virus type 2 infection, and against snake poison-induced myotoxicity. One derivative displaced flunitrazepam binding and showed benzodiazepine-like activity, suggesting novel neuroactive structural motifs.

Synthesis and preliminary pharmacological evaluation of coumestans with different patterns of oxygenation

Five coumestans with different patterns of oxygenation in rings A and D were synthesized from resorcinol and aromatic aldehydes, and screened for their antimyotoxic activity. The most potent compound (2b, IC50 = 1 microM) was selected for study of its pharmacological profile.

Searching for multi-target antipsychotics: Discovery of orally active heterocyclic N-phenylpiperazine ligands of D2-like and 5-HT1A receptors

We described herein the design, synthesis, and pharmacological evaluation of N-phenylpiperazine heterocyclic derivatives as multi-target compounds potentially useful for the treatment of schizophrenia. The isosteric replacement of the heterocyclic ring at the biaryl motif generating pyrazole, 1,2,3-triazole, and 2-methylimidazole[1,2-a]pyridine derivatives resulted in 21 analogues with different substitutions at the para-biaryl and para-phenylpiperazine positions. Among the compounds prepared, 4 (LASSBio-579) and 10 (LASSBio-664) exhibited an adequate binding profile and a potential for schizophrenia positive symptoms treatment without cataleptogenic effects. Structural features of this molecular scaffold are discussed regarding binding affinity and selectivity for D(2)-like, 5-HT(1A), and 5-HT(2A) receptors.

Cyclic GMP‐dependent vasodilatory properties of LASSBio 294 in rat aorta

The effects of LASSBio 294, a new 3,4‐methylenedioxybenzoyl‐2‐thienylhydrazone, on vascular tonus were investigated in isolated rat aortic rings. LASSBio 294 induced a concentration‐dependent relaxation of intact rat aortic rings with an inhibitory concentration (IC50) of 74 μM (95% confidence limits: 59 – 92). The mechanical removal of the endothelium abolished this effect. In aortic rings with intact endothelium the effect of 100 μM LASSBio 294 was not altered by the pharmacological inhibition of NOS and cyclo‐oxygenase pathways with 500 μM L‐NAME and 10 μM indomethacin, respectively. LASSBio 294 (100 μM) was able to relax aortic rings pre‐contracted with high extracellular K+ (KCl 100 mM). The relaxant effect of LASSBio 294 was fully reversed (and prevented) by the addition of 1 μM ODQ (1H‐(1,2,4)oxadiazolo[4,3‐a]quinoxaline‐1‐one), a selective inhibitor of soluble guanylate cyclase. LASSBio 294 (100 μM) had no direct effect on PDE3 and PDE4 activities, however, it increased by 150% cyclic GMP content in aortic rings pre‐treated with 100 μM L‐NAME and 10 μM indomethacin, as did 1 μM zaprinast, a selective PDE5 inhibitor. In conclusion, LASSBio 294 induced relaxation of isolated rat aorta probably by directly increasing cyclic GMP content, possibly as a consequence of PDE5 inhibition.

A comparison of the affinities of rat (Na+ + K+)-ATPase isozymes for cardioactive steroids, role of lactone ring, sugar moiety and KCl concentration.

Binding experiments at equilibrium were performed to study pharmacological properties of isozymes of (Na+ + K+)-ATPase from rat tissues. Experiments were performed on brain (alpha 3 isozyme), kidney (alpha 1 isozyme) and heart microsomes (alpha 1 and alpha 2 isozymes). Affinity of series of ouabain and digoxin derivatives was studied in competition experiments. It was observed that: (i) ouabain and digoxin had higher affinity (P less than 0.01) for alpha 3 isozyme (Kd of 0.071 +/- 0.004 and 0.066 +/- 0.001 microM, respectively) than for alpha 1 isozyme (Kd of 15.9 +/- 0.8 and 1.78 +/- 0.46 microM, respectively) and alpha 2 isozyme (Kd of 0.26 +/- 0.04 and 0.15 +/- 0.06 microM, respectively); (ii) saturation of the C20-C22 bond on the C17 beta lactone ring present in ouabain and digoxin markedly decreased the drug affinity for all isozymes (P less than 0.01); and (iii) suppression of the C3 beta osidic chain decreased the affinity of ouabain and digoxin to a higher extent for alpha 2 and alpha 3 than for alpha 1 (P less than 0.01). The presence of 10 mM KCl in the incubation medium decreased ouabain affinity for the alpha 1 isozyme to a much higher extent (Kd increase of about 20-fold) than for the other isozymes (Kd increase of about 2-fold). The results show that the isozymes of (Na+ + K+)-ATPase from rat tissue are differently sensitive to changes in the substituents of the cardioactive steroids and to the presence of 10 mM KCl.

Influence of development on Na(+)/K(+)-ATPase expression: isoform- and tissue-dependency.

The four isoforms of the catalytic subunit of Na(+)/K(+)-ATPase identified in rats differ in their affinities for ions and ouabain. Moreover, its expression is tissue-specific, developmentally and hormonally regulated. The aim of the present work was to evaluate the influence of age on the ratio and density of these isoforms in crude membrane preparations from rat brain hemispheres, brainstem, heart ventricles and kidneys. In all tissues investigated, Na(+)/K(+)-ATPase activity was higher in adults than in neonates but brain tissues presented the most remarkable differences. In these tissues, ouabain inhibition curves for Na(+)/K(+)-ATPase activity revealed the presence of two processes with different sensitivities to ouabain. An increase of approximately sixfold in the expression of the high affinity isoforms was observed between newborn and adult rats. In contrast, the low affinity isoform increased only approximately twofold in brainstem whereas it increased ninefold in brain hemispheres. Unlike brain tissues, a decrease (almost fourfold) in the number of high affinity ouabain binding sites was observed during ontogenesis of the heart. Although limited by the inability to resolve alpha(2) and alpha(3) isoforms, present data indicate that the influence of development on the expression of Na(+)/K(+)-ATPase depends not only on the isoform, but also on the tissue where the enzyme is expressed.

The inotropic effect of ouabain and its antagonism by dihydroouabain in rat isolated atria and ventricles in relation to specific binding sites.

1 The inotropic effect of ouabain has been studied in rat ventricles and atria. 2 The concentration‐effect curve of ouabain may be fitted by a model assuming the existence of two saturable components. The component with the higher sensitivity to ouabain accounted for 30% of the maximal increase in systolic tension in ventricles and for only 5% in atria. Increase in diastolic tension was only apparent at ouabain concentrations required to observe the low sensitivity component. 3 [3H]‐ouabain binding has been examined in microsomes prepared from atria and ventricles. High and low affinity binding sites have been observed. The ratio of high and low affinity ouabain binding sites was 4 fold lower in microsomes from rat atria than from rat ventricles. This could account for the difference in the response of these two tissues to the inotropic action of ouabain. 4 In ventricular strips the high sensitivity component was much less apparent in the presence of dihydroouabain than with ouabain. 5 When ventricular strips were preincubated in the presence of dihydroouabain 3 μm, the increase in systolic tension evoked by ouabain 1 μm was significantly reduced. Cumulative concentration‐effect curve studies showed dihydroouabain antagonism to the high sensitivity component.