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The protozoan parasite Leishmania causes a variety of sicknesses with different clinical manifestations known as leishmaniasis. The chemotherapy currently in use is not adequate because of their side effects, resistance occurrence, and recurrences. Investigations looking for new targets or new active molecules focus mainly on the disruption of parasite specific pathways. In this sense, ergosterol biosynthesis is one of the most attractive because it does not occur in mammals. Here, we report the synthesis of ergosterone coupled molecules and the characterization of their biological activity on Leishmania mexicana promastigotes. Molecule synthesis involved three steps: ergosterone formation using Jones oxidation, synthesis of Girard reagents, and coupling reaction. All compounds were obtained in good yield and high purity. Results show that ergosterone-triazol molecules (Erg-GTr and Erg-GTr2) exhibit an antiproliferative effect in low micromolar range with a selectivity index ~10 when compared to human dermic fibroblasts. Addition of Erg-GTr or Erg-GTr2 to parasites led to a rapid [Ca2+]cyt increase and acidocalcisomes alkalinization, indicating that Ca2+ was released from this organelle. Evaluation of cell death markers revealed some apoptosis-like indicators, as phosphatidylserine exposure, DNA damage, and cytosolic vacuolization and autophagy exacerbation. Furthermore, mitochondrion hyperpolarization and superoxide production increase were detected already 6 hours after drug addition, denoting that oxidative stress is implicated in triggering the observed phenotype. Taken together our results indicate that ergosterone-triazol coupled molecules induce a regulated cell death process in the parasite and may represent starting point molecules in the search of new chemotherapeutic agents to combat leishmaniasis.

The hemoparasite Trypanosomaequiperdum belongs to the Trypanozoon subgenus and includes several species that are pathogenic to animals and humans in tropical and subtropical areas across the world. As with all eukaryotic organisms, Ca2+ is essential for these parasites to perform cellular processes thus ensuring their survival across their life cycle. Despite the established paradigm to study proteins related to Ca2+ homeostasis as potential drug targets, so far little is known about Ca2+ entry into trypanosomes. Therefore, in the present study, the presence of a plasma membrane Ca2+-channel in T. equiperdum (TeCC), activated by sphingosine and inhibited by verapamil, is described. The TeCC was cloned and analyzed using bioinformatic resources, which confirmed the presence of several domains, motifs, and a topology similar to the Ca2+ channels found in higher eukaryotes. Biochemical and confocal microscopy assays using antibodies raised against an internal region of human L-type Ca2+ channels indicate the presence of a protein with similar predicted molar mass to the sequence analyzed, located at the plasma membrane of T. equiperdum. Physiological assays based on Fura-2 signals and Mn2+ quenching performed on whole parasites showed a unidirectional Ca2+ entry, which is activated by sphingosine and blocked by verapamil, with the distinctive feature of insensitivity to nifedipine and Bay K 8644. This suggests a second Ca2+ entry for T. equiperdum, different from the store-operated Ca2+ entry (SOCE) previously described. Moreover, the evidence presented here for the TeCC indicates molecular and pharmacological differences with their mammal counterparts, which deserve further studies to evaluate the potential of this channel as a drug target. [ABSTRACT FROM AUTHOR]

Originally developed as a chemotherapeutic agent, miltefosine (hexadecylphosphocholine) is an inhibitor of phosphatidylcholine synthesis with proven antiparasitic effects. It is the only oral drug approved for the treatment of Leishmaniasis and American Trypanosomiasis (Chagas disease). Although its precise mechanisms are not yet fully understood, miltefosine exhibits broad-spectrum anti-parasitic effects primarily by disrupting the intracellular Ca 2+ homeostasis of the parasites while sparing the human hosts. In addition to its inhibitory effects on phosphatidylcholine synthesis and cytochrome c oxidase, miltefosine has been found to affect the unique giant mitochondria and the acidocalcisomes of parasites. Both of these crucial organelles are involved in Ca 2+ regulation. Furthermore, miltefosine has the ability to activate a specific parasite Ca 2+ channel that responds to sphingosine, which is different to its L-type VGCC human ortholog. Here, we aimed to provide an overview of recent advancements of the anti-parasitic mechanisms of miltefosine. We also explored its multiple molecular targets and investigated how its pleiotropic effects translate into a rational therapeutic approach for patients afflicted by Leishmaniasis and American Trypanosomiasis. Notably, miltefosine's therapeutic effect extends beyond its impact on the parasite to also positively affect the host's immune system. These findings enhance our understanding on its multi-targeted mechanism of action. Overall, this review sheds light on the intricate molecular actions of miltefosine, highlighting its potential as a promising therapeutic option against these debilitating parasitic diseases.

A series of heterocyclic chloroquine hybrids, containing a chain of two carbon atoms at position four of the quinolinic chain and acting as a link between quinoline and several benzoyl groups, is synthesized and screened in vitro as an inhibitor of β-hematin formation and in vivo for its antimalarial activity against chloroquine-sensitive strains of Plasmodium berghei ANKA in this study. The compounds significantly reduced haeme crystallization, with IC 50 values < 10 µM. The values were comparable to chloroquine's, with an IC 50 of 1.50 ± 0.01 µM. The compounds 4c and 4e prolonged the average survival time of the infected mice to 16.7 ± 2.16 and 14.4 ± 1.20 days, respectively. We also studied the effect of the compounds 4b , 4c, and 4e on another important human parasite, Leishmania mexicana , which is responsible for cutaneous leishmaniasis, demonstrating a potential leishmanicidal effect against promasigotes, with an IC 50 < 10 µM. Concerning the possible mechanism of action of these compounds on Lesihmania mexicana , we performed experiments demonstrating that these three compounds could induce the collapse of the parasite mitochondrial electrochemical membrane potential (Δφ). The in vitro cytotoxicity assays against mammalian cancerous and noncancerous human cell lines showed that the studied compounds exhibit low cytotoxic effects. The ADME/Tox analysis predicted moderate lipophilicity values, low unbound fraction values, and a poor distribution for these compounds. Therefore, moderate bioavailability was expected. We calculated other molecular descriptors, such as the topological polar surface area, according to Veber's rules, and except for 2 and 4i , the rest of the compounds violated this descriptor, demonstrating the low antimalarial activity of our compounds in vivo.

Trichomonas vaginalis is a protozoan that causes human trichomoniasis, a sexually transmitted infection (STI) that affects approximately 278 million people worldwide. The current treatment for human trichomoniasis is based on 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole, known as Metronidazole (MTZ). Although effective in eliminating parasitic infection, MTZ is related to serious adverse effects and is not recommended during pregnancy. In addition, some strains are resistant to 5'-nitroimidazoles, prompting the development of alternative drugs for trichomoniasis. Here we show that SQ109 [N-adamantan-2-yl-N'-((E)-3,7-dimethyl-octa- 2,6-dienyl)-ethane-1,2-diamine], a drug under development (antitubercular drug candidate that completed Phase IIb/III) for the treatment of tuberculosis, and previously tested in Trypanosoma cruzi and Leishmania. SQ109 inhibited T.vaginalis growth with an IC50 of 3.15 μM. We used scanning and transmission electron microscopy to visualize the ultrastructural alterations induced by SQ109. The microscopy analysis showed morphological changes on the protozoan surface, where the cells became rounded with increasing surface projections. In addition, the hydrogenosomes increased their size and area occupied in the cell. Furthermore, the volume and a significant association of glycogen particles with the organelle were seen to be altered. A bioinformatics search was done about the compound to find its possible targets and mechanisms of action. Our observations identify SQ109 as a promising compound against T. vaginalis in vitro, suggesting its potential utility as an alternative chemotherapy for trichomoniasis., Competing Interests: Declaration of competing interest On behalf of all authors, the corresponding author states that there is no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The plasma membrane and autoinhibited Ca 2+ -ATPases contribute to the Ca 2+ homeostasis in a wide variety of organisms. The enzymatic activity of these pumps is stimulated by calmodulin, which interacts with the target protein through the calmodulin-binding domain (CaMBD). Most information about this region is related to all calmodulin modulated proteins, which indicates general chemical properties and there is no established relation between Ca 2+ pump sequences and taxonomic classification. Thus, the aim of this study was to perform an in silico analysis of the CaMBD from several Ca 2+ -ATPases, in order to determine their diversity and to detect specific patterns and amino acid selection in different species. Patterns related to potential and confirmed CaMBD were detected using sequences retrieved from the literature. The occurrence of these patterns was determined across 120 sequences from 17 taxonomical classes, which were analyzed by a phylogenetic tree to establish phylogenetic groups. Predicted physicochemical characteristics including hydropathy and net charge were calculated for each group of sequences. 22 Ca 2+ -ATPases sequences from animals, unicellular eukaryotes, and plants were retrieved from bioinformatic databases. These sequences allow us to establish the Patterns 1(GQILWVRGLTRLQTQ), 3(KNPSLEALQRW), and 4(SRWRRLQAEHVKK), which are present at the beginning of putative CaMBD of metazoan, parasites, and land plants. A pattern 2 (IRVVNAFR) was consistently found at the end of most analyzed sequences. The amino acid preference in the CaMBDs changed depending on the phylogenetic groups, with predominance of several aliphatic and charged residues, to confer amphiphilic properties. The results here displayed show a conserved mechanism to contribute to the Ca 2+ homeostasis across evolution and may help to detect putative CaMBDs., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

SQ109 is a tuberculosis drug candidate that has high potency against Mycobacterium tuberculosis and is thought to function at least in part by blocking cell wall biosynthesis by inhibiting the MmpL3 transporter. It also has activity against bacteria and protozoan parasites that lack MmpL3, where it can act as an uncoupler, targeting lipid membranes and Ca 2+ homeostasis. Here, we synthesized 18 analogs of SQ109 and tested them against M. smegmatis , M. tuberculosis , M. abscessus , Bacillus subtilis , and Escherichia coli , as well as against the protozoan parasites Trypanosoma brucei , T. cruzi , Leishmania donovani , L. mexicana , and Plasmodium falciparum . Activity against the mycobacteria was generally less than with SQ109 and was reduced by increasing the size of the alkyl adduct, but two analogs were ∼4-8-fold more active than SQ109 against M. abscessus , including a highly drug-resistant strain harboring an A309P mutation in MmpL3. There was also better activity than found with SQ109 with other bacteria and protozoa. Of particular interest, we found that the adamantyl C-2 ethyl, butyl, phenyl, and benzyl analogs had 4-10× increased activity against P. falciparum asexual blood stages, together with low toxicity to a human HepG2 cell line, making them of interest as new antimalarial drug leads. We also used surface plasmon resonance to investigate the binding of inhibitors to MmpL3 and differential scanning calorimetry to investigate binding to lipid membranes. There was no correlation between MmpL3 binding and M. tuberculosis or M. smegmatis cell activity, suggesting that MmpL3 is not a major target in mycobacteria. However, some of the more active species decreased lipid phase transition temperatures, indicating increased accumulation in membranes, which is expected to lead to enhanced uncoupler activity.

Trichomonas vaginalis is a protozoan that causes human trichomoniasis, the most common non-viral sexually transmitted infection (STI) affecting approximately 278 million people worldwide. The current treatment for trichomoniasis is based on 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole, known as metronidazole (MTZ). Although effective in clearing the parasite infection, MTZ is related to provoking severe side effects, and it is not recommended during pregnancy. In addition, some strains present resistance to 5'-nitroimidazoles, making urgent the development of alternative drugs for trichomoniasis. Amiodarone, an antiarrhythmic drug, exerts a significant anti-parasite effect, mainly due to its interference with calcium homeostasis and the biosynthesis of sterols. Therefore, we decided to test the effect of amiodarone and two other related compounds (amioder and dronedarone) on T. vaginalis. Our observations show that amiodarone stimulated, rather than inhibited, parasite growth, induced cell aggregation, and glycogen accumulation. Furthermore, the other two compounds displayed anti-parasite activity with IC50 of 3.15 and 11 µM, respectively, and the apoptosis-like process killed the cells. In addition, cells exhibited morphological changes, including an effect on hydrogenosomes structure., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The Trypanosomatidae family encompasses many unicellular organisms responsible of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by Trypanosoma brucei brucei (T. brucei), Trypanosoma equiperdum (T. equiperdum) and Trypanosoma evansi (T. evansi), have a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world. Similarly, to all organisms, the maintenance of Ca 2+ homeostasis is vital for these parasites, and the mechanism involved in the intracellular Ca 2+ regulation have been widely described. However, the evidences related to the mechanisms responsible for the Ca 2+ entry are scarce. Even more, to date the presence of a store-operated Ca 2+ channel (SOC) has not been reported. Despite the apparent absence of Orai and STIM-like proteins in these parasites, in the present work we demonstrate the presence of a store-operated Ca 2+ -entry (SOCE) in T. equiperdum, using physiological techniques. This Ca 2+ -entry is induced by thapsigargin (TG) and 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), and inhibited by 2-aminoethoxydiphenyl borate (2APB). Additionally, the use of bioinformatics techniques allowed us to identify putative transient receptor potential (TRP) channels, present in members of the Trypanozoon family, which would be possible candidates responsible for the SOCE described in the present work in T. equiperdum., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The repurposing or repositioning of previously-approved drugs has become an accepted strategy for the expansion of the pharmacopeia for neglected diseases. Accordingly, amiodarone, an inexpensive and extensively- used class III antiarrhythmic has been proposed as a treatment for Chagas' disease and leishmaniasis. Amiodarone has a potent trypanocidal and leishmanicidal action, mainly acting through the disruption of parasite intracellular Ca 2+ homeostasis, which is a recognized target of different drugs that have activity against trypanosomatids. Amiodarone collapses the mitochondrial electrochemical potential (Δφm) and induces the rapid alkalinization of parasite acidocalcisomes, driving a large increase in the intracellular Ca 2+ concentration. Amiodarone also inhibits oxidosqualene cyclase activity, a key enzyme in the ergosterol synthesis pathway that is essential for trypanosomatid survival. In combination, these three effects lead to parasite death. Dronedarone, a drug synthesized to minimize some of the adverse effects of amiodarone, displays trypanocidal and leishmanicidal activity through the same mechanisms, but curiously, being more potent on Leishmaniasis than its predecessor. In vitro studies suggest that other recently-synthesized benzofuran derivatives can act through the same mechanisms, and produce similar effects on different trypanosomatid species. Recently, the combination of amiodarone and itraconazole has been used successfully to treat 121 dogs naturally-infected by T. cruzi, strongly supporting the potential therapeutic use of this combination against human trypanosomatid infections., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Ca 2+ is an essential signaling messenger in all eukariotic cells, playing a pivotal role in many cellular functions as cell growth control (differentiation, fertilization and apoptosis), secretion, gene expression, enzyme regulation, among many others. This basic premise includes trypanosomatids as Trypanosoma cruzi and various species of Leishmania , the causative agents of Chagas disease and leishmaniasis respectively, where intracellular Ca 2+ concentration ([Ca 2+ ] i ) has been demonstrated to be finely regulated. Nevertheless [Ca 2+ ] i has been difficult to measure because of its very low cytoplasmic concentration (typically around 50-100 nM), when compared to the large concentration in the outside milieu (around 2 mM in blood). The development of intracellular fluorescent Ca 2+ -sensitive indicators has been of paramount importance to achieve this goal. The success was based on the synthesis of acetoximethylated derivative precursors, which allow the fluorescent molecules typically composed of many hydrophilic carboxyl groups responsible for its high affinity Ca 2+ -binding (and therefore very hydrophilic), to easily cross the plasma membrane. Once in the cell interior, unspecific esterases split the hydrophobic moiety from the fluorescent backbone structure, releasing the carboxyl groups, transforming it in turn to the acid form of the molecule, which remain trapped in the cytoplasm and regain its ability to fluoresce in a Ca 2+ -dependent manner. Among them, Fura-2 is by far the most used, because it is a ratiometric (two different wavelength excitation and one emission) Ca 2+ indicator with a Ca 2+ affinity compatible with the [Ca 2+ ] i . This protocol essentially consists in loading exponential phase parasites with Fura-2 and recording changes in [Ca 2+ ] i by mean of a double wavelength spectrofluorometer. This technique allows the acquisition of valuable information about [Ca 2+ ] i changes in real time, as a consequence of diverse stimuli or changes in conditions, as addition of drugs or different natural modulators., Competing Interests: Competing interestsThere are no conflicts of interest or competing interest., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

There is no effective cure for Chagas disease, which is caused by infection with the arthropod-borne parasite, Trypanosoma cruzi . In the search for new drugs to treat Chagas disease, potential therapeutic targets have been identified by exploiting the differences between the mechanisms involved in intracellular Ca 2+ homeostasis, both in humans and in trypanosomatids. In the trypanosomatid, intracellular Ca 2+ regulation requires the concerted action of three intracellular organelles, the endoplasmic reticulum, the single unique mitochondrion, and the acidocalcisomes. The single unique mitochondrion and the acidocalcisomes also play central roles in parasite bioenergetics. At the parasite plasma membrane, a Ca 2+ - - ATPase (PMCA) with significant differences from its human counterpart is responsible for Ca 2+ extrusion; a distinctive sphingosine-activated Ca 2+ channel controls Ca 2+ entrance to the parasite interior. Several potential anti-trypansosomatid drugs have been demonstrated to modulate one or more of these mechanisms for Ca 2+ regulation. The antiarrhythmic agent amiodarone and its derivatives have been shown to exert trypanocidal effects through the disruption of parasite Ca 2+ homeostasis. Similarly, the amiodarone-derivative dronedarone disrupts Ca 2+ homeostasis in T. cruzi epimastigotes, collapsing the mitochondrial membrane potential (ΔΨ m ), and inducing a large increase in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) from this organelle and from the acidocalcisomes in the parasite cytoplasm. The same general mechanism has been demonstrated for SQ109, a new anti-tuberculosis drug with potent trypanocidal effect. Miltefosine similarly induces a large increase in the [Ca 2+ ] i acting on the sphingosine-activated Ca 2+ channel, the mitochondrion and acidocalcisomes. These examples, in conjunction with other evidence we review herein, strongly support targeting Ca 2+ homeostasis as a strategy against Chagas disease., (Copyright © 2020 Benaim, Paniz-Mondolfi, Sordillo and Martinez-Sotillo.)

Leishmania donovani is the causative agent of visceral leishmaniasis. Annually, 500 million new cases of infection are reported mainly in poor communities, decreasing the interest of the pharmaceutical industries. Therefore, the repositioning of new drugs is an ideal strategy to fight against these parasites. SQ109, a compound in phase IIb/III of clinical trials to treat resistant Mycobacterium tuberculosis, has a potent effect against Trypanosoma cruzi, responsible for Chagas' disease, and on Leishmania mexicana, the causative agent of cutaneous and muco-cutaneous leishmaniasis. In the latter, the toxic dose against intramacrophagic amastigotes is very low (IC 50 ~ 11 nM). The proposed mechanism of action on L. mexicana involves the disruption of the parasite intracellular Ca 2+ homeostasis through the collapse of the mitochondrial electrochemical potential (ΔΨ m ). In the present work, we show a potent effect of SQ109 on L. donovani, the parasite responsible for visceral leishmaniasis, the more severe and uniquely lethal form of these infections, obtaining a toxic effect on amastigotes inside macrophages even lower to that obtained in L. mexicana (IC 50 of 7.17 ± 0.09 nM) and with a selectivity index > 800, even higher than in L. mexicana. We also demonstrated for first time that SQ109, besides collapsing ΔΨ m of the parasite, induced a very rapid damage to the parasite acidocalcisomes, essential organelles involved in the bioenergetics and many other important functions, including Ca 2+ homeostasis. Both effects of the drug on these organelles generated a dramatic increase in the intracellular Ca 2+ concentration, causing parasite death.

Trypanosoma cruzi is the causative agent of Chagas disease. The only two drugs accepted for the treatment of this infection are benznidazole and nifurtimox, which are of limited use in the predominant chronic phase. On the search for new drugs, the intracellular Ca 2+ regulation has been postulated as a possible target, due to differences found between host cells and the parasite. The mechanisms involved in the intracellular Ca 2+ regulation of T. cruzi have been partially elucidated. However, nothing is known about a putative channel responsible for the Ca 2+ entry into this parasite. In contrast, in Leishmania spp., a closely related hemoflagelate, a sphingosine-activated plasma membrane Ca 2+ channel has been recently described. The latter resembles the L-type voltage-gated Ca 2+ channel present in humans, but with distinct characteristics. This channel is one of the main targets concerning the mechanism of action of miltefosine, the unique oral drug approved against leishmaniasis. In the present work, we describe for the first time, the electrophysiological characterization of a sphingosine-activated Ca 2+ channel of T. cruzi by reconstituting plasma membrane vesicles into giant liposomes and patch clamp. This channel shares some characteristic as activation by Bay K8644 and inhibition by channel blockers such as nifedipine. However, the T. cruzi channel differs from the L-type VGCC in its activation by sphingosine and/or miltefosine. Albeit the conductance for each, Ba 2+ , Ca 2+ and Sr 2+ was similar, the parasite channel appears not to be voltage dependent. A gene that presents homology in critical amino acids with its human ortholog Ca 2+ channel was identified., (© 2019 Federation of European Biochemical Societies.)

Leishmaniasis is a parasitic disease representing an important problem of public health. Visceral leishmaniasis, resulting from infection with Leishmania donovani, causes considerable mortality and morbidity in the poorest region of the word. At present there is no current effective treatment, since the approved, drugs are expensive and are not free of undesirable side effects. Therefore, there is a need for the identification of new drugs. In this context, the parasite Ca 2+ regulatory mechanisms in which mitochondria and acidocalcisomes are involved have been postulated as important targets for several trypanocidal drugs. Thus, amiodarone and dronedarone, common human antiarrythmics, exert its known action on these parasites through the disruption of the intracellular Ca 2+ homeostasis. AMIODER is a benzofuran derivate based on the structure of amiodarone that recently demonstrates a significant effect on Trypanosoma cruzi. We now report the effect of AMIODER on Leishmania donovani demonstrating that it inhibit the growth of promastigotes and also of amastigotes inside macrophages, the clinically relevant stage of the parasite, obtaining IC 50 values significantly lower than those reported for T. cruzi. We also show that this compound disrupted Ca 2+ homeostasis in L. donovani, through its action on two organelles involved in the intracellular Ca 2+ regulation and on the bioenergetics of the parasite. AMIODER totally collapsed the electrochemical membrane potential of the unique giant mitochondrion and simultaneously induced the alkalinization of acidocalcisomes, driving together to a large increase in the intracellular Ca 2+ concentration of the parasite as the main mechanism of action of this benzofurane derivative., (Copyright © 2019. Published by Elsevier B.V.)

In the past 5-10 years, Venezuela has faced a severe economic crisis, precipitated by political instability and declining oil revenue. Public health provision has been affected particularly. In this Review, we assess the impact of Venezuela's health-care crisis on vector-borne diseases, and the spillover into neighbouring countries. Between 2000 and 2015, Venezuela witnessed a 359% increase in malaria cases, followed by a 71% increase in 2017 (411 586 cases) compared with 2016 (240 613). Neighbouring countries, such as Brazil, have reported an escalating trend of imported malaria cases from Venezuela, from 1538 in 2014 to 3129 in 2017. In Venezuela, active Chagas disease transmission has been reported, with seroprevalence in children (<10 years), estimated to be as high as 12·5% in one community tested (n=64). Dengue incidence increased by more than four times between 1990 and 2016. The estimated incidence of chikungunya during its epidemic peak is 6975 cases per 100 000 people and that of Zika virus is 2057 cases per 100 000 people. The re-emergence of many vector-borne diseases represents a public health crisis in Venezuela and has the possibility of severely undermining regional disease elimination efforts. National, regional, and global authorities must take action to address these worsening epidemics and prevent their expansion beyond Venezuelan borders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The present study evaluates in vitro the effect of two synthetic compounds of the 7-chloro-4-aryloxyquinoline series, QI (C 17 H 12 ClNO 3 ) and QII (C 18 H 15 ClN 4 O 2 S), on Leishmania donovani parasites. The results obtained demonstrate that these compounds are able to inhibit the proliferation of L. donovani promastigotes in a dose-dependent way (QI IC 50  = 13.03 ± 3.4 and QII IC 50  = 7.90 ± 0.6 μM). Likewise, these compounds significantly reduced the percentage of macrophage infection by amastigotesand the number of amastigotes within macrophage phagolysosomes, the clinical relevant phase of these parasites. Compound QI showed an IC 50 value of 0.66 ± 0.2 μM, while for derivative QII, the corresponding IC 50 was 1.02 ± 0.17 μM. Interestingly, the amastigotes were more susceptible to the drug treatment when compared to promastigotes. Furthermore, no cytotoxic effect of these compounds was observed on the macrophage cell line at the concentrations tested. The combination of these compounds with miltefosine and amphotericin B on both parasite morphotypes was evaluated. The isobolograms showed a synergistic effect for both combinations; with a Fractional Inhibitory Concentration (FIC) Index lower than 1 for promastigotes and less than 0.3 for intracellular amastigotes. The effect of QI and QII on mitochondrial membrane potential was also studied. The combination of quinolone derivatives compounds with miltefosine and amphotericin B showed 5-8-fold stronger depolarization of membrane mitochondrial potential when compared to drugs alone. The present work validates the combination of drugs as an effective alternative to potentiate the action of anti-Leishmania agents and points to the quinoline compounds studied here as possible leishmanicidal drugs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The plasma membrane Ca 2+ -ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1-18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain. We demonstrated that P14 and a synthetic peptide corresponding to residues 1037-1062 (which contains the predicted 1-18 binding motif) competed efficiently for binding to TcCaM, exhibiting similar IC 50 s of 200 nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca 2+ , as determined by native-polyacrylamide gel electrophoresis. A predicted structure obtained by molecular docking showed an interaction of the 1-18 binding motif with the Ca 2+ /CaM complex. Moreover, when the peptide was incubated with CaM and Ca 2+ , a blue shift in the tryptophan fluorescence spectrum (from 350 to 329 nm) was observed. Substitutions at W 1039 and F 1056 , strongly decreased both CaM-peptide interaction and the complex assembly. Our results demonstrated the presence of a functional 1-18 motif at the TePMCA C-terminal domain. Furthermore, on the basis of spectrofluorometric assays and the resulting structure modeled by docking we propose that the L 1042 and W 1060 residues might also participate as anchors to form a 1-4-18-22 motif., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chagas disease is a neglected tropical affection caused by the protozoan parasite Trypanosoma cruzi. There is no current effective treatment since the only two available drugs have a limited efficacy and produce side effects. Thus, investigation efforts have been directed to the identification of new drug leads. In this context, Ca 2+ regulating mechanisms have been postulated as targets for antiparasitic compounds, since they present paramount differences when compared to host cells. Amiodarone is an antiarrhythmic with demonstrated trypanocidal activity acting through the disruption of the parasite intracellular Ca 2+ homeostasis. We now report the effect of a benzofuran derivative based on the structure of amiodarone on T. cruzi. This derivative was able to inhibit the growth of epimastigotes in culture and of amastigotes inside infected cells, the clinically relevant phase. We also show that this compound, similarly to amiodarone, disrupts Ca 2+ homeostasis in T. cruzi epimastigotes, via two organelles involved in the intracellular Ca 2+ regulation and the bioenergetics of the parasite. We demonstrate that the benzofuran derivative was able to totally collapse the membrane potential of the unique giant mitochondrion of the parasite and simultaneously produced the alkalinization of the acidocalcisomes. Both effects are evidenced by a large increase in the intracellular Ca 2+ concentration of T. cruzi., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Leishmania donovani is the causing agent of visceral leishmaniasis, a common infection that affects millions of people from the most underdeveloped countries. Miltefosine is the only oral drug to treat infections caused by L. donovani Nevertheless, its mechanism of action is not well understood. While miltefosine inhibits the synthesis of phosphatidylcholine and also affects the parasite mitochondrion, inhibiting the cytochrome c oxidase, it is to be expected that this potent drug also produces its effect through other targets. In this context, it has been reported that the disruption of the intracellular Ca 2+ homeostasis represents an important object for the action of drugs in trypanosomatids. Recently, we have described a plasma membrane Ca 2+ channel in Leishmania mexicana , which is similar to the L-type voltage-gated Ca 2+ channel (VGCC) present in humans. Remarkably, the parasite Ca 2+ channel is activated by sphingosine, while the L-type VGCC is not affected by this sphingolipid. In the present work we demonstrated that, similarly to sphingosine, miltefosine is able to activate the plasma membrane Ca 2+ channel from L. donovani Interestingly, nifedipine, the classical antagonist of the human channel, was not able to fully block the parasite plasma membrane Ca 2+ channel, indicating that the mechanism of interaction is not identical to that of sphingosine. In this work we also show that miltefosine is able to strongly affect the acidocalcisomes from L. donovani , inducing the rapid alkalinization of these important organelles. In conclusion, we demonstrate two new mechanisms of action of miltefosine in L. donovani , both related to disruption of parasite Ca 2+ homeostasis., (Copyright © 2017 American Society for Microbiology.)

Trypanosoma equiperdum belongs to the subgenus Trypanozoon, which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca 2+ regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca 2+ homeostasis. Therefore, the plasma membrane Ca 2+ -ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca 2+ translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1-18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1-18 motif., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We report that the tuberculosis drug SQ109 [N-adamantan-2-yl-N'-((E)-3,7-dimethyl-octa-2,6-dienyl)-ethane-1,2-diamine] has potent activity against the intracellular amastigote form of Leishmania mexicana (50% inhibitory concentration [IC50], ∼11 nM), with a good selectivity index (>500). It is also active against promastigotes (IC50, ∼500 nM) and acts as a protonophore uncoupler, in addition to disrupting Ca(2+) homeostasis by releasing organelle Ca(2+) into the cytoplasm, and as such, it is an interesting new leishmaniasis drug hit candidate., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The increase in the intracellular Ca(2+) concentration ([Ca(2+)]i) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca(2+)]i by inhibiting the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca(2+) pump. The results showed that addition of sphingosine produced a release of Ca(2+) from the endoplasmic reticulum followed by a Ca(2+) entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca(2+)]I in mammalian cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Neglected tropical diseases represent a major sanitary problem and a huge economic burden to endemic countries, and are currently expanding to non-endemic countries owing to migration currents. Though long abandoned in the past, recent research on novel therapeutics has already started to show results. Drug repositioning is one of the prominent, more successful strategies to approach the development of new treatments for these diseases. Here we present an overview on the limitations of the current available medications to treat African trypanosomiasis, Chagas disease and Leishmaniasis, along with a review on drug candidates presently undergoing clinical trials and drug candidates identified through drug repositioning initiatives.

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca(2+), but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca(2+). This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca(2+), absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site ((645)R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q(660)) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells., (© 2015 Authors; published by Portland Press Limited.)

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.

We tested the antituberculosis drug SQ109, which is currently in advanced clinical trials for the treatment of drug-susceptible and drug-resistant tuberculosis, for its in vitro activity against the trypanosomatid parasite Trypanosoma cruzi, the causative agent of Chagas disease. SQ109 was found to be a potent inhibitor of the trypomastigote form of the parasite, with a 50% inhibitory concentration (IC50) for cell killing of 50 ± 8 nM, but it had little effect (50% effective concentration [EC50], ∼80 μM) in a red blood cell hemolysis assay. It also inhibited extracellular epimastigotes (IC50, 4.6 ± 1 μM) and the clinically relevant intracellular amastigotes (IC50, ∼0.5 to 1 μM), with a selectivity index of ∼10 to 20. SQ109 caused major ultrastructural changes in all three life cycle forms, as observed by light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). It rapidly collapsed the inner mitochondrial membrane potential (Δψm) in succinate-energized mitochondria, acting in the same manner as the uncoupler FCCP [carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone], and it caused the alkalinization of internal acidic compartments, effects that are likely to make major contributions to its mechanism of action. The compound also had activity against squalene synthase, binding to its active site; it inhibited sterol side-chain reduction and, in the amastigote assay, acted synergistically with the antifungal drug posaconazole, with a fractional inhibitory concentration index (FICI) of 0.48, but these effects are unlikely to account for the rapid effects seen on cell morphology and cell killing. SQ109 thus most likely acts, at least in part, by collapsing Δψ/ΔpH, one of the major mechanisms demonstrated previously for its action against Mycobacterium tuberculosis. Overall, the results suggest that SQ109, which is currently in advanced clinical trials for the treatment of drug-susceptible and drug-resistant tuberculosis, may also have potential as a drug lead against Chagas disease., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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.