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Rodriguez-Angulo H.O.,CNR Institute of Biophysics | Toro-Mendoza J.,Venezuelan Institute for Scientific Research | Marques J.A.,Central University of Venezuela | Concepcion J.L.,University of Los Andes, Venezuela | And 6 more authors.
PLoS Neglected Tropical Diseases | Year: 2015

Chagas cardiomyopathy, caused by the protozoan Trypanosoma cruzi, is characterized by alterations in intracellular ion, heart failure and arrhythmias. Arrhythmias have been related to sudden death, even in asymptomatic patients, and their molecular mechanisms have not been fully elucidated. The aim of this study is to demonstrate the effect of proteins secreted by T. cruzi on healthy, isolated beating rat heart model under a non-damage-inducing protocol. We established a non-damage-inducing recirculation-reoxygenation model where ultrafiltrate fractions of conditioned medium control or conditioned infected medium were perfused at a standard flow rate and under partial oxygenation. Western blotting with chagasic patient serum was performed to determine the antigenicity of the conditioned infected medium fractions. We observed bradycardia, ventricular fibrillation and complete atrioventricular block in hearts during perfusion with >50 kDa conditioned infected culture medium. The preincubation of conditioned infected medium with chagasic serum abolished the bradycardia and arrhythmias. The proteins present in the conditioned infected culture medium of >50 kDa fractions were recognized by the chagasic patient sera associated with arrhythmias. These results suggest that proteins secreted by T. cruzi are involved in Chagas disease arrhythmias and may be a potential biomarker in chagasic patients. © 2015 Rodríguez-Angulo et al.


Benaim G.,Institute Estudios Avanzados IDEA | Benaim G.,Central University of Venezuela | Casanova P.,Institute Estudios Avanzados IDEA | Casanova P.,Central University of Venezuela | And 9 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2014

Dronedarone and amiodarone are cationic lipophilic benzofurans used to treat cardiac arrhythmias. They also have activity against the parasitic protozoan Trypanosoma cruzi, the causative agent of Chagas' disease. They function by disrupting intracellular Ca2+ homeostasis of the parasite and by inhibiting membrane sterol (ergosterol) biosynthesis. Amiodarone also has activity against Leishmania mexicana, suggesting that dronedarone might likewise be active against this organism. This might be of therapeutic interest, since dronedarone is thought to have fewer side effects in humans than does amiodarone. We show here that dronedarone effectively inhibits the growth of L. mexicana promastigotes in culture and, more importantly, has excellent activity against amastigotes inside infected macrophages (the clinically relevant form) without affecting the host cell, with the 50% inhibitory concentrations against amastigotes being 3 orders of magnitude lower than those obtained previously with T. cruzi amastigotes (0.65 nM versus 0.75 μM). As with amiodarone, dronedarone affects intracellular Ca2+ homeostasis in the parasite, inducing an elevation of intracellular Ca2+ levels. This is achieved by rapidly collapsing the mitochondrial membrane potential and inducing an alkalinization of acidocalcisomes at a rate that is faster than that observed with amiodarone. We also show that dronedarone inhibits parasite oxidosqualene cyclase, a key enzyme in ergosterol biosynthesis known to be vital for survival. Overall, our results suggest the possibility of repurposing dronedarone as a treatment for cutaneous, and perhaps other, leishmaniases. Copyright © 2014, American Society for Microbiology. All Rights Reserved.


Stateva S.R.,Autonomous University of Madrid | Salas V.,Autonomous University of Madrid | Salas V.,Central University of Venezuela | Benaim G.,Central University of Venezuela | And 4 more authors.
PLoS ONE | Year: 2015

Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro. © 2015 Stateva et al.


Benaim G.,Institute Estudios Avanzados IDEA | Benaim G.,Central University of Venezuela | Garcia C.R.S.,University of Sao Paulo
Tropical Biomedicine | Year: 2011

Ca 2+ has been largely recognized as an essential messenger in all eukaryotes, from mammals to parasites. The disruption of Ca 2+ homeostasis in any cell usually drives to lethal effects resulting in cell death by apoptosis or necrosis. This appears also to be the case in human trypanosomatids, such as Trypanosoma cruzi, the causative agent of Chagas' disease, Trypanosoma brucei, which produces "sleeping sickness" and Leishmania sp, responsible for leishmaniasis. The aim of this review is to describe the intracellular Ca 2+ regulation and the cytotoxic effect of new drugs regarding the disruption of Ca 2+ homeostasis in these parasites. With regard to intracellular Ca 2+ regulation, all these trypanosomatids possess a single mitochondrion that occupies 12% of the total volume of the parasite which is able to accumulate large amounts of Ca 2+. The endoplasmic reticulum is also involved in Ca 2+ regulation. These parasites also possess acidocalcisomes, an unusual organelle involved in the bioenergetics of these cells in accumulating large amounts of polyphosphates together with Ca 2+ ions. Trypanosomatids possess relatively large amounts of calmodulin. While this well conserved protein is identical among all vertebrates, there is 89% amino acid sequence identity between T. cruzi and vertebrate calmodulin. Recently, this protein has been cloned and expressed from T. cruzi, allowing a further characterization corroborating significant differences between calmodulin from T. cruzi and mammals. It has also been reported that a commonly used antiarrhytmic, amiodarone, which is used in chronic Chagas' patients with heart problems, is able to produce a large trypanocidal effect. The intracellular compartments responsible for the increase in the intracellular Ca 2+ concentration ([Ca 2+] i) upon the addition of amiodarone are the single large mitochondrion and the acidocalcisomes. Amiodarone is also capable to inhibit the oxidosqualene cyclase, a key enzyme in the synthesis of ergosterol. The effect of amiodarone was highly synergistic with posaconazole, a known potent inhibitor of the synthesis of ergosterol. Interestingly, posaconazole by itself is able to produce an increase in the [Ca 2+] i. Concerning putative treatments in humans, amiodarone was reported to induce the cure of a patient with Chagas' disease, when used in combination with itraconazole. Seemingly, a recent case indicated the cure of a patient with Chagas' disease by the administration of posaconazole. Miltefosine, an orally active alkyl-lysophospholipid with potent anti-Leishmania activity, represents a major advance in the treatment of leishmaniasis. Recently it was reported that miltefosine also disrupts the parasite's intracellular Ca 2+ homeostasis, by inducing a large increase in [Ca 2+] i, through the activation of a plasma membrane Ca 2+ channel. It has been found that the combination of miltefosine and amiodarone have synergistic effects on the proliferation of amastigotes growing inside macrophages and this led to 90% of parasitological cure in a murine model of leishmaniasis, as revealed by a PCR assay. More recently, posaconazole has been used successfully in a case of a human Old World cutaneous leishmaniasis. All these findings strongly suggest that the alteration of the intracellular Ca 2+ homeostasis of these parasites is a promising strategy as a target of new as well as repurposed old-known drugs.


Benaim G.,Institute Estudios Avanzados IDEA | Benaim G.,Central University of Venezuela | Hernandez-Rodriguez V.,Institute Estudios Avanzados IDEA | Hernandez-Rodriguez V.,Central University of Venezuela | And 8 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2012

Amiodarone, a commonly used antiarrhythmic, is also a potent and selective anti-Trypanosoma cruzi agent. Dronedarone is an amiodarone derivative in which the 2,5-diiodophenyl moiety of the parental drug has been replaced with an unsubstituted phenyl group aiming to eliminate the thyroid toxicity frequently observed with amiodarone treatment. Dronedarone has been approved by the Food and Drug Administration (FDA), and its use as a safe antiarrhythmic has been extensively documented. We show here that dronedarone also has potent anti-T. cruzi activity, against both extracellular epimastigotes and intracellular amastigotes, the clinically relevant form of the parasite. The 50% inhibitory concentrations against both proliferative stages are lower than those previously reported for amiodarone. The mechanism of action of dronedarone resembles that of amiodarone, as it induces a large increase in the intracellular Ca 2+ concentration of the parasite, which results from the release of this ion from intracellular storage sites, including a direct effect of the drug on the mitochondrial electrochemical potential, and through alkalinization of the acidocalcisomes. Our results suggest a possible future repurposed use of dronedarone for the treatment of Chagas' disease. Copyright © 2012, American Society for Microbiology. All Rights Reserved.


Benaim G.,Institute Estudios Avanzados IDEA | Benaim G.,Central University of Venezuela | Paniz Mondolfi A.E.,Institute Biomedicina SAIB | Paniz Mondolfi A.E.,St Lukes Roosevelt Hospital Center
Nature Reviews Cardiology | Year: 2012

Chagas disease has emerged as an important health problem in the Americas and, with globalization, in other parts of the world. Drug therapy for this parasitic infection has remained largely ineffective, especially in chronic stages of the disease. However, developments in experimental therapy might signal an important advance for the management of patients with Chagas disease. Herein, we review studies on the potential use of the benzofuran derivatives amiodarone and dronedarone in patients with Chagas disease. These agents have a dual role, not only as primary antiarrhythmic drugs, but also as antiparasitic agents. We believe that this 'kill two birds with one stone' approach represents a new tactic for the treatment of Chagas disease using currently approved drugs. © 2012 Macmillan Publishers Limited. All rights reserved.


Rojas H.,Venezuelan Institute for Scientific Research | Colina C.,Venezuelan Institute for Scientific Research | Ramos M.,Venezuelan Institute for Scientific Research | Benaim G.,Institute Estudios Avanzados IDEA | And 3 more authors.
Advances in Experimental Medicine and Biology | Year: 2013

We have previously demonstrated that rat type-1 cerebellar astrocytes express a very active Na+/Ca2+ exchanger which accounts for most of the total plasma membrane Ca2+ fluxes and for the clearance of Ca i 2+ induced by physiological agonist. In this chapter, we have explored the mechanism by which the reverse Na +/Ca2+ exchange is involved in agonist-induced Ca 2+ signalling in rat cerebellar astrocytes. Laser-scanning confocal microscopy experiments using immunofluorescence labelling of Na +/Ca2+ exchanger and RyRs demonstrated that they are highly co-localized. The most important finding presented in this chapter is that L-glutamate activates the reverse mode of the Na+/Ca 2+ exchange by inducing a Na+ entry through the electrogenic Na+-glutamate co-transporter and not through the ionophoric L-glutamate receptors as confirmed by pharmacological experiments with specific blockers of ionophoric L-glutamate receptors, electrogenic glutamate transporters and the Na/Ca exchange. © Springer Science+Business Media New York 2013.


Benaim G.,Institute Estudios Avanzados IDEA | Benaim G.,Central University of Venezuela | Garcia-Marchan Y.,Institute Estudios Avanzados IDEA | Reyes C.,Venezuelan Institute for Scientific Research | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2013

The disruption of the intracellular Ca2+ homeostasis of Leishmania mexicana represents a major target for the action of drugs, such as amiodarone and miltefosine. However, little is known about the mechanism of Ca2+ entry to these cells. Here we show the presence of a Ca2+ channel in the plasma membrane of these parasites. This channel has many characteristics similar to the human L-type voltage-gated Ca2+ channel. Thus, Ca2+ entry is blocked by verapamil, nifedipine and diltiazem while Bay K 8644 opened this channel. However, different to its human counterpart, sphingosine was able to open this channel, while other well known sphingolipids had no effect. This fact could have important pharmacological implications. © 2012 Elsevier Inc.


PubMed | Institute Estudios Avanzados IDEA, Central University of Venezuela and Simon Bolivar University of Venezuela
Type: | Journal: European journal of medicinal chemistry | Year: 2017

With the aim to identify a potential drug candidate to treat cutaneous leishmaniasis, a series of 1-phthalazinyl hydrazones were synthesized and tested against Leishmania braziliensis parasite, one of the main responsible of this disease in the world. A structure-activity relationship permitted to identify two phthalazines containing nitroheterocyclic moiety 3l and 3m as promising new lead compounds. These compounds showed a significant antileishmanial activity against promastigote form of L.braziliensis, with EC


Recio-Pinto E.,NYU Langone Medical Center | Castillo C.,Institute Estudios Avanzados IDEA
Techniques in Regional Anesthesia and Pain Management | Year: 2010

The role of N-methyl-D-aspartate receptors (NMDArs) in pain sensation was initially uncovered in 1987 when the hyperexcitability of spinal cord dorsal horn nociceptive neurons evoked by C-fiber stimulation was found to be blocked by spinal delivery of NMDAr antagonist. Since then, many studies have focused on the role of central NMDArs in pain sensation. It is now apparent that peripheral NMDArs also play a role not only in the initiation but also in the maintenance of chronic pain states, particularly those following peripheral nerve injuries. Peripheral NMDArs are an attractive target for treating chronic pain, because under normal (nonpainful) stimulation NMDArs in dorsal root ganglia (DRG) neurons do not activate; in addition, some of the NMDArs isoforms are predominantly expressed in DRG neurons, and NMDArs have various regulatory sites that are isoform-dependent. This article concentrates on reviewing the possible role of peripheral NMDArs in initiating and maintaining chronic pain states. Of particular interest is the role of NMDArs not only on peripheral DRG neurons but also on their surrounding glia, since neuronal-glial interactions have been shown to contribute to injury-evoked neuronal hyperexcitability. Drugs that would target selectively peripheral NMDArs would improve treatment of chronic pain states. This review is divided into 5 sections: NMDAr structure and function; the role of peripheral NMDArs in pain perception; modulation of NMDArs during pain states; modulation of NMDAr activity by Substance P; and role of glia in DRG neuronal hyperexcitability. © 2010.

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