Quinoline derivatives: Synthesis, leishmanicidal activity and involvement of mitochondrial oxidative stress as mechanism of action.

Leishmaniasis comprise a spectrum of diseases caused by protozoa parasites from the genus Leishmania, affecting millions of people worldwide, mainly in subtropical countries. Most antileishmanial drugs are highly toxic, present resistance issues or require long-term treatment. Consequently, new drugs are urgently needed. Quinoline-containing compounds have displayed an impressive array of biological properties over the years, including antileishmanial activity. In the present study, we report the synthesis and evaluation of novel quinoline derivatives (QuinDer) against Leishmania species and cytotoxic effect on mammalian cells. The ROS production and mitochondrial membrane potential analyses were also studied. The compound QuinDer1 showed activity on L. amazonensis and L. braziliensis promastigotes and this compound exhibited a strong inhibition of the proliferation of L. amazonensis amastigotes at nM concentration (IC50 of 0.0911 µM), being 139 times more active than miltefosine (IC50 of 12.7 µM), used as reference drug. This compound presents low cytotoxicity toward murine macrophages and human erythrocytes. In addition, promastigotes of L. amazonensis treated with the compound QuinDer1 present high generation of ROS levels with low alterations in mitochondrial membrane potential and maintenance of parasite membrane integrity. No substantial NO production in infected-macrophages treated with this compound was detected. These results suggest that the compound QuinDer 1 is a potent and selective antileishmanial agent by mitochondrial oxidative stress.

Anti-tuberculosis evaluation and conformational study of N-acylhydrazones containing the thiophene nucleus.

A series of N-acylhydrazonyl-thienyl derivatives (compounds 2 and 3), mainly of the type 2-(aryl-CH=N-NHCOCH2 )-thiene (2: aryl = substituted-phenyl; 3: aryl = heteroaryl) were evaluated against Mycobacterium tuberculosis. Particularly active compound was 3 (heteroaryl = 5-nitrothien-2-yl or 5-nitrofuran-2-yl) with MIC values of 8.5 and 9.0 µM, respectively. Moderately active compounds were compound 3 (heteroaryl = pyridin-2-yl) and compound 2 containing aryl = 2- or 4-hydroxyphenyl groups, with MIC values between 170 and 408 µM. Compound 2 containing OMe, H, F, Cl, Br, CN, and NO2 substituents and compound 3 (heteroaryl = furan-2-yl, thien-2-yl, pyrrol-2-yl, imidazol-2-yl, pyridin-3-yl, and pyridin-4-yl) were all inactive. Clearly, there is no correlation of activity with the electronic effects of the substituents. The activities suggest different modes of biological action of the compounds having nitro-heteroaryl groups, on the one hand, and the 2-hydroxyphenyl or pyridin-2-yl substituents, on the other hand. Compounds having 2- or 4-hydroxyphenyl, 2-hydroxy-5-nitrophenyl, or 4-hydroxy-3-chlorophenyl were less cytotoxic than ethambutol. It is important to notice that compound 3 (aryl = 5-NO2 -furan-2-yl) exhibited a promising therapeutic index (TI = 1093.90), with a value 4.4 less than that of ethambutol. Compounds 2 and 3 exist in DMSO or MeOD solutions as mixtures of EC(O)N /EC=N and ZC(O)N /EC=N conformers.

Molecular characterization of the hexose transporter gene in benznidazole resistant and susceptible populations of Trypanosoma cruzi.

BACKGROUND: Hexose transporters (HT) are membrane proteins involved in the uptake of energy-supplying glucose and other hexoses into the cell. Previous studies employing the Differential Display technique have shown that the transcription level of the HT gene from T. cruzi (TcrHT) is higher in an in vitro-induced benznidazole (BZ)-resistant population of the parasite (17 LER) than in its susceptible counterpart (17 WTS). METHODS: In the present study, TcrHT has been characterized in populations and strains of T. cruzi that are resistant or susceptible to BZ. We investigated the copy number and chromosomal location of the gene, the levels of TcrHT mRNA and of TcrHT activity, and the phylogenetic relationship between TcrHT and HTs from other organisms. RESULTS: In silico analyses revealed that 15 sequences of the TcrHT gene are present in the T. cruzi genome, considering both CL Brener haplotypes. Southern blot analyses confirmed that the gene is present as a multicopy tandem array and indicated a nucleotide sequence polymorphism associated to T. cruzi group I or II. Karyotype analyses revealed that TcrHT is located in two chromosomal bands varying in size from 1.85 to 2.6 Mb depending on the strain of T. cruzi. The sequence of amino acids in the HT from T. cruzi is closely related to the HT sequences of Leishmania species according to phylogenetic analysis. Northern blot and quantitative real-time reverse transcriptase polymerase chain reaction analyses revealed that TcrHT transcripts are 2.6-fold higher in the resistant 17 LER population than in the susceptible 17 WTS. Interestingly, the hexose transporter activity was 40% lower in the 17 LER population than in all other T. cruzi samples analyzed. This phenotype was detected only in the in vitro-induced BZ resistant population, but not in the in vivo-selected or naturally BZ resistant T. cruzi samples. Sequencing analysis revealed that the amino acid sequences of the TcrHT from 17WTS and 17LER populations are identical. This result suggests that the difference in glucose transport between 17WTS and 17LER populations is not due to point mutations, but probably due to lower protein expression level. CONCLUSION: The BZ resistant population 17 LER presents a decrease in glucose uptake in response to drug pressure.

Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps.

Neutrophils are short-lived leukocytes that die by apoptosis, necrosis, and NETosis. Upon death by NETosis, neutrophils release fibrous traps of DNA, histones, and granule proteins named neutrophil extracellular traps (NETs), which can kill bacteria and fungi. Inoculation of the protozoan Leishmania into the mammalian skin causes local inflammation with neutrophil recruitment. Here, we investigated the release of NETs by human neutrophils upon their interaction with Leishmania parasites and NETs' ability to kill this protozoan. The NET constituents DNA, elastase, and histones were detected in traps associated to promastigotes by immunofluorescence. Electron microscopy revealed that Leishmania was ensnared by NETs released by neutrophils. Moreover, Leishmania and its surface lipophosphoglycan induced NET release by neutrophils in a parasite number- and dose-dependent manner. Disruption of NETs by DNase treatment during Leishmania-neutrophil interaction increased parasite survival, evidencing NETs' leishmanicidal effect. Leishmania killing was also elicited by NET-rich supernatants from phorbol 12-myristate 13-acetate-activated neutrophils. Immunoneutralization of histone during Leishmania-neutrophil interaction partially reverted Leishmania killing, and purified histone killed the parasites. Meshes composed of DNA and elastase were evidenced in biopsies of human cutaneous leishmaniasis. NET is an innate response that might contribute to diminish parasite burden in the Leishmania inoculation site.

Synthesis and antimalarial activity of semicarbazone and thiosemicarbazone derivatives.

Seventeen semicarbazone and thiosemicarbazone derivatives were prepared and tested in vitro against a chloroquine resistant strain of Plasmodium falciparum (W2) to evaluate their antiplasmodial potential. Three thiosemicarbazones were found to be active against the parasite and non-toxic to human peripheral blood mononuclear cells (PBMC). Among these, compound 5b presented the lowest IC50 value against P. falciparum (7.2 microM) and was the least toxic in the PBMC proliferation assay (IC50=73.5 microM). It was selected for in vivo tests on mice infected with Plasmodium berghei (strain NK-65). The thiosemicarbazone 5b was able to reduce the parasitaemia by 61% at 20 mg/kg on day 7 after infection without any sign of toxicity to the animals. In comparison, the standard drug chloroquine at 15 mg/kg showed a reduction around 95%. These in vitro and in vivo results make 5b an interesting lead for further development.

Antimalarial activity of 4-(5-trifluoromethyl-1H-pyrazol-1-yl)-chloroquine analogues.

The antimalarial activity of chloroquine-pyrazole analogues, synthesized from the reaction of 1,1,1-trifluoro-4-methoxy-3-alken-2-ones with 4-hydrazino-7-chloroquinoline, has been evaluated in vitro against a chloroquine resistant Plasmodium falciparum clone. Parasite growth in the presence of the test drugs was measured by incorporation of [(3)H]hypoxanthine in comparison to controls with no drugs. All but one of the eight (4,5-dihydropyrazol-1-yl) chloroquine 2 derivatives tested showed a significant activity in vitro, thus, are a promising new class of antimalarials. The three most active ones were also tested in vivo against Plasmodium berghei in mice. However, the (pyrazol-1-yl) chloroquine 3 derivatives were mostly inactive, suggesting that the aromatic functionality of the pyrazole ring was critical.

Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis.

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.

Leishmania tropica: intraspecific polymorphisms in lipophosphoglycan correlate with transmission by different Phlebotomus species.

Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes which has been shown to be critical for parasite-sand fly vector interactions. To provide additional evidence for its importance in transmission, the LPGs from three Leishmania tropica strains that differ in their capability to infect sand flies, were biochemically characterized. One of these strains, ISER/IL/98/LRC-L747, was isolated from a Phlebotomus sergenti female collected in the Judean Desert close to Jerusalem. The other strains originated from a different focus in the Galilee region of northern Israel. One was isolated from a patient (MHOM/IL/02/Ofri-LRC-L863) and the other from a naturally infected Phlebotomus arabicus female (IARA/IL/00/Amnunfly1-LRC-L810). The LPG structures of the isolates from the Galilee (L863 and L810) were similar to each other, but differed in the LPG repeat units from the Judean Desert isolate (L747). The terminal sugar in the side chains of the repeat units of LPG purified from L863 and L810 was beta-galactose and was not capped with glucose, unlike L747 and a previously characterized L. tropica strain from Iraq (L36). Since L810 was isolated from P. arabicus and L747 from P. sergenti, variations in the structure of their LPGs may explain their capacity to infect different sand fly species.

Interpopulation variability among Panstrongylus megistus (Hemiptera: Reduviidae) from Brazil.

Comparisons were made among Panstrongylus megistus (Burmeister) from three areas of Brazil (Bahia, Minas Gerais, and Santa Catarina), where populations differ with regard to their degree of association with human dwellings. The following characters were studied: morphology of the eggs and the male genitalia; morphometry of the head and thorax; isoenzyme profile; enzymatic activity of the alpha-glycerophosphate dehydrogenase (alpha-GPDH); and cytogenetics. In general, differences were observed in the weight, diameter, and length of the egg among Bahia, Minas Gerais, and Santa Catarina populations. Differences were not observed in the architecture of the egg exochorion. The size of the median process of the pygophore of the male genitalia of individuals from Bahia differed from the other two populations. The Minas Gerais population presented the largest number of denticles in the endosome process. The morphometry of the head and thorax differentiated Santa Catarina from the Bahia and Minas Gerais populations. Phosphoglucomutase (EC 5.4.2.2., PGM) was the only enzyme out of 11 that showed polymorphism; the population from Minas Gerais was most polymorphic, whereas the population from Santa Catarina was monomorphic. Study of the alpha-GPDH activity and cytogenetics did not reveal differences among the three populations. Analysis of all the characters studied together with information on these three populations from previous publications allowed a phenogram to be constructed. Two distinct groups were evident, one represented by Santa Catarina and the other by Bahia and Minas Gerais. Considering the greater variability of the Minas Gerais and Bahia populations, we propose the inclusion of these states within the area of origin of P. megistus.

Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae): a review

Lutzomyia longipalpis is the most important vector of AmericanVisceral Leishmaniasis (AVL) due to Leishmania chagasi in the New World. Despite its importance, AVL, a disease primarily of rural areas, has increased its prevalence and became urbanized in some large cities in Brazil and other countries in Latin America. Although the disease is treatable, other control measures include elimination of infected dogs and the use of insecticides to kill the sand flies. A better understanding of vector biology could also account as one more tool for AVL control. A wide variety of papers about L. longipalpis have been published in the recent past years. This review summarizes our current information of this particular sand fly regarding its importance, biology, morphology, pheromones genetics, saliva, gut physiology and parasite interactions

Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae): a review.

Lutzomyia longipalpis is the most important vector of AmericanVisceral Leishmaniasis (AVL) due to Leishmania chagasi in the New World. Despite its importance, AVL, a disease primarily of rural areas, has increased its prevalence and became urbanized in some large cities in Brazil and other countries in Latin America. Although the disease is treatable, other control measures include elimination of infected dogs and the use of insecticides to kill the sand flies. A better understanding of vector biology could also account as one more tool for AVL control. A wide variety of papers about L. longipalpis have been published in the recent past years. This review summarizes our current information of this particular sand fly regarding its importance, biology, morphology, pheromones genetics, saliva, gut physiology and parasite interactions.

Leishmania chagasi: lipophosphoglycan characterization and binding to the midgut of the sand fly vector Lutzomyia longipalpis.

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterized by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This glycoconjugate is polymorphic among species with variations in sugars that branch off the conserved Gal(beta 1,4)Man(alpha 1)-PO(4) backbone of repeat units and the oligosaccharide cap. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly. These adaptations were explored in the context of the structure and function of LPG for the first time on a New World species, Leishmania chagasi. The distinguishing feature of LPG of procyclic L. chagasi consisted of beta 1,3-glucose residues that branch off the disaccharide-phosphate repeat units and also are present in the cap. Importantly, metacyclic L. chagasi significantly down-regulate the glucose substitutions in the LPG. The significance of these modifications was demonstrated in the interaction of L. chagasi with its vector Lutzomyia longipalpis. In contrast to procyclic parasites and procyclic LPG, metacyclic parasites and metacyclic LPG were unable to bind to the insect midgut. These results are consistent with the proposal that a New World Leishmania species, similar to Old World species, adapts the expression of terminally exposed sugars of its LPG to mediate parasite-sand fly interactions.

Experimental evidence for a demographic cline in Panstrongylus megistus populations

The population biology of three populations of Panstrongylus megistus was compared to determine possible influence on the behaviour and epidemiological importance of this species. The results demonstrated differences in terms of egg eclosion time, nymphal mortality and development rates, and feeding and defaecation rates. These differences appeared to follow a geographical cline, primarily reflecting different degrees of adaptation to domestic habitats