Statins and selective inhibition of Rho kinase protect small conductance calcium-activated potassium channel function (KCa2.3) in cerebral arteries

Background: In rat middle cerebral and mesenteric arteries the KCa2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, KCa2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain KCa2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA). Methods: MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence. Results: Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of KCa2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. KCa2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation. Conclusions: Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell KCa2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of KCa2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.

Diminished neural processing of aversive and rewarding stimuli during selective serotonin reuptake inhibitor treatment

Background Selective serotonin reuptake inhibitors (SSRIs) are popular medications for anxiety and depression, but their effectiveness, particularly in patients with prominent symptoms of loss of motivation and pleasure, has been questioned. There are few studies of the effect of SSRIs on neural reward mechanisms in humans. Methods We studied 45 healthy participants who were randomly allocated to receive the SSRI citalopram, the noradrenaline reuptake inhibitor reboxetine, or placebo for 7 days in a double-blind, parallel group design. We used functional magnetic resonance imaging to measure the neural response to rewarding (sight and/or flavor of chocolate) and aversive stimuli (sight of moldy strawberries and/or an unpleasant strawberry taste) on the final day of drug treatment. Results Citalopram reduced activation to the chocolate stimuli in the ventral striatum and the ventral medial/orbitofrontal cortex. In contrast, reboxetine did not suppress ventral striatal activity and in fact increased neural responses within medial orbitofrontal cortex to reward. Citalopram also decreased neural responses to the aversive stimuli conditions in key “punishment” areas such as the lateral orbitofrontal cortex. Reboxetine produced a similar, although weaker effect. Conclusions Our findings are the first to show that treatment with SSRIs can diminish the neural processing of both rewarding and aversive stimuli. The ability of SSRIs to decrease neural responses to reward might underlie the questioned efficacy of SSRIs in depressive conditions characterized by decreased motivation and anhedonia and could also account for the experience of emotional blunting described by some patients during SSRI treatment.

Classical and hologram QSAR studies on a series of inhibitors of trypanosomatid Glyceraldehyde-3-Phosphate Dehydrogenase

Leishmaniasis and trypanosomiasis are major causes of morbidity and mortality in both tropical and subtropical regions of the world. The current available drugs are limited, ineffective, and require long treatment regimens. Due to the high dependence of trypanosomatids on glycolysis as a source of energy, some glycolytic enzymes have been identified as attractive targets for drug design. In the present work, classical Two-Dimensional Quantitative Structure -Activity Relationships (2D QSAR) and Hologram QSAR (HQSAR) studies were performed on a series of adenosine derivatives as inhibitors of Leishmania mexicana Glyceraldehyde-3-Phosphate Dehydrogenase (LmGAPDH). Significant correlation coefficients (classical QSAR, r(2)=0.83 and q(2) =0.81; HQSAR, r(2)=0.91 and q(2) =0.86) were obtained for the 56 training set compounds, indicating the potential of the models for untested compounds. The models were then externally validated using a test set of 14 structurally related compounds and the predicted values were in good agreement with the experimental results (classical QSAR, r(pred)(2) = 0.94; HQSAR, r(pred)(2) = 0.92).

Comparative molecular field analysis of a series of inhibitors of HIV-1 protease

Several protease inhibitors have reached the world market in the last fifteen years, dramatically improving the quality of life and life expectancy of millions of HIV-infected patients. In spite of the tremendous research efforts in this area, resistant HIV-1 variants are constantly decreasing the ability of the drugs to efficiently inhibit the enzyme. As a consequence, inhibitors with novel frameworks are necessary to circumvent resistance to chemotherapy. In the present work, we have created 3D QSAR models for a series of 82 HIV-1 protease inhibitors employing the comparative molecular field analysis (CoMFA) method. Significant correlation coefficients were obtained (q(2) = 0.82 and r(2) = 0.97), indicating the internal consistency of the best model, which was then used to evaluate an external test set containing 17 compounds. The predicted values were in good agreement with the experimental results, showing the robustness of the model and its substantial predictive power for untested compounds. The final QSAR model and the information gathered from the CoMFA contour maps should be useful for the design of novel anti-HIV agents with improved potency.

Discovery of New Inhibitors of Schistosoma mansoni PNP by Pharmacophore-Based Virtual Screening

Schistosomiasis is considered the second most important tropical parasitic disease, with severe socioeconomic consequences for millions of people worldwide. Schistosoma monsoni, one of the causative agents of human schistosomiasis, is unable to synthesize purine nucleotides de novo, which makes the enzymes of the purine salvage pathway important targets for antischistosomal drug development. In the present work, we describe the development of a pharmacophore model for ligands of S. mansoni purine nucleoside phosphorylase (SmPNP) as well as a pharmacophore-based virtual screening approach, which resulted in the identification of three thioxothiazolidinones (1-3) with substantial in vitro inhibitory activity against SmPNP. Synthesis, biochemical evaluation, and structure activity relationship investigations led to the successful development of a small set of thioxothiazolidinone derivatives harboring a novel chemical scaffold as new competitive inhibitors of SmPNP at the low-micromolar range. Seven compounds were identified with IC(50) values below 100 mu M. The most potent inhibitors 7, 10, and 17 with 1050 of 2, 18, and 38 mu M, respectively, could represent new potential lead compounds for further development of the therapy of schistosomiasis.

Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: Kinetic and structural studies

Selectivity plays a crucial role in the design of enzyme inhibitors as novel antiparasitic agents, particularly in cases where the target enzyme is also present in the human host. Purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive target for the discovery of potential antischistosomal agents. In the present work, kinetic studies were carried out in order to determine the inhibitory potency, mode of action and enzyme selectivity of a series of inhibitors of SmPNP. In addition, crystallographic studies provided important structural insights for rational inhibitor design, revealing consistent structural differences in the binding mode of the inhibitors in the active sites of the SmPNP and human PNP (HsPNP) structures. The molecular information gathered in this work should be useful for future medicinal chemistry efforts in the design of new inhibitors of SmPNP having increased affinity and selectivity. (C) 2010 Elsevier Ltd. All rights reserved.

MPO Inhibitors Selected by Virtual Screening

The hemeprotein myeloperoxidase (MPO) participates in innate immune defense through its ability to generate potent microbicidal oxidants. However, these oxidants are also key mediators of the tissue damage associated with many inflammatory diseases. Thus, there is considerable interest in developing therapeutically useful MPO inhibitors. Here, we used structure-based drug design (SBDD) and ligand-based drug design (LBDD) to select for potentially new and selective MPO inhibitors. A pharmacophore model was developed based on the crystal structure of human MPO in complex with salicylhydroxamic acid (SHA), a known inhibitor of the enzyme. The pharmacophore model was used to screen the ZINC database for potential ligands, which were further filtered on the basis of their physical-chemical properties and docking score. The filtered compounds were visually inspected, and nine were purchased for experimental studies. Surprisingly, almost all of the selected compounds belonged to the aromatic hydrazide class, which had been previously described as MPO inhibitors. The compounds selected by virtual screening were shown to inhibit the chlorinating activity of MPO; the top four compounds displayed IC(50) values ranging from 1.0 to 2.8 mM. MPO inactivation by the most effective compound was shown to be irreversible. Overall, our results show that SBDD and LBDD may be useful for the rational development of new MPO inhibitors.

Immobilized analogues of sunflower trypsin inhibitor-1 constitute a versatile group of affinity sorbents for selective isolation of serine proteases

Sunflower trypsin inhibitor-1 (SFI-1), a natural 14-residue cyclic peptide, and some of its synthetic acyclic variants are potent protease inhibitors displaying peculiar inhibitory profiles. Here we describe the synthesis and use of affinity sorbents prepared by coupling SFTI-1 analogues to agarose resin. Chymotrypsinand trypsin-like proteases could then be selectively isolated from pancreatin; similarly, other proteases were obtained from distinct biological sources. The binding capacity of [Lys5]-SFTI-1-agarose for trypsin was estimated at over 10 mg/mL of packed gel. SFTI-1-based resins could find application either to improve the performance of current purification protocols or as novel protease-discovery tools in different areas of biological investigation. (C) 2009 Elsevier B.V. All rights reserved.

Selective activity of butyrylcholinesterase in serum by a chemiluminescent assay

In a previous study, we showed that purified commercial esterase activity can be detected in a chemiluminescent assay based on the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) to 2-methyl-1-propenol, which is subsequently oxidized by the horseradish peroxidase (HRP)-H2O2 system. The purpose of this study was to verify the applicability of this assay to human serum. The existence of an esterase activity capable of hydrolysing MPB is indicated by the fact that the MPB-scruin-HRP-H2O2 System consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at less than or equal to4 degreesC. Addition of aliesterase inhibitors, such as fluoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specific substrate benzoylcholine causes a delay in both O-2 uptake and light emission, while the specific acetylcholinesterase substrate, acetyl-beta -methylcholine, had practically no effect. Purified butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The finding that butyryleholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specific chemiluminescent assay for this enzyme. Copyright (C) 2001 John Wiley & Sons, Ltd.

Effect of selective cyclooxygenase-2 inhibition on the development of ligature-induced periodontitis in rats

Background: the purpose of this study was to evaluate the effect of a selective cyclooxygenase-2 inhibitor on the progression of alveolar bone loss in an experimental periodontitis model in rats.Methods: One hundred eighty (180) Wistar rats were separated into 3 experimental groups. Cotton ligatures were placed at the gingival margin level of lower right first molars. The rats were randomly assigned to one of the following groups that received: a daily oral dose of 10 mg/kg body weight of celecoxib (Ce1); 20 mg/kg body weight of celecoxib (Ce2); or 10 ml/kg of saline solution (C). Serum levels of celecoxib and white blood cell count were determined. Standardized digital radiographs were taken after sacrifice at 3, 5, 10, 18, and 30 days to measure the amount of bone loss around the mesial root surface of the first molar tooth in each rat.Results: Two-way analysis of variance (ANOVA) indicated that groups treated with celecoxib had significantly less bone loss compared to controls (P <0.0001) and that there was a significant interaction between treatment with celecoxib and time (P <0.03). Post-hoc comparisons showed that in both groups treated with celecoxib, the bone loss became significant only after 10 days of ligature placement, while in the control group it was already significant after 5 days. However, differences in mean bone loss between control and Ce1 were significant only at 18 days and, between control and Ce2, at 5 and 18 days. There was no significant difference in bone loss among experimental groups at the end of the experimental period.Conclusion: These data provide evidence that systemic therapy with celecoxib can modify the progression of experimentally induced periodontitis in rats.

Subfornical organ mediates pressor effect of angiotensin: Influence of nitric oxide synthase inhibitors, AT(1) and AT(2) angiotensin antagonist's receptors

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

New anti-Alzheimer drugs from biodiversity: the role of the natural acetylcholinesterase inhibitors

Alzheimer's disease (AD) is a progressive neurodegenerative pathology with severe economic and social impact. There is currently no cure, although cholinesterase inhibitors provide effective temporary relief of symptoms in some patients. Nowadays, drug research and development are based on the cholinergic hypothesis that supports the cognition improvement by regulation of the synthesis and release of acetylcholine in the brain. There are only four commercial medicines approved for treatment of AD, and natural products have played an important alternative role in the research for new acetylcholinesterase inhibitors, as exemplified through the discovery of galantamine. This profile conducts us to give in this paper an overview relating the several classes of natural products with anti-cholinesterasic activity as potential templates to the design of new selective and powerful anti-Alzheimer drugs.

Eucalyptus ESTs associated with resistance to herbicide inhibitors of aromatic and branched-chain amino acid synthesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Endothelial and neuronal nitric oxide synthase inhibitors influences angiotensin II pressor effect in central nervous system

The present study investigated the central role of angiotensin II and nitric oxide on arterial blood pressure (MAP) in rats. Losartan and PD123349 AT 1 and AT 2 (selective no peptides antagonists angiotensin receptors), as well as FK 409 (a nitric oxide donor), N W-nitro-L-arginine methyl ester (L-NAME) a constituve nitric oxide synthase inhibitor endothelial (eNOSI) and 7-nitroindazol (7NI) a specific neuronal nitric oxide synthase inhibitor (nNOSI) were used. Holtzman strain, (Rattus norvergicus) weighting 200-250 g were anesthetized with zoletil 50 mg kg -1 (tiletamine chloridrate 125 mg and zolazepan chloridrate 125 mg) into quadriceps muscle anda stainless steel cannula was stereotaxically implanted into their Lateral Ventricle (LV). Controls were injected with a 0.5 μl volume of 0.15 M NaCl. Angiotensin II injected into LV increased MAP (19±3 vs. control 3±1 mm Hg), which is potentiated by prior injection of L-NAME in the same site 26±2 mm Hg. 7NI injected prior to ANG II into LV also potentiated the pressor effect of ANG II but with a higher intensity than L-NAME 32±3 mm Hg. FK 409 inhibited the pressor effect of ANG II (6±1 mm Hg). Losartan injected into LV before ANG II influences the pressor effect of ANG II (8±1 mm Hg). The PD 123319 decreased the pressor effects of ANG II (16±1 mm Hg). Losartan injected simultaneously with FK 409 blocked the pressor effect of ANG II (3±1 mm Hg). L-NAME produced an increase in the pressor effect of ANG II, may be due to local vasoconstriction and all at once by neuronal NOS inhibition but the main effect is of the 7-NIT an specific nNOS inhibitor. The AT 1 antagonist receptors improve basal nitric oxide (NO) production and release. These data suggest the involvement of constitutive and neuronal NOS in the control of arterial blood pressure induced by ANG II centrally, evolving AT 1 receptor-mediated vasoconstriction and AT 2 receptor-mediated vasodilatation. These results were confirmed by the experiment using FK 409. © 2006 Asian Network for Scientific Information.

Treatment of experimental periodontal disease by a selective inhibitor of cyclooxygenase-2 with scaling and root planing (SRP)

Lumiracoxib is a selective inhibitor of cyclooxygenase-2 (COX-2) approved for the relief of symptoms of chronic inflammatory conditions. The aim of this study was to evaluate the effects of this specific inhibitor of COX-2 as adjunctive treatment on induced periodontitis in rats. Periodontal disease was induced at the first mandibular molar of 60 rats. After 7 days, the ligature was removed and all animals were submitted to scaling and root planing (SRP) along with local irrigation with saline solution and were divided into 2 groups: SRP (n = 30)-received subcutaneous injections of 1 mg/kg of body weight/day of saline solution for 3 days and; SRP + L (n = 30)-received subcutaneous injections of 1 mg/kg of body weight/day of Lumiracoxib for 3 days. Ten animals in each group were killed at 7, 15, and 30 days. The histological description was performed and the histometric values were statistically analyzed. In Group SRP + L, the histometric analysis (0.58 ± 0.08, 0.64 ± 0.06, and 0.56 ± 0.10 mm 2) showed less bone loss (p < 0.05) than Group SRP (1.52 ± 0.08, 1.55 ± 0.09, and 1.49 ± 0.24 mm 2) at 7, 15, and 30 days, respectively. Within the limits of this study it can be concluded that subcutaneous application of specific inhibitor of COX-2 was a beneficial adjunctive treatment for periodontal diseases induced in rats. © 2010 Springer Basel AG.