Discovery of new Schistosoma mansoni aspartyl protease inhibitors by structure-based virtual screening.

BACKGROUND: Schistosomiasis is a neglected tropical disease caused by trematodes of the genus Schistosoma, with a limited treatment, mainly based on the use of praziquantel (PZQ). Currently, several aspartic proteases genes have already been identified within the genome of Schistosoma species. At least one enzyme encoded from this gene family (SmAP), named SmCD1, has been validated for the development of schistosomicidal drugs, since it has a key role in haemoglobin digestion by worms. OBJECTIVE: In this work, we integrated a structure-based virtual screening campaign, enzymatic assays and adult worms ex vivo experiments aiming to discover the first classes of SmCD1 inhibitors. METHODS: Initially, the 3D-structures of SmCD1, SmCD2 and SmCD3 were generated using homology modelling approach. Using these models, we prioritised 50 compounds from 20,000 compounds from ChemBridge database for further testing in adult worm aqueous extract (AWAE) and recombinant SmCD1 using enzymatic assays. FINDINGS: Seven compounds were confirmed as hits and among them, two compounds representing new chemical scaffolds, named 5 and 19, had IC50 values against SmCD1 close to 100 µM while presenting binding efficiency indexes comparable to or even higher than pepstatin, a classical tight-binding peptide inhibitor of aspartyl proteases. Upon activity comparison against mammalian enzymes, compound 50 was selective and the most potent against the AWAE aspartic protease activity (IC50 = 77.7 µM). Combination of computational and experimental results indicate that compound 50 is a selective inhibitor of SmCD2. Compounds 5, 19 and 50 tested at low concentrations (10 uM) were neither cytotoxic against WSS-1 cells (48 h) nor could kill adult worms ex-vivo, although compounds 5 and 50 presented a slight decrease on female worms motility on late incubations times (48 or 72 h). MAIN CONCLUSION: Overall, the inhibitors identified in this work represent promising hits for further hit-to-lead optimisation.

Neurological impairment caused by Schistosoma mansoni systemic infection exhibits early features of idiopathic neurodegenerative disease.

Schistosomiasis, a neglected tropical disease caused by trematodes of the Schistosoma genus, affects over 250 million people around the world. This disease has been associated with learning and memory deficits in children, whereas reduced attention levels, impaired work capacity, and cognitive deficits have been observed in adults. Strongly correlated with poverty and lack of basic sanitary conditions, this chronic endemic infection is common in Africa, South America, and parts of Asia and contributes to inhibition of social development and low quality of life in affected areas. Nonetheless, studies on the mechanisms involved in the neurological impairment caused by schistosomiasis are scarce. Here, we used a murine model of infection with Schistosoma mansoni in which parasites do not invade the central nervous system to evaluate the consequences of systemic infection on neurologic function. We observed that systemic infection with S. mansoni led to astrocyte and microglia activation, expression of oxidative stress-induced transcription factor Nrf2, oxidative damage, Tau phosphorylation, and amyloid-ß peptide accumulation in the prefrontal cortex of infected animals. We also found impairment in spatial learning and memory as evaluated by the Morris water maze task. Administration of anthelmintic (praziquantel) and antioxidant (N-acetylcysteine plus deferoxamine) treatments was effective in inhibiting most of these phenotypes, and the combination of both treatments had a synergistic effect to prevent such changes. These data demonstrate new perspectives toward the understanding of the pathology and possible therapeutic approaches to counteract long-term effects of systemic schistosomiasis on brain function.

COVID-19: molecular targets, drug repurposing and new avenues for drug discovery.

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious infection that may break the healthcare system of several countries. Here, we aimed at presenting a critical view of ongoing drug repurposing efforts for COVID-19 as well as discussing opportunities for development of new treatments based on current knowledge of the mechanism of infection and potential targets within. Finally, we also discuss patent protection issues, cost effectiveness and scalability of synthetic routes for some of the most studied repurposing candidates since these are key aspects to meet global demand for COVID-19 treatment.

N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES: To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS: Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS: In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1ß, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS: L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.

Increased tau phosphorylation and receptor for advanced glycation endproducts (RAGE) in the brain of mice infected with Leishmania amazonensis.

Leishmaniasis is a parasitosis caused by several species of the genus Leishmania, an obligate intramacrophagic parasite. Although neurologic symptoms have been observed in human cases of leishmaniasis, the manifestation of neurodegenerative processes is poorly studied. The aim of the present work was to investigate if peripheral infection of BALB/c mice with Leishmania amazonensis affects tau phosphorylation and RAGE protein content in the brain, which represent biochemical markers of neurodegenerative processes observed in diseases with a pro-inflammatory component, including Alzheimer's disease and Down syndrome. Four months after a single right hind footpad subcutaneous injection of L. amazonensis, the brain cortex of BALB/c mice was isolated. Western blot analysis indicated an increase in tau phosphorylation (Ser(396)) and RAGE immunocontent in infected animals. Brain tissue TNF-α, IL-1ß, and IL-6 levels were not different from control animals; however, increased protein carbonylation, decreased IFN-γ levels and impairment in antioxidant defenses were detected. Systemic antioxidant treatment (NAC 20mg/kg, i.p.) inhibited tau phosphorylation and recovered IFN-γ levels. These data, altogether, indicate an association between impaired redox state, tau phosphorylation and RAGE up-regulation in the brain cortex of animals infected with L. amazonensis. In this context, it is possible that neurologic symptoms associated to chronic leishmaniasis are associated to disruptions in the homeostasis of CNS proteins, such as tau and RAGE, as consequence of oxidative stress. This is the first demonstration of alterations in biochemical parameters of neurodegeneration in an experimental model of Leishmania infection.

Investigation into effects of antipsychotics on ectonucleotidase and adenosine deaminase in zebrafish brain.

Antipsychotic agents are used for the treatment of psychotic symptoms in patients with several brain disorders, such as schizophrenia. Atypical and typical antipsychotics differ regarding their clinical and side-effects profile. Haloperidol is a representative typical antipsychotic drug and has potent dopamine receptor antagonistic functions; however, atypical antipsychotics have been developed and characterized an important advance in the treatment of schizophrenia and other psychotic disorders. Purine nucleotides and nucleosides, such as ATP and adenosine, constitute a ubiquitous class of extracellular signaling molecules crucial for normal functioning of the nervous system. Indirect findings suggest that changes in the purinergic system, more specifically in adenosinergic activity, could be involved in the pathophysiology of schizophrenia. We investigated the effects of typical and atypical antipsychotics on ectonucleotidase and adenosine deaminase (ADA) activities, followed by an analysis of gene expression patterns in zebrafish brain. Haloperidol treatment (9 µM) was able to decrease ATP hydrolysis (35%), whereas there were no changes in hydrolysis of ADP and AMP in brain membranes after antipsychotic exposure. Adenosine deamination in membrane fractions was inhibited (38%) after haloperidol treatment when compared to the control; however, no changes were observed in ADA soluble fractions after haloperidol exposure. Sulpiride (250 µM) and olanzapine (100 µM) did not alter ectonucleotidase and ADA activities. Haloperidol also led to a decrease in entpd2_mq, entpd3 and adal mRNA transcripts. These findings demonstrate that haloperidol is an inhibitor of NTPDase and ADA activities in zebrafish brain, suggesting that purinergic signaling may also be a target of pharmacological effects promoted by this drug.

Kinetics and molecular modeling studies on the inhibition mechanism of GH13 α-glycosidases by small molecule ligands.

Alpha-glucosidase inhibitors play an important role in Diabetes Mellitus (DM) treatment since they prevent postprandial hyperglycemia. The Glycoside Hydrolase family 13 (GH13) is the major family of enzymes acting on substrates containing α-glucoside linkages, such as maltose and amylose/amylopectin chains in starch. Previously, our group identified glycoconjugate 1H-1,2,3-triazoles (GCTs) inhibiting two GH13 α-glycosidases: yeast maltase (MAL12) and porcine pancreatic amylase (PPA). Here, we combined kinetic studies and computational methods on nine GCTs to characterize their inhibitory mechanism. They all behaved as reversible inhibitors, and kinetic models encompassed noncompetitive and various mechanisms of mixed-type inhibition for both enzymes. Most potent inhibitors displayed Ki values of 30 µM for MAL12 (GPESB16) and 37 µM for PPA (GPESB15). Molecular dynamics and docking simulations indicated that on MAL12, GPESB15 and GPESB16 bind in a cavity adjacent to the active site, while on the PPA, GPESB15 was predicted to bind at the entrance of the catalytic site. Notably, despite its putative location within the active site, the binding of GPESB15 does not obstruct the substrate's access to the cleavage site. Our study contributes to paving the way for developing novel therapeutic strategies for managing DM-2 through GH13 α-glycosidases inhibition.

Kinetics studies on the inhibition mechanism of pancreatic α-amylase by glycoconjugated 1H-1,2,3-triazoles: a new class of inhibitors with hypoglycemiant activity.

Glycoconjugated 1H-1,2,3-triazoles (GCTs) comprise a new class of glycosidase inhibitors that are under investigation as promising therapeutic agents for a variety of diseases, including type 2 diabetes mellitus. However, few kinetics studies have been performed to clarify the mode of inhibition of GCTs with their target glycosidases. Our group has previously shown that some methyl-ß-D-ribofuranosyl-1H-1,2,3-triazoles that inhibit baker's yeast maltase were also able to reduce post-prandial glucose levels in normal rats. We hypothesized that this hypoglycemiant activity was attributable to inhibition of mammalian α-glucosidases involved in sugar metabolism, such as pancreatic α-amylase. Hence, the aim of this work was to test a series of 26 GCTs on porcine pancreatic α-amylase (PPA) and to characterize their inhibition mechanisms. Six GCTs, all ribofuranosyl-derived GCTs, significantly inhibited PPA, with IC(50) values in the middle to high micromolar range. Our results also demonstrated that ribofuranosyl-derived GCTs are reversible, noncompetitive inhibitors when using 2-chloro-4-nitrophenyl-α-D-maltotrioside as a substrate. E/ES affinity ratios (α) ranged from 0.3 to 1.1, with the majority of ribofuranosyl-derived GCTs preferentially forming stable ternary ESI complexes. Competition assays with acarbose showed that ribofuranosyl-derived GCTs bind to PPA in a mutually exclusive fashion. The data presented here show that pancreatic α-amylase is one of the possible molecular targets in the pharmacological activity of ribofuranosyl-derived GCTs. Our results also provide important mechanistic insight that can be of major help to develop this new class of synthetic small molecules into more potent compounds with anti-diabetic activity through rational drug design.

Aluminum exposure alters behavioral parameters and increases acetylcholinesterase activity in zebrafish (Danio rerio) brain.

Aluminum is a metal that is known to impact fish species. The zebrafish has been used as an attractive model for toxicology and behavioral studies, being considered a model to study environmental exposures and human pathologies. In the present study, we have investigated the effect of aluminum exposure on brain acetylcholinesterase activity and behavioral parameters in zebrafish. In vivo exposure of zebrafish to 50 µg/L AlCl(3) for 96 h at pH 5.8 significantly increased (36%) acetylthiocholine hydrolysis in zebrafish brain. There were no changes in acetylcholinesterase (AChE) activity when fish were exposed to the same concentration of AlCl(3) at pH 6.8. In vitro concentrations of AlCl(3) varying from 50 to 250 µM increased AChE activity (28% to 33%, respectively). Moreover, we observed that animals exposed to AlCl(3) at pH 5.8 presented a significant decrease in locomotor activity, as evaluated by the number of line crossings (25%), distance traveled (14.1%), and maximum speed (24%) besides an increase in the absolute turn angle (12.7%). These results indicate that sublethal levels of aluminum might modify behavioral parameters and acetylcholinesterase activity in zebrafish brain.

Efficient identification of novel anti-glioma lead compounds by machine learning models.

Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumor. Pharmacological treatment of this malignance is limited by the selective permeability of the blood-brain barrier (BBB) and relies on a single drug, temozolomide (TMZ), thus making the discovery of new compounds challenging and urgent. Therefore, aiming to discover new anti-glioma drugs, we developed robust machine learning models for predicting anti-glioma activity and BBB penetration ability of new compounds. Using these models, we prioritized 41 compounds from our in-house library of compounds, for further in vitro testing against three glioma cell lines and astrocytes. Subsequently, the most potent and selective compounds were resynthesized and tested in vivo using an orthotopic glioma model. This approach revealed two lead candidates, 4m and 4n, which efficiently decreased malignant glioma development in mice, probably by inhibiting thioredoxin reductase activity, as shown by our enzymological assays. Moreover, these two compounds did not promote body weight reduction, death of animals, or altered hematological and toxicological markers, making then good candidates for lead optimization as anti-glioma drug candidates.

Dealing with frequent hitters in drug discovery: a multidisciplinary view on the issue of filtering compounds on biological screenings.

Introduction: The timely identification biologically active chemicals, in disease relevant screening assays, is a major endeavor in drug discovery. The existence of frequent hitters (FHs) in non-related assays poses a formidable challenge in terms of whether to consider these molecules as chemical gold or promiscuous non-selective reactive trash (also known as PAINS - pan assay interference compounds).Areas covered: In this review, the authors bring together expertize in synthetic chemistry, cheminformatics and biochemistry, three key areas for dealing with FHs. They discuss synthetic methods facilitating preparation of chemically diverse molecular libraries, while favoring activity in the biological space. They also survey and discuss recent computational advances in the prediction of PAINS from chemical structures. Finally, they review experimental approaches for the validation of the biological activity of screening hits and discuss alternatives for exploiting promiscuity and chemical reactivity.Expert opinion: It's essential to develop more efficient computational methods to reliably recognize PAINS in distinct molecular environments. Accordingly, advances in synthetic chemistry hold the promise to provide a better quality of chemical matter for drug discovery. Medicinal chemists should be more open to screening for hits showing biologically complex mechanisms of action rather than discarding molecules that may prove valuable as innovative disease treatments.

Synthesis of new lophine-carbohydrate hybrids as cholinesterase inhibitors: cytotoxicity evaluation and molecular modeling.

In this study, we synthesized nine novel hybrids derived from d-xylose, d-ribose, and d-galactose sugars connected by a methylene chain with lophine. The compounds were synthesized by a four-component reaction to afford the substituted imidazole moiety, followed by the displacement reaction between sugar derivatives with an appropriate N-alkylamino-lophine. All the compounds were found to be the potent and selective inhibitors of BuChE activity in mouse serum, with compound 9a (a d-galactose derivative) being the most potent inhibitor (IC50 = 0.17 µM). According to the molecular modeling results, all the compounds indicated that the lophine moiety existed at the bottom of the BuChE cavity and formed a T-stacking interaction with Trp231, a residue accessible exclusively in the BuChE cavity. Noteworthily, only one compound exhibited activity against AChE (8b; IC50 = 2.75 µM). Moreover, the in silico ADME predictions indicated that all the hybrids formulated in this study were drug-likely, orally available, and able to reach the CNS. Further, in vitro studies demonstrated that the two most potent compounds against BuChE (8b and 9a) had no cytotoxic effects in the Vero (kidney), HepG2 (hepatic), and C6 (astroglial) cell lines.

Ethanol and acetaldehyde alter NTPDase and 5'-nucleotidase from zebrafish brain membranes.

Alcohol abuse is an acute health problem throughout the world and alcohol consumption is linked to the occurrence of several pathological conditions. Here we tested the acute effects of ethanol on NTPDases (nucleoside triphosphate diphosphohydrolases) and 5'-nucleotidase in zebrafish (Danio rerio) brain membranes. The results have shown a decrease on ATP (36.3 and 18.4%) and ADP (30 and 20%) hydrolysis after 0.5 and 1% (v/v) ethanol exposure during 60 min, respectively. In contrast, no changes on 5'-nucleotidase activity were observed in zebrafish brain membranes. Ethanol in vitro did not alter ATP and ADP hydrolysis, but AMP hydrolysis was inhibited at 0.5, and 1% (23 and 28%, respectively). Acetaldehyde in vitro, in the range 0.5-1%, inhibited ATP (40-85%) and ADP (28-65%) hydrolysis, whereas AMP hydrolysis was reduced (52, 58 and 64%) at 0.25, 0.5 and 1%, respectively. Acetate in vitro did not alter these enzyme activities. Semi-quantitative expression analysis of NTPDase and 5'-nucleotidase were performed. Ethanol treatment reduced NTPDase1 and three isoforms of NTPDase2 mRNA levels. These findings demonstrate that acute ethanol intoxication may influence the enzyme pathway involved in the degradation of ATP to adenosine, which could affect the responses mediated by adenine nucleotides and nucleosides in zebrafish central nervous system.

Ecto-nucleotidase pathway is altered by different treatments with fluoxetine and nortriptyline.

Depression is one of the most disabling diseases and causes a significant burden to both individual and society. Selective serotonin reuptake inhibitors and tricyclic antidepressants, such as fluoxetine and nortriptyline, respectively, are commonly used in treatment for depression. These antidepressants were tested on cerebral cortex and hippocampal synaptosomes after acute and chronic in vivo and in vitro treatments. In chronic treatment, fluoxetine and nortriptyline decreased ATP hydrolysis (17.8% and 16.3%, respectively) in hippocampus. In cerebral cortex, nortriptyline increased ATP (32.3%), ADP (51.8%), and AMP (59.5%) hydrolysis. However, fluoxetine decreased ATP (25.5%) hydrolysis and increased ADP (80.1%) and AMP (33.3%) hydrolysis. Significant activation of ADP hydrolysis was also observed in acute treatment with nortriptyline (49.8%) in cerebral cortex. However, in hippocampus, ATP (24.7%) and ADP (46.1%) hydrolysis were inhibited. Fluoxetine did not alter enzyme activities in acute treatment for both structures. In addition, there were significant changes in NTPDase activities when fluoxetine and nortriptyline (100, 250, and 500 microM) were tested in vitro. There was no inhibitory effect of fluoxetine and nortriptyline on AMP hydrolysis in cerebral cortex and hippocampus. The findings showed that these antidepressant drugs can affect the ecto-nucleotidase pathway, suggesting that the extracellular adenosine levels could be modulated by these drugs.

Kinetic characterization of adenosine deaminase activity in zebrafish (Danio rerio) brain.

Adenosine deaminase (ADA; EC 3.5.4.4) activity is responsible for cleaving adenosine to inosine. In this study we described the biochemical properties of adenosine deamination in soluble and membrane fractions of zebrafish (Danio rerio) brain. The optimum pH for ADA activity was in the range of 6.0-7.0 in soluble fraction and reached 5.0 in brain membranes. A decrease of 31.3% on adenosine deamination in membranes was observed in the presence of 5 mM Zn(2+), which was prevented by 5 mM EDTA. The apparent K(m) values for adenosine deamination were 0.22+/-0.03 and 0.19+/-0.04 mM for soluble and membrane fractions, respectively. The apparent V(max) value for soluble ADA activity was 12.3+/-0.73 nmol NH(3) min(-1) mg(-1) of protein whereas V(max) value in brain membranes was 17.5+/-0.51 nmol NH(3) min(-1) mg(-1) of protein. Adenosine and 2'-deoxyadenosine were deaminated in higher rates when compared to guanine nucleosides in both fractions. Furthermore, a significant inhibition on adenosine deamination in both soluble and membrane fractions was observed in the presence of 0.1 mM of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The presence of ADA activity in zebrafish brain may be important to regulate the adenosine/inosine levels in the CNS of this species.

Discovery of new Schistosoma mansoni aspartyl protease inhibitors by structure-based virtual screening

BACKGROUND Schistosomiasis is a neglected tropical disease caused by trematodes of the genus Schistosoma, with a limited treatment, mainly based on the use of praziquantel (PZQ). Currently, several aspartic proteases genes have already been identified within the genome of Schistosoma species. At least one enzyme encoded from this gene family (SmAP), named SmCD1, has been validated for the development of schistosomicidal drugs, since it has a key role in haemoglobin digestion by worms. OBJECTIVE In this work, we integrated a structure-based virtual screening campaign, enzymatic assays and adult worms ex vivo experiments aiming to discover the first classes of SmCD1 inhibitors. METHODS Initially, the 3D-structures of SmCD1, SmCD2 and SmCD3 were generated using homology modelling approach. Using these models, we prioritised 50 compounds from 20,000 compounds from ChemBridge database for further testing in adult worm aqueous extract (AWAE) and recombinant SmCD1 using enzymatic assays. FINDINGS Seven compounds were confirmed as hits and among them, two compounds representing new chemical scaffolds, named 5 and 19, had IC50 values against SmCD1 close to 100 μM while presenting binding efficiency indexes comparable to or even higher than pepstatin, a classical tight-binding peptide inhibitor of aspartyl proteases. Upon activity comparison against mammalian enzymes, compound 50 was selective and the most potent against the AWAE aspartic protease activity (IC50 = 77.7 μM). Combination of computational and experimental results indicate that compound 50 is a selective inhibitor of SmCD2. Compounds 5, 19 and 50 tested at low concentrations (10 uM) were neither cytotoxic against WSS-1 cells (48 h) nor could kill adult worms ex-vivo, although compounds 5 and 50 presented a slight decrease on female worms motility on late incubations times (48 or 72 h). MAIN CONCLUSION Overall, the inhibitors identified in this work represent promising hits for further hit-to-lead optimisation.

Acute and subchronic copper treatments alter extracellular nucleotide hydrolysis in zebrafish brain membranes.

Copper is a divalent cation with physiological importance since deficiency of copper homeostasis can cause serious neurological diseases. ATP is an important signalling molecule stored at nerve endings and its inactivation is promoted by ecto-nucleotidases. In this study, we verified the effect of acute and subchronic copper treatments on ecto-nucleotidase activities in zebrafish brain membranes. Treatment with copper sulfate (15 microg/L) during 24h inhibited ATP hydrolysis (16%), whereas ADP and AMP hydrolysis were not altered. Nevertheless, a 96-h exposure with the copper concentration mentioned above inhibited NTPDase (31% and 42% for ATP and ADP hydrolysis, respectively) and ecto-5'-nucleotidase (40%) activities. NTPDase1, NTPDase2_mg and NTPDase2_mv transcripts were decreased after copper exposures during 24 and 96 h. Subchronic copper treatment also reduced the NTPDase2_mq and ecto-5'-nucleotidase expression. In vitro assays demonstrated that NTPDase activities were reduced after copper exposure during 40 min. ATP hydrolysis was inhibited at 0.25, 0.5 and 1mM (13%, 31% and 48%, respectively) and ADP hydrolysis also had a significant decrease at these same copper concentrations (41%, 63% and 68%, respectively). In contrast to the subchronic exposure, no significant changes on ecto-5'-nucleotidase were observed after in vitro assays. Lineweaver-Burk plots suggested that both inhibitory effects on nucleotide hydrolysis may occur in a non-competitive manner. Altogether, these findings indicate that copper is able to promote distinct changes on ecto-nucleotidases after in vivo and in vitro treatments and, consequently, it could control the nucleotide and nucleoside levels, modulating the purinergic signalling.

Fluoxetine and nortriptyline affect NTPDase and 5'-nucleotidase activities in rat blood serum.

Depression is a serious condition associated with considerable morbidity and mortality. Selective serotonin reuptake inhibitors and tricyclic antidepressants, such as fluoxetine and nortriptyline, respectively, were commonly used in treatment for depression. Selective serotonin reuptake inhibitors have been associated with increased risk of bleeding complications, possibly as a result of inhibition of platelet aggregation. ATP, ADP and adenosine are signaling molecules in the vascular system and nucleotidases activities are considered an important thromboregulatory system which functions in the maintenance of blood fluidity. Therefore, here we investigate the effect of in vivo (acute and chronic) and in vitro treatments with the antidepressant drugs on nucleotidases activities in rat blood serum. In acute treatment, nortriptyline decreased ATP hydrolysis (41%), but not altered ADP and AMP hydrolysis. In contrast, fluoxetine did not alter NTPDase and ecto-5'-nucleotidase activities. A significant inhibition of ATP, ADP, and AMP hydrolysis were observed in chronic treatment with fluoxetine (60%, 32%, and 42% for ATP, ADP, and AMP hydrolysis, respectively). Similar effects were shown in chronic treatment with nortriptyline (37%, 41%, and 30% for ATP, ADP, and AMP hydrolysis, respectively). In addition, there were no significant changes in NTPDase and ecto-5'-nucleotidase activities when fluoxetine and nortriptyline (100, 250, and 500 microM) were tested in vitro. Our results have shown that fluoxetine and nortriptyline changed the nucleotide catabolism, suggesting that homeostasis of vascular system can be altered by antidepressant treatments.

Sertraline and clomipramine inhibit nucleotide catabolism in rat brain synaptosomes.

The effects of sertraline, a selective serotonin reuptake inhibitor, and clomipramine, a tricyclic antidepressant, were tested on ecto-nucleotidases from synaptosomes of cerebral cortex and hippocampus of rats. Sertraline and clomipramine (100-500 microM) inhibited NTPDase, but not ecto-5'-nucleotidase activity in both cerebral cortex and hippocampus. In cortical synaptosomes, sertraline inhibited both ATP and ADP hydrolysis in the concentrations tested. The inhibitory effect varied from 21% to 83% for ATP hydrolysis and 48% to 75% for ADP hydrolysis. The inhibition promoted by sertraline in hippocampal synaptosomes varied from 38% to 89% for ATP hydrolysis and 45% to 77% for ADP hydrolysis. A significant inhibition of cortical NTPDase activity by clomipramine was observed in the all concentrations tested (35-72% and 36-87% for ATP and ADP hydrolysis, respectively). Similar effects were observed in hippocampus (29-91% and 48-83% for ATP and ADP hydrolysis, respectively). There was no inhibitory effect of sertraline and clomipramine on AMP hydrolysis in cerebral cortex and hippocampus. Our results have shown that classical antidepressants inhibit the extracellular catabolism of ATP. Therefore, it is possible to suggest that changes induced by antidepressants on bilayer membrane could affect NTPDase activities and consequently, modulating ATP and adenosine levels in the synaptic cleft.

Exposure to Hg2+ and Pb2+ changes NTPDase and ecto-5'-nucleotidase activities in central nervous system of zebrafish (Danio rerio).

Neurotransmission can be affected by exposure to heavy metals, such as mercury and lead. ATP is a signaling molecule that can be metabolized by a group of enzymes called ecto-nucleotidases. Here we investigated the effects of mercury chloride (HgCl(2)) and lead acetate (Pb(CH(3)COO)(2)) on NTPDase (nucleoside triphosphate diphosphohydrolase) and ecto-5'-nucleotidase activities in zebrafish brain membranes. In vitro exposure to HgCl(2) decreased ATP and ADP hydrolysis in an uncompetitive mechanism and AMP hydrolysis in a non-competitive manner. Pb(CH(3)COO)(2) inhibited ATP hydrolysis in an uncompetitive manner, but not ADP and AMP hydrolysis. In vivo exposure of zebrafish to HgCl(2) or Pb(CH(3)COO)(2) (20mug/L, during 24, 96h and 30 days) caused differential effects on nucleotide hydrolysis. HgCl(2), during 96h, inhibited the hydrolysis of ATP, ADP and AMP. After 30 days of exposure to HgCl(2), ATP hydrolysis returned to the control levels, ADP hydrolysis was strongly increased and AMP hydrolysis remained inhibited. Exposure to Pb(CH(3)COO)(2) during 96h caused a significant decrease only on ATP hydrolysis. After 30 days, Pb(CH(3)COO)(2) promoted the inhibition of ATP, ADP and AMP hydrolysis. Semi-quantitative RT-PCR analysis showed no changes in the expression of NTPDase1 and 5'-nucleotidase, following 30 days of exposure to both metals. This study demonstrated that Hg(2+) and Pb(2+) affect the ecto-nucleotidase activities, an important enzymatic pathway for the control of purinergic signaling.