RESUMO
In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation.
Assuntos
Leishmania mexicana , Proteínas de Protozoários , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Leishmania mexicana/genética , Leishmania mexicana/enzimologia , Leishmania mexicana/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , ImunoprecipitaçãoRESUMO
Endogenous nucleotides produced by various group of cells under inflammatory conditions act as potential danger signals in vivo. Extracellularly released nucleotides such as ATP are rapidly hydrolyzed to adenosine by the coordinated ectonucleotidase activities of CD39 and CD73. Leishmania is an obligate intracellular parasite of macrophages and capable of modulating host immune response in order to survive and multiply within host cells. In this study, the activity of CD73 induced by Leishmania amazonensis in infected macrophages has been investigated and correlated with parasite survival and infection in vitro. For this, the expression of CD39 and CD73, by flow cytometry, in murine peritoneal macrophages infected with metacyclic promastigotes of L. amazonensis has been analyzed. Our results showed that L. amazonensis-infected macrophages, unlike LPS-treated macrophages, increased CD73 expression. It was also noted that when CD73 enzymatic activity was blocked by α, ß-methyleneadenosine 5'-diphosphate sodium salt (APCP), macrophage parasitism was significantly decreased. Interestingly, these effects were not associated with the production of TNF-α, IL-10, or nitric oxide (NO). Together, these data demonstrate that L. amazonensis induces a regulatory phenotype in macrophages, which by activating the CD39/CD73 pathway allows parasite survival through the action of immunomodulatory adenosine receptors.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina Trifosfatases/metabolismo , Leishmania mexicana/enzimologia , Macrófagos Peritoneais/parasitologia , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Leishmaniasis is a disease caused by parasites of the Leishmania genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people are at risk. The use of the actual treatments is limited due to toxicity concerns and the apparition of resistance strains. Therefore, there is an urgent necessity to find new drugs for the treatment of this disease. In this context, enzymes from the polyamine biosynthesis pathway, such as arginase, have been considered a good target. In the present work, a chemical library of benzimidazole derivatives was studied performing computational, enzyme kinetics, biological activity, and cytotoxic effect characterization, as well as in silico ADME-Tox predictions, to find new inhibitors for arginase from Leishmania mexicana (LmARG). The results show that the two most potent inhibitors (compounds 1 and 2) have an I50 values of 52 µM and 82 µM, respectively. Moreover, assays with human arginase 1 (HsARG) show that both compounds are selective for LmARG. According to molecular dynamics simulation studies these inhibitors interact with important residues for enzyme catalysis. Biological activity assays demonstrate that both compounds have activity against promastigote and amastigote, and low cytotoxic effect in murine macrophages. Finally, in silico prediction of their ADME-Tox properties suggest that these inhibitors support the characteristics to be considered drug candidates. Altogether, the results reported in our study suggest that the benzimidazole derivatives are an excellent starting point for design new drugs against leishmanisis.
Assuntos
Antiprotozoários/farmacologia , Arginase/antagonistas & inibidores , Benzimidazóis/farmacologia , Leishmania mexicana/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/química , Arginase/metabolismo , Benzimidazóis/química , Linhagem Celular , Descoberta de Drogas , Humanos , Leishmania mexicana/enzimologia , Leishmania mexicana/fisiologia , Leishmaniose Cutânea/tratamento farmacológico , Camundongos , Modelos Moleculares , Proteínas de Protozoários/metabolismoRESUMO
The compound 3-bromopyruvate (3-BrPA) is well-known and studies from several researchers have demonstrated its involvement in tumorigenesis. It is an analogue of pyruvic acid that inhibits ATP synthesis by inhibiting enzymes from the glycolytic pathway and oxidative phosphorylation. In this work, we investigated the effect of 3-BrPA on energy metabolism of L. amazonensis. In order to verify the effect of 3-BrPA on L. amazonensis glycolysis, we measured the activity level of three glycolytic enzymes located at different points of the pathway: (i) glucose kinases, step 1, (ii) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), step 6, and (iii) enolase, step 9. 3-BrPA, in a dose-dependent manner, significantly reduced the activity levels of all the enzymes. In addition, 3-BrPA treatment led to a reduction in the levels of phosphofruto-1-kinase (PFK) protein, suggesting that the mode of action of 3-BrPA involves the downregulation of some glycolytic enzymes. Measurement of ATP levels in promastigotes of L. amazonensis showed a significant reduction in ATP generation. The O2 consumption was also significantly inhibited in promastigotes, confirming the energy depletion effect of 3-BrPA. When 3-BrPA was added to the cells at the beginning of growth cycle, it significantly inhibited L. amazonensis proliferation in a dose-dependent manner. Furthermore, the ability to infect macrophages was reduced by approximately 50% when promastigotes were treated with 3-BrPA. Taken together, these studies corroborate with previous reports which suggest 3-BrPA as a potential drug against pathogenic microorganisms that are reliant on glucose catabolism for ATP supply.
Assuntos
Leishmania mexicana/efeitos dos fármacos , Leishmaniose Tegumentar Difusa/parasitologia , Piruvatos/farmacologia , Animais , Western Blotting , Brasil , Cricetinae , Humanos , Leishmania mexicana/enzimologia , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/metabolismo , Macrófagos/parasitologia , Camundongos , Consumo de Oxigênio/efeitos dos fármacos , Fosfopiruvato Hidratase/metabolismo , Células RAW 264.7RESUMO
Leishmania is an obligate intracellular parasite that primarily inhabits macrophages. The destruction of the parasite in the host cell is a fundamental mechanism for infection control. In addition, inhibition of the leishmanicidal activity of macrophages seems to be related to the ability of some species to inhibit the production of nitric oxide (NO) by depleting arginine. Some species of Leishmania have the ability to produce NO from a constitutive nitric oxide synthase-like enzyme (cNOS-like). However, the localization of cNOS-like in Leishmania has not been described before. As such, this study was designed to locate cNOS-like enzyme and NO production in promastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. NO production was initially quantified by flow cytometry, which indicated a significant difference in NO production between L. (L.) amazonensis (GMFC = 92.17 +/- 4.6) and L. (V.) braziliensis (GMFC = 18.89 +/- 2.29) (P < 0.05). Analysis of cNOS expression by immunoblotting showed more expression in L. (L.) amazonensis versus L. (V.) braziliensis. Subsequently, cNOS-like immunolabeling was observed in promastigotes in regions near vesicles, the flagellar pocket and mitochondria, and small clusters of particles appeared to be fusing with vesicles suggestive of glycosomes, peroxisome-like-organelles that compartmentalize the glycolytic pathway in trypanosomatid parasites. In addition, confocal microscopy analysis demonstrated colocalization of cNOS-like and GAPDH, a specific marker for glycosomes. Thus, L. (L.) amazonensis produces greater amounts of NO than L. (V.) braziliensis, and both species present the cNOS-like enzyme inside glycosomes.
Assuntos
Leishmania braziliensis/enzimologia , Leishmania mexicana/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/biossíntese , Proteínas de Protozoários/metabolismo , Leishmaniose Cutânea/metabolismo , Leishmaniose Mucocutânea/metabolismo , Especificidade da EspécieRESUMO
Leishmania mexicana is an obligate intracellular protozoan parasite that causes the cutaneous form of leishmaniasis affecting South America and Mexico. The cysteine protease LmCPB is essential for the virulence of the parasite and therefore, it is an appealing target for antiparasitic therapy. A library of nitrile-based cysteine protease inhibitors was screened against LmCPB to develop a treatment of cutaneous leishmaniasis. Several compounds are sufficiently high-affinity LmCPB inhibitors to serve both as starting points for drug discovery projects and as probes for target validation. A 1.4 Å X ray crystal structure, the first to be reported for LmCPB, was determined for the complex of this enzyme covalently bound to an azadipeptide nitrile ligand. Mapping the structure-activity relationships for LmCPB inhibition revealed superadditive effects for two pairs of structural transformations. Therefore, this work advances our understanding of azadipeptidyl and dipeptidyl nitrile structure-activity relationships for LmCPB structure-based inhibitor design. We also tested the same series of inhibitors on related cysteine proteases cathepsin L and Trypanosoma cruzi cruzain. The modulation of these mammalian and protozoan proteases represents a new framework for targeting papain-like cysteine proteases.
Assuntos
Compostos Aza/farmacologia , Catepsina B/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Leishmania mexicana/efeitos dos fármacos , Tripanossomicidas/farmacologia , Compostos Aza/síntese química , Compostos Aza/química , Catepsina B/metabolismo , Cristalografia por Raios X , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Dipeptídeos/síntese química , Dipeptídeos/química , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Leishmania mexicana/enzimologia , Simulação de Dinâmica Molecular , Estrutura Molecular , Nitrilas/síntese química , Nitrilas/química , Nitrilas/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/químicaRESUMO
Leishmania parasites are of great relevance to public health because they are the causative agents of various long-term and health-threatening diseases in humans. Dependent on the manifestation, drugs either require difficult and lengthy administration, are toxic, expensive, not very effective or have lost efficacy due to the resistance developed by these pathogens against clinical treatments. The intermediary metabolism of Leishmania parasites is characterized by several unusual features, among which whether the Krebs cycle operates in a cyclic and/or in a non-cyclic mode is included. Our survey of the genomes of Leishmania species and monoxenous parasites such as those of the genera Crithidia and Leptomonas (http://www.tritrypdb.org) revealed that two genes encoding putative isocitrate dehydrogenases (IDHs) -with distantly related sequences- are strictly conserved among these parasites. Thus, in this study, we aimed to functionally characterize the two leishmanial IDH isoenzymes, for which we selected the genes LmxM10.0290 (Lmex_IDH-90) and LmxM32.2550 (Lmex_IDH-50) from L. mexicana. Phylogenetic analysis showed that Lmex_IDH-50 clustered with members of Subfamily I, which contains mainly archaeal and bacterial IDHs, and that Lmex_IDH-90 was a close relative of eukaryotic enzymes comprised within Subfamily II IDHs. 3-D homology modeling predicted that both IDHs exhibited the typical folding motifs recognized as canonical for prokaryotic and eukaryotic counterparts, respectively. Both IDH isoforms displayed dual subcellular localization, in the cytosol and the mitochondrion. Kinetic studies showed that Lmex_IDH-50 exclusively catalyzed the reduction of NAD+, while Lmex_IDH-90 solely used NADP+ as coenzyme. Besides, Lmex_IDH-50 differed from Lmex_IDH-90 by exhibiting a nearly 20-fold lower apparent Km value towards isocitrate (2.0 µM vs 43 µM). Our findings showed, for the first time, that the genus Leishmania differentiates not only from other trypanosomatids such as Trypanosoma cruzi and Trypanosoma brucei, but also from most living organisms, by exhibiting two functional homo-dimeric IDHs, highly specific towards NAD+ and NADP+, respectively. It is tempting to argue that any or both types of IDHs might be directly or indirectly linked to the Krebs cycle and/or to the de novo synthesis of glutamate. Our results about the biochemical and structural features of leishmanial IDHs show the relevance of deepening our knowledge of the metabolic processes in these pathogenic parasites to potentially identify new therapeutic targets.
Assuntos
Clonagem Molecular , Expressão Gênica , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Leishmania mexicana/enzimologia , Leishmania mexicana/genética , Sequência de Aminoácidos , Ativação Enzimática , Humanos , Isocitrato Desidrogenase/química , Isoenzimas , Cinética , Leishmania mexicana/classificação , Leishmaniose Cutânea/parasitologia , NAD/metabolismo , NADP/metabolismo , Filogenia , Transporte Proteico , Análise de Sequência de DNA , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. A series of 26 new compounds were designed, synthesized, and tested against the recombinant cruzain (Cz) to map its S1/S1´ subsites. The same series was evaluated on a panel of four human cysteine proteases (CatB, CatK, CatL, CatS) and Leishmania mexicana CPB, which is a potential target for the treatment of cutaneous leishmaniasis. The synthesized compounds are dipeptidyl nitriles designed based on the most promising combinations of different moieties in P1 (ten), P2 (six), and P3 (four different building blocks). Eight compounds exhibited a Ki smaller than 20.0 nM for Cz, whereas three compounds met these criteria for LmCPB. Three inhibitors had an EC50 value of ca. 4.0 µM, thus being equipotent to benznidazole according to the antitrypanosomal effects. Our mapping approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cysteine proteases.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Leishmania mexicana/enzimologia , Nitrilas/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Cisteína Endopeptidases , Cisteína Proteases/metabolismo , HumanosRESUMO
BACKGROUND: Leishmaniosis is a neglected tropical disease and glyceraldehyde 3- phosphate dehydrogenase (GAPDH) is a key enzyme in the design of new drugs to fight this disease. OBJECTIVE: The present study aimed to evaluate potential inhibitors of GAPDH enzyme found in Leishmania mexicana (L. mexicana). METHODS: A search for novel antileishmanial molecules was carried out based on similarities from the pharmacophoric point of view related to the binding site of the crystallographic enzyme using the ZINCPharmer server. The molecules selected in this screening were subjected to molecular docking and molecular dynamics simulations. RESULTS: Consensual analysis of the docking energy values was performed, resulting in the selection of ten compounds. These ligand-receptor complexes were visually inspected in order to analyze the main interactions and subjected to toxicophoric evaluation, culminating in the selection of three compounds, which were subsequently submitted to molecular dynamics simulations. The docking results showed that the selected compounds interacted with GAPDH from L. mexicana, especially by hydrogen bonds with Cys166, Arg249, His194, Thr167, and Thr226. From the results obtained from molecular dynamics, it was observed that one of the loop regions, corresponding to the residues 195-222, can be related to the fitting of the substrate at the binding site, assisting in the positioning and the molecular recognition via residues responsible for the catalytic activity. CONCLUSION: The use of molecular modeling techniques enabled the identification of promising compounds as inhibitors of the GAPDH enzyme from L. mexicana, and the results obtained here can serve as a starting point to design new and more effective compounds than those currently available.
Assuntos
Antipruriginosos/síntese química , Antipruriginosos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Leishmania mexicana/enzimologia , Desenho de Fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Software , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Leishmanicidal drugs have many side effects, and drug resistance to all of them has been documented. Therefore, the development of new drugs and the identification of novel therapeutic targets are urgently needed. Leishmania mexicana trypanothione reductase (LmTR), a NADPH-dependent flavoprotein oxidoreductase important to thiol metabolism, is essential for parasite viability. Its absence in the mammalian host makes this enzyme an attractive target for the development of new anti-Leishmania drugs. Herein, a tridimensional model of LmTR was constructed and the molecular docking of 20 molecules from a ZINC database was performed. Five compounds (ZINC04684558, ZINC09642432, ZINC12151998, ZINC14970552, and ZINC11841871) were selected (docking scores -10.27 kcal/mol to -5.29 kcal/mol and structurally different) and evaluated against recombinant LmTR (rLmTR) and L. mexicana promastigote. Additionally, molecular dynamics simulation of LmTR-selected compound complexes was achieved. The five selected compounds inhibited rLmTR activity in the range of 32.9% to 40.1%. The binding of selected compounds to LmTR involving different hydrogen bonds with distinct residues of the molecule monomers A and B is described. Compound ZINC12151998 (docking score -10.27 kcal/mol) inhibited 32.9% the enzyme activity (100 µM) and showed the highest leishmanicidal activity (IC50 = 58 µM) of all the selected compounds. It was more active than glucantime, and although its half-maximal cytotoxicity concentration (CC50 = 53 µM) was higher than that of the other four compounds, it was less cytotoxic than amphotericin B. Therefore, compound ZINC12151998 provides a promising starting point for a hit-to-lead process in our search for new anti-Leishmania drugs that are more potent and less cytotoxic.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/enzimologia , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/química , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Relação Dose-Resposta a Droga , Ligação de Hidrogênio , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Farmacocinética , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
Leishmania mexicana is one of the causative agents of cutaneous leishmaniasis in humans. There is an urgent need to identify new drug targets to combat the disease. Cysteine peptidases play crucial role in pathogenicity and virulence in Leishmania spp. and are promising targets for developing new anti-leishmanial drugs. Genetic drug target validation has been performed on a number of cysteine peptidases, but others have yet to be characterized. We targeted 16 L. mexicana cysteine peptidases for gene deletion and tagging using CRISPR-Cas9 in order to identify essential genes and ascertain their cellular localization. Our analysis indicates that two clan CA, family C2 calpains (LmCAL27.1, LmCAL31.6) and clan CD, family C11 PNT1 are essential for survival in the promastigote stage. The other peptidases analysed, namely calpains LmCAL4.1, LmCAL25.1, and members of clan CA C51, C78, C85 and clan CP C97 were found to be non-essential. We generated a gene deletion mutant (Δpnt1) which was severely compromised in its cell growth and a conditional gene deletion mutant of PNT1 (Δpnt1: PNT1flox/Δ pnt1:HYG [SSU DiCRE]). PNT1 localizes to distinct foci on the flagellum and on the surface of the parasite. The conditional gene deletion of PNT1 induced blebs and pits on the cell surface and eventual cell death. Over-expression of PNT1, but not an active site mutant PNT1C134A, was lethal, suggesting that active PNT1 peptidase is required for parasite survival. Overall, our data suggests that PNT1 is an essential gene and one of a number of cysteine peptidases that are potential drug targets in Leishmania.
Assuntos
Cisteína Endopeptidases/genética , Cisteína Endopeptidases/fisiologia , Leishmania mexicana/enzimologia , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Deleção de Genes , Genes Essenciais , Humanos , Leishmania mexicana/genética , Leishmania mexicana/patogenicidade , Virulência/genéticaRESUMO
Triose-phosphate isomerase (TIM) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Two catalytic mechanisms have been proposed based on two reaction-intermediate analogues, 2-phosphoglycolate (2PG) and phosphoglycolohydroxamate (PGH), that have been used as mimics of the cis-enediol(ate) intermediate in several studies of TIM. The protonation states that are critical for the mechanistic interpretation of these structures are generally not visible in the X-ray structures. To resolve these questions, it is necessary to determine the hydrogen positions using neutron crystallography. Neutron crystallography requires large crystals and benefits from replacing all hydrogens with deuterium. Leishmania mexicana triose-phosphate isomerase was therefore perdeuterated and large crystals with 2PG and PGH were produced. Neutron diffraction data collected from two crystals with different volumes highlighted the importance of crystal volume, as smaller crystals required longer exposures and resulted in overall worse statistics.
Assuntos
Deutério/química , Leishmania mexicana/enzimologia , Proteínas Mutantes/química , Difração de Nêutrons , Triose-Fosfato Isomerase/química , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de PoliacrilamidaRESUMO
Cyclooxygenase-2 (COX-2) is an enzyme responsible of prostaglandins production, such as prostaglandin E2 (PGE2), an immune response modulator that regulates the immune system to inhibit Th1 and to promote Th2 cytokines production. Many parasites modulate their host immune response through PGE2 effects; however, in parasites, only one protein with COX activity has been described, the α-actinin of Entamoeba histolytica. Prostanoids production has been reported in some species of Leishmania but not the enzymes responsible of their production. To identify the protein responsible for COX activity in Leishmania mexicana, we examined total extracts of promastigotes and samples with COX activity were subjected to ion exchange column purification and precipitation with ammonium sulphate; fractions with activity were analyzed by SDS-PAGE and Western blot using an anti-mouse COX-2 polyclonal antibody. Results showed that in those samples with enzymatic activity, the anti-mouse COX-2 polyclonal antibody recognized a protein with an approximate molecular weight of 66â¯KDa. Bands recognized by the antibody were subjected to mass spectrometry analysis and the results showed that several peptides from the bands purified by two different methods, and that were recognized by the anti-mouse COX-2 polyclonal antibody corresponded to the Leishmania mexicana gp63 surface protease. L. mexicana gp63 was purified by a Concanavalin A (Con-A) affinity column and subjected to immunoprecipitation with a commercial anti-Leishmania gp63 polyclonal antibody; the immunoprecipitated sample was analyzed for COX activity showing that the anti-gp63 antibody did immunoprecipitate the COX activity. The presence of COX activity was further confirmed in amastigotes extracts of L. mexicana. Moreover, a recombinant gp63 protein was produced and its COX activity tested, confirming that gp63 is the molecule responsible for COX activity.
Assuntos
Leishmania mexicana/enzimologia , Metaloendopeptidases/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Dinoprostona/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Imunoprecipitação , Espectrometria de Massas , Metaloendopeptidases/química , Metaloendopeptidases/isolamento & purificação , Camundongos Endogâmicos BALB C , Prostaglandina-Endoperóxido Sintases/química , Prostaglandina-Endoperóxido Sintases/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Chemotherapy is currently the only effective approach to treat all forms of leishmaniasis. However, its effectiveness is severely limited due to high toxicity, long treatment length, drug resistance, or inadequate mode of administration. As a consequence, there is a need to identify new molecular scaffolds and targets as potential therapeutics for the treatment of this disease. We report a small series of 1,2-substituted-1H-benzo[d]imidazole derivatives (9a-d) showing affinity in the submicromolar range (Ki = 0.15-0.69 µM) toward Leishmania mexicanaCPB2.8ΔCTE, one of the more promising targets for antileishmanial drug design. The compounds confirmed activity in vitro against intracellular amastigotes of Leishmania infantum with the best result being obtained with derivative 9d (IC50 = 6.8 µM), although with some degree of cytotoxicity (CC50 = 8.0 µM on PMM and CC50 = 32.0 µM on MCR-5). In silico molecular docking studies and ADME-Tox properties prediction were performed to validate the hypothesis of the interaction with the intended target and to assess the drug-likeness of these derivatives.
Assuntos
Benzimidazóis/química , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Leishmania mexicana/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Antiprotozoários/síntese química , Antiprotozoários/metabolismo , Antiprotozoários/uso terapêutico , Antiprotozoários/toxicidade , Benzimidazóis/metabolismo , Benzimidazóis/uso terapêutico , Benzimidazóis/toxicidade , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cisteína Proteases/química , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/uso terapêutico , Inibidores de Cisteína Proteinase/toxicidade , Avaliação Pré-Clínica de Medicamentos , Ensaios Enzimáticos , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Leishmaniose/tratamento farmacológico , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismoRESUMO
In this study, we synthesized eight new compounds containing the 2-amino-cycloalkyl[b]thiophene and acridine moieties (ACT01 and ACS01 -ACS07 ). None tested compounds presented human erythrocyte cytotoxicity. The new compounds presented antipromastigote activity, where ACS01 and ACS02 derivatives presented significant antileishmanial activity, with better performance than the reference drugs (tri and pentavalent antimonials), with respective IC50 values of 9.60 ± 3.19 and 10.95 ± 3.96 µm. Additionally, these two derivatives were effective against antimony-resistant Leishmania (Leishmania) amazonensis strains. In addition, binding and fragmentation DNA assays were performed. It was observed that the antileishmanial activity of ACS01 is not associated with DNA fragmentation of the promastigote forms. However, it interacted with DNA with a binding constant of 104 m-1 . In partial least-squares studies, it was observed that the most active compounds (ACS01 and ACS02 ) showed lower values of amphiphilic moment descriptor, but there was a correlation between the lipophilicity of the molecules and antileishmanial activity. Furthermore, the docking molecular studies showed interactions between thiophene-acridine derivatives and the active site of pyruvate kinase enzyme with the major contribution of asparagine 152 residue for the interaction with thiophene moiety. Thus, the results suggested that the new thiophene-acridine derivatives are promising molecules as potential drug candidates.
Assuntos
Acridinas/química , Antiprotozoários/síntese química , DNA de Protozoário/química , Simulação de Acoplamento Molecular , Tiofenos/química , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Sítios de Ligação , Domínio Catalítico , DNA de Protozoário/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Análise dos Mínimos Quadrados , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Relação Estrutura-AtividadeRESUMO
The identification of specific therapeutic targets and the development of new drugs against leishmaniasis are urgently needed, since chemotherapy currently available for its treatment has several problems including many adverse side effects. In an effort to develop new antileishmanial drugs, in the present study a series of 28 N-benzyl-1H-benzimidazol-2-amine derivatives was synthesized and evaluated in vitro against Leishmania mexicana promastigotes. Compounds 7 and 8 with the highest antileishmanial activity (micromolar) and lower cytotoxicity than miltefosine and amphotericin B were selected to evaluate their activity against L. braziliensis 9and L. donovani, species causative of mucocutaneous and visceral leishmaniasis, respectively. Compound 7 showed significantly higher activity against L. braziliensis promastigotes than compound 8 and slightly lower than miltefosine. Compounds 7 and 8 had IC50 values in the micromolar range against the amastigote of L. mexicana and L. braziliensis. However, both compounds did not show better activity against L. donovani than miltefosine. Compound 8 showed the highest SI against both parasite stages of L. mexicana. In addition, compound 8 inhibited 68.27% the activity of recombinant L. mexicana arginase (LmARG), a therapeutic target for the treatment of leishmaniasis. Docking studies were also performed in order to establish the possible mechanism of action by which this compound exerts its inhibitory effect. Compound 8 shows promising potential for the development of more potent antileishmanial benzimidazole derivatives.
Assuntos
Antiprotozoários/farmacologia , Benzimidazóis/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania donovani/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Sequência de Aminoácidos , Anfotericina B/farmacologia , Animais , Antiprotozoários/toxicidade , Arginase/antagonistas & inibidores , Arginase/química , Benzimidazóis/síntese química , Benzimidazóis/química , Benzimidazóis/toxicidade , Linhagem Celular , Concentração Inibidora 50 , Leishmania mexicana/enzimologia , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Leishmaniose Mucocutânea/tratamento farmacológico , Leishmaniose Mucocutânea/parasitologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Macrófagos/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Alinhamento de SequênciaRESUMO
In an effort to identify novel molecular warheads able to inhibit Leishmania mexicana cysteine protease CPB2.8ΔCTE, fused benzo[b]thiophenes and ß,ß'-triketones emerged as covalent inhibitors binding the active site cysteine residue. Enzymatic screening showed a moderate-to-excellent activity (12%-90% inhibition of the target enzyme at 20 µm). The most promising compounds were selected for further profiling including in vitro cell-based assays and docking studies. Computational data suggest that benzo[b]thiophenes act immediately as non-covalent inhibitors and then as irreversible covalent inhibitors, whereas a reversible covalent mechanism emerged for the 1,3,3'-triketones with a Y-topology. Based on the predicted physicochemical and ADME-Tox properties, compound 2b has been identified as a new drug-like, non-mutagen, non-carcinogen, and non-neurotoxic lead candidate.
Assuntos
Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Leishmania mexicana/enzimologia , Proteínas de Protozoários/metabolismo , Tiofenos/química , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cisteína Proteases/química , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Concentração Inibidora 50 , Leishmania mexicana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Proteínas de Protozoários/antagonistas & inibidores , Relação Estrutura-Atividade , Tiofenos/metabolismo , Tiofenos/farmacologiaRESUMO
Leishmania parasites cause a broad spectrum of clinical manifestations in humans and the available clinical treatments are far from satisfactory. Since these pathogens require large amounts of NADPH to maintain intracellular redox homeostasis, oxidoreductases that catalyze the production of NADPH are considered as potential drug targets against these diseases. In the sequenced genomes of most Leishmania spp. two putative malic enzymes (MEs) with an identity of about 55% have been identified. In this work, the ME from L. major (LmjF24.0770, Lmj_ME-70) and its less similar homolog from L. mexicana (LmxM.24.0761, Lmex_ME-61) were cloned and functionally characterized. Both MEs specifically catalyzed NADPH production, but only Lmex_ME-61 was activated by l-aspartate. Unlike the allosterically activated human ME, Lmex_ME-61 exhibited typical hyperbolic curves without any sign of cooperativity in the absence of l-aspartate. Moreover, Lmex_ME-61 and Lmj_ME-70 differ from higher eukaryotic homologs in that they display dimeric instead of tetrameric molecular organization. Homology modeling analysis showed that Lmex_ME-61 and Lmj_ME-70 notably differ in their surface charge distribution; this feature encompasses the coenzyme binding pockets as well. However, in both isozymes, the residues directly involved in the coenzyme binding exhibited a good degree of conservation. Besides, only Lmex_ME-61 and its closest homologs were immunodetected in cell-free extracts from L. mexicana, L. amazonensis and L. braziliensis promastigotes. Our findings provide a first glimpse into the biochemical properties of leishmanial MEs and suggest that MEs could be potentially related to the metabolic differences among the species of Leishmania parasites.
Assuntos
Leishmania major/enzimologia , Leishmania mexicana/enzimologia , Malato Desidrogenase (NADP+)/genética , Malato Desidrogenase (NADP+)/metabolismo , Ácido Aspártico/metabolismo , Sítios de Ligação , Clonagem Molecular , Coenzimas/metabolismo , Biologia Computacional , Expressão Gênica , Leishmania major/genética , Leishmania mexicana/genética , Malato Desidrogenase (NADP+)/química , Modelos Moleculares , NADP/metabolismo , Multimerização ProteicaRESUMO
The aims of the present work were to test the effect of tamoxifen administered topically and the therapeutic efficacy of tamoxifen and pentavalent antimonial combinations in an experimental model of cutaneous leishmaniasis. BALB/c mice infected with a luciferase expressing line of Leishmania amazonensis were treated with topical tamoxifen in two different formulations (ethanol or oil-free cream) as monotherapy or in co-administration with pentavalent antimonial. Treatment efficacy was evaluated by lesion size and parasite burden, quantified through luminescence, at the end of treatment and 4 weeks later. Topical tamoxifen, formulated in ethanol or as a cream, was shown to be effective. The interaction between tamoxifen and pentavalent antimonial was additive in vitro. Treatment with combined schemes containing tamoxifen and pentavalent antimonial was effective in reducing lesion size and parasite burden. Co-administration of tamoxifen and pentavalent antimonial was superior to monotherapy with antimonial.
Assuntos
Antiprotozoários/uso terapêutico , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Antimoniato de Meglumina/uso terapêutico , Pele/efeitos dos fármacos , Tamoxifeno/uso terapêutico , Administração Tópica , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/efeitos adversos , Quimioterapia Combinada/efeitos adversos , Etanol/química , Feminino , Leishmania mexicana/enzimologia , Leishmania mexicana/genética , Leishmaniose Cutânea/parasitologia , Luciferases/genética , Luminescência , Antimoniato de Meglumina/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Pele/parasitologia , Creme para a Pele/administração & dosagem , Creme para a Pele/efeitos adversos , Creme para a Pele/uso terapêutico , Tamoxifeno/administração & dosagem , Tamoxifeno/efeitos adversos , Tamoxifeno/químicaRESUMO
BACKGROUND: Leishmania is a protozoan parasite that alternates its life cycle between the sand-fly vector and the mammalian host. This alternation involves environmental changes and leads the parasite to dynamic modifications in morphology, metabolism, cellular signaling and regulation of gene expression to allow for a rapid adaptation to new conditions. The L-arginine pathway in L. amazonensis is important during the parasite life cycle and interferes in the establishment and maintenance of the infection in mammalian macrophages. Host arginase is an immune-regulatory enzyme that can reduce the production of nitric oxide by activated macrophages, directing the availability of L-arginine to the polyamine pathway, resulting in parasite replication. In this work, we performed transcriptional profiling to identify differentially expressed genes in L. amazonensis wild-type (La-WT) versus L. amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes. METHODOLOGY/PRINCIPAL FINDINGS: A total of 8253 transcripts were identified in La-WT and La-arg- promastigotes and axenic amastigotes, about 60% of them codifying hypothetical proteins and 443 novel transcripts, which did not match any previously annotated genes. Our RNA-seq data revealed that 85% of genes were constitutively expressed. The comparison of transcriptome and metabolome data showed lower levels of arginase and higher levels of glutamate-5-kinase in La-WT axenic amastigotes compared to promastigotes. The absence of arginase activity in promastigotes increased the levels of pyrroline 5-carboxylate reductase, but decreased the levels of arginosuccinate synthase, pyrroline 5-carboxylate dehydrogenase, acetylornithine deacetylase and spermidine synthase transcripts levels. These observations can explain previous metabolomic data pointing to the increase of L-arginine, citrulline and L-glutamate and reduction of aspartate, proline, ornithine and putrescine. Altogether, these results indicate that arginase activity is important in Leishmania gene expression modulation during differentiation and adaptation to environmental changes. Here, we confirmed this hypothesis with the identification of differential gene expression of the enzymes involved in biosynthesis of amino acids, arginine and proline metabolism and arginine biosynthesis. CONCLUSIONS/SIGNIFICANCE: All data provided information about the transcriptomic profiling and the expression levels of La-WT and La-arg- promastigotes and axenic amastigotes. These findings revealed the importance of arginase in parasite survival and differentiation, and indicated the existence of a coordinated response in the absence of arginase activity related to arginine and polyamine pathways.