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1.
Exp Parasitol ; 221: 108059, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33338468

RESUMO

Treatment for visceral leishmaniasis (VL) is hindered mainly by the toxicity and/or high cost of therapeutic drugs. In addition, parasite resistance has been registered. Thus, there is an urgent need for the identification of novel, effective and low-cost antileishmanial agents. Since drug discovery is a long and expensive process, drug repositioning for treatment of leishmaniasis should be considered. In the present study, Ivermectin (IVE), a broad-spectrum drug used for treatment of parasitic diseases, was evaluated in vitro and in vivo against Leishmania infantum species. Results in vitro showed that IVE presented 50% Leishmania and macrophage inhibitory concentrations (IC50 and CC50, respectively) of 3.64 ± 0.48 µM and 427.50 ± 17.60 µM, respectively, with a selectivity index (SI) of 117.45; whereas Amphotericin B (AmpB), which was used as control, showed IC50 and CC50 values of 0.12 ± 0.05 µM and 1.06 ± 0.23 µM, respectively, with a corresponding SI of 8.90. Treatment with IVE effectively reduced the infection percentage and parasite burden in infected and treated macrophages and displayed a prophylactic activity by inhibiting macrophage infection with pre-treated parasites. Furthermore, preliminary studies suggested that IVE targets the parasite's mitochondria. Activity of IVE in its free format or incorporated into Pluronic® F127-based polymeric micelles (IVE/Mic) was also evaluated in vivo as a treating drug for L. infantum-infected BALB/c mice. Miltefosine was used as a control. Results showed that Miltefosine, IVE and IVE/Mic-treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as well as development of an antileishmanial Th1-type immune response one and 15 days after treatment. Notably, IVE/Mic showed a better parasitological and immunological response in comparison to other alternative treatments. In conclusion, results suggest that IVE/Mic could be considered in future studies as a therapeutic alternative to treat VL.


Assuntos
Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Ivermectina/farmacologia , Ivermectina/uso terapêutico , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Anfotericina B/farmacologia , Animais , Antiprotozoários/toxicidade , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Concentração Inibidora 50 , Ivermectina/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Baço/parasitologia
2.
PLoS Negl Trop Dis ; 14(12): e0008947, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33338041

RESUMO

Leishmaniasis is among the world's most neglected diseases. Dogs are the main reservoirs/hosts of Leishmania infantum, causative agent of both canine and human visceral leishmaniosis. Canine leishmaniasis (CanL) represents a public health problem as one of the most prevalent zoonotic diseases worldwide. Current therapeutics present drawbacks; thus, there is a need for more effective, safer, and cheaper drugs. The aim of this study was to evaluate and to compare the efficacy of oral administration of artesunate or meglumine antimoniate/allopurinol in dogs with clinical leishmaniasis. Forty-two dogs with naturally occurring clinical leishmaniasis were included in this open-label, simple randomized positive-control clinical field trial with 6 months of follow-up. Dogs received meglumine antimoniate 100 mg/kg/day and allopurinol 30 mg/kg/day for 28 days (control group, n = 26) or artesunate 25 mg/kg/day for 6 days (test group, n = 16). The animals were evaluated for their clinical evolution, parasite load (by qPCR) and humoral response at different time points: 0, 30, 90, and 180 days after treatment. Data analyses showed a significant improvement in both groups in clinical scores, parasitemia and antibody titers after treatment. Compared to the control group, the artesunate group showed significantly lower clinical score (P = 0.0001), lower parasitemia (P = 0.0001) and antibody titers after 6 months of follow-up. Compared to baseline values, a rapid, significant reduction (P < 0.012) in antibody levels, 2.28- versus 3.04-fold for the control versus artesunate groups, respectively, was observed 30 days after treatment. Antibody levels continued to decrease further in the artesunate group, where 58% of cases became seronegative at the 6-month follow-up. All qPCR-positive dogs were negative after treatment with artesunate, while 14.3% remained positive with the appearance of two new cases in the control group. Artesunate was well tolerated, and no side effects were recorded. Treatment failures were similar in both groups with 27.27% (6/22), including 18.18% (4/22) mortality in the control group, versus 26.66% (4/15), including 13.33% (2/15) mortality in the artesunate group. This is the first report showing the potential of artesunate in the treatment of dogs with clinical leishmaniasis. Artesunate showed higher efficacy than the current first-line treatment for CanL without any adverse effects. It could be a good alternative chemotherapy for CanL, and may be considered for further studies in human leishmaniases. Further clinical trials are needed to confirm these findings, to determine if there are relapses after treatment and if dogs remain infective to sandflies, to define the ideal therapeutic dosage and duration of treatment with artesunate.


Assuntos
Alopurinol/uso terapêutico , Antiprotozoários/uso terapêutico , Artesunato/uso terapêutico , Doenças do Cão/tratamento farmacológico , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/veterinária , Antimoniato de Meglumina/uso terapêutico , Animais , Doenças do Cão/parasitologia , Cães , Feminino , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Masculino , Carga Parasitária/veterinária , Parasitemia/tratamento farmacológico , Zoonoses
3.
PLoS One ; 15(9): e0238601, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881961

RESUMO

Canines are proven reservoir hosts of Leishmania infantum, the causative agent of human zoonotic visceral leishmaniasis, and therefore domestic dogs play a central role in transmitting the disease to humans. Studies on the effect of insecticide-impregnated dog collars for controlling canine visceral leishmaniasis (CanL) have been increasing; however, meta-analysis has not been conducted. This study assessed the effectiveness of insecticide-impregnated dog collars for preventing CanL. We searched (PubMed, Web of Science, Scopus, Embase, Ovid Medline(R), and Cochrane library), from inception until 2 May 2020. Two authors independently performed articles screening and data extraction. We applied the RoB 2.0 tool to evaluate the risk of bias in randomized trials, while the ROBINS-I tool was used for non-randomized trials. I-squared statistics(I2) and funnel plot and Egger's test, respectively, were used to assesses heterogeneity between studies and publication bias. Relative Risk (RR) and 95% Confidence Interval (CI) were calculated using the random-effects model in Stata 14 software. Out of 242 citations identified, 14 studies comprising 3786 collared dogs and 3428 uncollared dogs were eligible for meta-analysis. The use of deltamethrin-impregnated dog collars(DMC) showed an overall effectiveness of 54% (95%CI: 35-65%, I2 = 63.2%, P = 0.002) in decreasing incidence of CanL, while 10% imidacloprid and 4.5% flumethrin collars provided an overall effectiveness of 90% (95%CI: 80-96%, I2 = 0.0%, P = 0.376). DMC efficacy stratified by follow-up duration was estimated to be 58% (RR = 0.42, 95%CI: 0.20-0.87), 54% (RR = 0.46, 95%CI: 0.31-0.68), 53% (RR = 0.47, 95%CI: 0.29-0.82) for follow-up periods of 5 to 6 months, 1 year and 2 years, respectively. The current evidence indicates that using insecticide-impregnated dog collars can reduce the risk of CanL caused by L. infantum. Therefore, insecticide-impregnated dog collars could be a viable alternative for inclusion as a public health measure for controlling CanL.


Assuntos
Doenças do Cão/prevenção & controle , Inseticidas/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/prevenção & controle , Administração Cutânea , Animais , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/veterinária , Neonicotinoides/farmacologia , Nitrilos/farmacologia , Nitrocompostos/farmacologia , Piretrinas/farmacologia
4.
Mem Inst Oswaldo Cruz ; 115: e190469, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32638832

RESUMO

BACKGROUND Oxidative stress is responsible for generating DNA lesions and the 8-oxoguanine (8-oxoG) is the most commonly lesion found in DNA damage. When this base is incorporated during DNA replication, it could generate double-strand DNA breaks and cellular death. MutT enzyme hydrolyzes the 8-oxoG from the nucleotide pool, preventing its incorporation during DNA replication. OBJECTIVES To investigate the importance of 8-oxoG in Leishmania infantum and L. braziliensis, in this study we analysed the impact of heterologous expression of Escherichia coli MutT (EcMutT) enzyme in drug-resistance phenotype and defense against oxidative stress. METHODS Comparative analysis of L. braziliensis and L. infantum H2O2 tolerance and cell cycle profile were performed. Lines of L. braziliensis and L. infantum expressing EcMutT were generated and evaluated using susceptibility tests to H2O2 and SbIII, cell cycle analysis, γH2A western blotting, and BrdU native detection assay. FINDINGS Comparative analysis of tolerance to oxidative stress generated by H2O2 showed that L. infantum is more tolerant to exogenous H2O2 than L. braziliensis. In addition, cell cycle analysis showed that L. infantum, after treatment with H2O2, remains in G1 phase, returning to its normal growth rate after 72 h. In contrast, after treatment with H2O2, L. braziliensis parasites continue to move to the next stages of the cell cycle. Expression of the E. coli MutT gene in L. braziliensis and L. infantum does not interfere in parasite growth or in susceptibility to SbIII. Interestingly, we observed that L. braziliensis EcMutT-expressing clones were more tolerant to H2O2 treatment, presented lower activation of γH2A, a biomarker of genotoxic stress, and lower replication stress than its parental non-transfected parasites. In contrast, the EcMutT is not involved in protection against oxidative stress generated by H2O2 in L. infantum. MAIN CONCLUSIONS Our results showed that 8-oxoG clearance in L. braziliensis is important to avoid misincorporation during DNA replication after oxidative stress generated by H2O2.


Assuntos
Antimônio/toxicidade , Proteínas de Escherichia coli/genética , Escherichia coli , Guanina/análogos & derivados , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Pirofosfatases , Superóxido Dismutase/metabolismo , Animais , Antiprotozoários/farmacologia , Proteínas de Escherichia coli/metabolismo , Guanina/farmacologia , Humanos , Peróxido de Hidrogênio/toxicidade , Leishmania braziliensis/enzimologia , Leishmania infantum/enzimologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , Coelhos , Ratos , Superóxido Dismutase/genética
5.
PLoS Negl Trop Dis ; 14(7): e0008439, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628683

RESUMO

Leishmaniasis constitutes the 9th largest disease burden among all infectious diseases. Control of this disease is based on a short list of chemotherapeutic agents headed by pentavalent antimonials, followed by miltefosine and amphotericin B; drugs that are far from ideal due to host toxicity, elevated cost, limited access, and high rates of drug resistance. Knowing that the composition of extracellular vesicles (EVs) can vary according to the state of their parental cell, we hypothesized that EVs released by drug-resistant Leishmania infantum parasites could contain unique and differently enriched proteins depending on the drug-resistance mechanisms involved in the survival of their parental cell line. To assess this possibility, we studied EV production, size, morphology, and protein content of three well-characterized drug-resistant L. infantum cell lines and a wild-type strain. Our results are the first to demonstrate that drug-resistance mechanisms can induce changes in the morphology, size, and distribution of L. infantum EVs. In addition, we identified L. infantum's core EV proteome. This proteome is highly conserved among strains, with the exception of a handful of proteins that are enriched differently depending on the drug responsible for induction of antimicrobial resistance. Furthermore, we obtained the first snapshot of proteins enriched in EVs released by antimony-, miltefosine- and amphotericin-resistant parasites. These include several virulence factors, transcription factors, as well as proteins encoded by drug-resistance genes. This detailed study of L. infantum EVs sheds new light on the potential roles of EVs in Leishmania biology, particularly with respect to the parasite's survival in stressful conditions. This work outlines a crucial first step towards the discovery of EV-based profiles capable of predicting response to antileishmanial agents.


Assuntos
Antiprotozoários/farmacologia , Resistência a Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/metabolismo , Biologia Computacional , Vesículas Extracelulares , Regulação da Expressão Gênica/fisiologia , Proteoma , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
6.
Exp Parasitol ; 214: 107903, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32360142

RESUMO

The aim of this 6-month, randomized, blinded, controlled clinical trial was to compare the efficacy and safety of aminosidine-allopurinol combination with that of meglumine antimoniate-allopurinol combination for the treatment of leishmaniosis in dogs without stage III or IV chronic kidney disease. Forty client-owned dogs were randomly assigned to group A [n = 20; aminosidine (15 mg/kg, subcutaneously, once daily, for 28 days) and allopurinol (10 mg/kg, per os, twice daily, for 6 months)] or group B [(n = 20; meglumine antimoniate (100 mg/kg SC, once daily, for 28 days) and allopurinol (10 mg/kg, per os, twice daily, for 6 months)]. Clinical and clinicopathological evaluations, parasitic load measurement (lymph node and bone marrow microscopy, bone marrow real-time PCR), specific serology and leishmanin skin test (LST) were performed at baseline (time 1) and after 14 (time 2), 28 (time 3), 60 (time 4) and 180 (time 5) days. Both treatments were safe and resulted in significant clinical and clinicopathological improvement, reduction of parasitic load and of indirect immunofluorescence antibody test (IFAT) titer and induction of positive LST. There was no significant difference between groups with regards to the primary outcome measures of the trial that included the proportion of dogs that presented severe treatment-related side effects, were cured and were parasitologically negative at time 5. However, some (proportion of dogs that presented no clinical signs, no hyperglobulinemia and negative serology at time 5) secondary outcome measures showed significant differences in favor of the meglumine antimoniate-allopurinol treatment arm. Treatment-related death occurred in one dog in each group, while injection site reactions appeared at a similar frequency in both groups. Due to the differences in some secondary outcome measures in association with the low power of this trial, it cannot be definitively concluded that the two treatments are equally effective. Therefore, the aminisodine-allopurinol combination cannot be proposed as a first-line treatment of CanL but rather as a second-line treatment that may be particularly useful to avoid repeated administration of meglumine antimoniate and in countries where the latter is not available or registered.


Assuntos
Alopurinol/uso terapêutico , Doenças do Cão/tratamento farmacológico , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Antimoniato de Meglumina/uso terapêutico , Paromomicina/uso terapêutico , Tripanossomicidas/uso terapêutico , Animais , Cães , Quimioterapia Combinada , Feminino , Injeções Subcutâneas/veterinária , Masculino
7.
Parasitol Res ; 119(7): 2263-2274, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32462293

RESUMO

Leishmaniasis is responsible for approximately 65,000 annual deaths. Despite the mortality data, drugs available for the treatment of patients are insufficient and have moderate therapeutic efficacy in addition to serious adverse effects, which makes the development of new drugs urgent. To achieve this goal, the integration of kinetic and DSF assays against parasitic validated targets, along with phenotypic assays, can help the identification and optimization of bioactive compounds. Pteridine reductase 1 (PTR1), a validated target in Leishmania sp., is responsible for the reduction of folate and biopterin to tetrahydrofolate and tetrahydrobiopterin, respectively, both of which are essential for cell growth. In addition to the in vitro evaluation of 16 thiazolidine-2,4-dione derivatives against Leishmania major PTR1 (LmPTR1), using the differential scanning fluorimetry (ThermoFluor®), phenotypic assays were employed to evaluate the compound effect over Leishmania braziliensis (MHOM/BR/75/M2903) and Leishmania infantum (MHOM/BR/74/PP75) promastigotes viability. The ThermoFluor® results show that thiazolidine-2,4-dione derivatives have micromolar affinity to the target and equivalent activity on Leishmania cells. 2b is the most potent compound against L. infantum (EC50 = 23.45 ± 4.54 µM), whereas 2a is the most potent against L. braziliensis (EC50 = 44.16 ± 5.77 µM). This result suggests that lipophilic substituents on either-meta and/or-para positions of the benzylidene ring increase the potency against L. infantum. On the other hand, compound 2c (CE50 = 49.22 ± 7.71 µM) presented the highest selectivity index.


Assuntos
Antiprotozoários/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Animais , Antiprotozoários/química , Humanos , Leishmania braziliensis/enzimologia , Leishmania infantum/enzimologia , Oxirredutases/antagonistas & inibidores , Testes de Sensibilidade Parasitária , Tiazolidinedionas/química
8.
J Med Chem ; 63(11): 5734-5751, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32392053

RESUMO

Leishmania (L.) infantum causes visceral, cutaneous, and mucosal leishmaniasis in humans and canine leishmaniasis in dogs. Herein, we describe that O-alkyl hydroxamate derivatives displayed potent and selective in vitro activity against the amastigote stage of L. infantum while no activity was observed against promastigotes. Compound 5 showed potent in vivo activity against L. infantum. Moreover, the combination of compound 5 supported on gold nanoparticles and meglumine antimoniate was also effective in vivo and improved the activity of these compounds compared to that of the individual treatment. Docking studies showed that compound 5 did not reach highly conserved pocket C and established interactions with the semiconserved residues V44, A45, R242, and E243 in pocket A of LiSIR2rp1. The surface space determined by these four amino acids is not conserved in human sirtuins. Compound 5 represents a new class of selective ligands with antileishmanial activity.


Assuntos
Antiprotozoários/farmacologia , Ácidos Hidroxâmicos/química , Leishmania infantum/efeitos dos fármacos , Animais , Antiprotozoários/química , Sítios de Ligação , Feminino , Ouro/química , Histona Desacetilase 1/química , Histona Desacetilase 1/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Leishmania infantum/crescimento & desenvolvimento , Estágios do Ciclo de Vida/efeitos dos fármacos , Antimoniato de Meglumina/farmacologia , Nanopartículas Metálicas/química , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Baço/parasitologia
9.
Mem Inst Oswaldo Cruz ; 115: e190408, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32321156

RESUMO

BACKGROUND: The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host. OBJECTIVES: In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated. METHODS: Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry. FINDINGS: The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(ß1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines. MAIN CONCLUSIONS: Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.


Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Leishmania braziliensis/imunologia , Leishmania infantum/imunologia , Monócitos/parasitologia , Óxido Nítrico/biossíntese , Fagocitose/imunologia , Adulto , Resistência a Medicamentos , Feminino , Citometria de Fluxo , Humanos , Imunidade Inata , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Masculino , Monócitos/imunologia , Adulto Jovem
10.
Molecules ; 25(6)2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32204358

RESUMO

Leishmaniasis affects around 12 million people worldwide and is estimated to cause the ninth-largest disease burden. There are three main forms of the disease, visceral (VL), cutaneous (CL), and mucocutaneous (MCL), leading to more than one million new cases every year and several thousand deaths. Current treatments based on chemically synthesized molecules are far from ideal. In this study, we have tested the in vitro and in vivo efficacy of ursolic acid (UA), a multifunctional triterpenoid with well-known antitumoral, antioxidant, and antimicrobial effects on different Leishmania strains. The in vitro antileishmanial activity against the intracellular forms was six and three-fold higher compared to extracellular forms of L. amazonensis and L. infantum, respectively. UA also showed to be a potent antileishmanial drug against both VL and CL manifestations of the disease in experimental models. UA parenterally administered at 5 mg/kg for seven days significantly reduced the parasite burden in liver and spleen not only in murine acute infection but also in a chronic-infection model against L. infantum. In addition, UA ointment (0.2%) topically administered for four weeks diminished (50%) lesion size progression in a chronic infection model of CL caused by L. amazonensis, which was much greater than the effect of UA formulated as an O/W emulsion. UA played a key role in the immunological response modulating the Th1 response. The exposure of Leishmania-infected macrophages to UA led to a significant different production in the cytokine levels depending on the Leishmania strain causing the infection. In conclusion, UA can be a promising therapy against both CL and VL.


Assuntos
Antiprotozoários/administração & dosagem , Leishmania infantum/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Triterpenos/administração & dosagem , Administração Tópica , Animais , Antiprotozoários/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Humanos , Infusões Parenterais , Leishmaniose Visceral/parasitologia , Fígado/efeitos dos fármacos , Fígado/parasitologia , Masculino , Mesocricetus , Camundongos , Phytolaccaceae/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Baço/efeitos dos fármacos , Baço/parasitologia , Triterpenos/farmacologia
11.
Med Chem ; 16(3): 422-430, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30931864

RESUMO

BACKGROUND: The World Health Organization catalogues illnesses such as Leishmaniasis as neglected diseases, due to low investment in new drugs to fight them. The search of novel and non-side effects anti-parasitic compounds is one of the urgent needs for the Third World. The use of triazolopyrimidines and their metallic complexes has demonstrated hopeful results in this field. OBJECTIVE: This work studies the antiparasitic efficacy of a series of 5,7-dimethyl-1,2,4- triazolo[1,5-a]pyrimidine first row transition metal complexes against three leishmania spp. strains. METHODS: The in vitro antiproliferation of promastigote forms of different strains of leishmania spp. (L. infantum, L. braziliensis and L donovani) and the cytotoxicity in macrophage host cells are reported here. The antiparasitic assays have been complemented with enzymatic tests to elucidate the mechanisms of action. New crystal structure description, thermal analysis, magnetic susceptibility and magnetization experiments have also been carried out in order to present a whole characterization of the studied compounds and interesting physical properties besides the biological tests. RESULTS: The results of antiproliferation screening and cytotoxicity show great antiparasitic efficacy in the studied complexes. The superoxide dismutase enzymatic assays exhibit a different behaviour according to the thermochromic triazolopyrimidine form tested. CONCLUSION: Antiproliferative assays and enzymatic tests corroborate the synergetic leishmanicidal effect present in coordination triazolopyrimidine complexes. The changes in coordination sphere derived from thermochromism affect the physical properties as well as the biological efficacy.


Assuntos
Complexos de Coordenação/farmacologia , Pirimidinas/farmacologia , Triazóis/farmacologia , Tripanossomicidas/farmacologia , Animais , Linhagem Celular , Cor , Complexos de Coordenação/síntese química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania donovani/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Parasitária , Pirimidinas/síntese química , Superóxido Dismutase/metabolismo , Temperatura , Triazóis/síntese química , Tripanossomicidas/síntese química
12.
Amino Acids ; 52(2): 247-259, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31037461

RESUMO

Leishmania protozoans are the causative agent of leishmaniasis, a neglected tropical disease consisting of three major clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis, and mucocutaneous leishmaniasis. VL is caused by Leishmania donovani in East Africa and the Indian subcontinent and by Leishmania infantum in Europe, North Africa, and Latin America, and causes an estimated 60,000 deaths per year. Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against leishmaniasis. This enzyme is fundamental for parasite survival in the human host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize the hydrogen peroxide produced by host macrophages during infection. Recently, we solved the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine), which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466', Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777-TR complex, we synthesized structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase.


Assuntos
Antiprotozoários/química , Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Sulfetos/química , Sulfetos/farmacologia , Motivos de Aminoácidos , Domínio Catalítico , Glutationa/análogos & derivados , Glutationa/metabolismo , Humanos , Leishmania infantum/enzimologia , Leishmania infantum/metabolismo , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Modelos Moleculares , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Espermidina/análogos & derivados , Espermidina/metabolismo
13.
Chem Biol Interact ; 315: 108899, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31738906

RESUMO

Parasitic diseases still represent serious public health problems, since the high and steady emergence of resistant strains is evident. Because parasitic infections are distributed predominantly in developing countries, less toxic, more efficient, safer and more accessible drugs have become desirable in the treatment of the infected population. This is the case of leishmaniasis, an infectious disease caused by a protozoan of the genus Leishmania sp., responsible for triggering pathological processes from the simplest to the most severe forms leading to high rates of morbidity and mortality throughout the world. In the search for new leishmanicidal drugs, the thiosemicarbazones and the indole fragments have been identified as promising structures for leishmanicidal activity. The present study proposes the synthesis and structural characterization of new indole-thiosemicarbazone derivatives (2a-j), in addition to performing in vitro evaluations through cytotoxicity assays using macrophages (J774) activity against forms of Leishmania infantum and Leishmania amazonensis promastigote as well as ultrastructural analyzes in promastigotes of L. infantum. Results show that the indole-thiosemicarbazone derivatives were obtained with yield values varying from 32.09 to 94.64%. In the evaluation of cytotoxicity, the indole-thiosemicarbazone compounds presented CC50 values between 53.23 and 357.97 µM. Concerning the evaluation against L. amazonensis promastigote forms, IC50 values ranged between 12.31 and  > 481.52 µM, while the activity against L. infantum promastigotes obtained IC50 values between 4.36 and 23.35 µM. The compounds 2d and 2i tested against L. infantum were the most promising in the series, as they showed the lowest IC50 values: 5.60 and 4.36 respectively. The parasites treated with the compounds 2d and 2i showed several structural alterations, such as shrinkage of the cell body, shortening and loss of the flagellum, intense mitochondrial swelling and vacuolization of the cytoplasm leading the parasite to cellular unviability. Therefore, the indole-thiosemicarbazone compounds are promising because they yield considerable synthesis, have low cytotoxicity to mammalian cells and act as leishmanicidal agents.


Assuntos
Antiprotozoários/farmacologia , Indóis/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Tiossemicarbazonas/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Leishmaniose/parasitologia , Macrófagos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos
14.
Molecules ; 24(24)2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31847066

RESUMO

Banana inflorescences are a byproduct of banana cultivation consumed in various regions of Brazil as a non-conventional food. This byproduct represents an alternative food supply that can contribute to the resolution of nutritional problems and hunger. This product is also used in Asia as a traditional remedy for the treatment of various illnesses such as bronchitis and dysentery. However, there is a lack of chemical and pharmacological data to support its consumption as a functional food. Therefore, this work aimed to study the anti-inflammatory action of Musa acuminata blossom by quantifying the cytokine levels (NOx, IL-1ß, TNF-α, and IL-6) in peritoneal neutrophils, and to study its antiparasitic activities using the intracellular forms of T. cruzi, L. amazonensis, and L. infantum. This work also aimed to establish the chemical profile of the inflorescence using UPLC-ESI-MS analysis. Flowers and the crude bract extracts were partitioned in dichloromethane and n-butanol to afford four fractions (FDCM, FNBU, BDCM, and BNBU). FDCM showed moderate trypanocidal activity and promising anti-inflammatory properties by inhibiting IL-1ß, TNF-α, and IL-6. BDCM significantly inhibited the secretion of TNF-α, while BNBU was active against IL-6 and NOx. LCMS data of these fractions revealed an unprecedented presence of arylpropanoid sucroses alongside flavonoids, triterpenes, benzofurans, stilbenes, and iridoids. The obtained results revealed that banana inflorescences could be used as an anti-inflammatory food ingredient to control inflammatory diseases.


Assuntos
1-Butanol/farmacologia , Anti-Inflamatórios/farmacologia , Cloreto de Metileno/farmacologia , Musa/química , Tripanossomicidas/farmacologia , 1-Butanol/química , Animais , Anti-Inflamatórios/química , Sobrevivência Celular/efeitos dos fármacos , Flores/química , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Leishmania/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Cloreto de Metileno/química , Camundongos , NADPH Oxidases/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Células THP-1 , Tripanossomicidas/química , Fator de Necrose Tumoral alfa/metabolismo
15.
Turkiye Parazitol Derg ; 43(4): 158-164, 2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31865648

RESUMO

Objective: Current in-silico research was designed and administered for the screening of 20000 Food and Drug Administration-approved drug compounds with the goal of finding promising drugs against lipophosphoglycan (LPG) and γ-glutamylcysteine synthetase (γ-GCS) of Leishmania infantum. Methods: After the protein sequence of both targets was taken, the 3D structures of protein of interest were predicted and validated. Molecular docking was done among the two putative targets (LPG and γ-GCS) and approved compounds were selected using AutoDock 4.2 program to predict ligand-receptor interactions. Results: After docking experiment was done on 20000 drug compounds, a total number of seven ligands, two for γ-GCS receptor and five for LPG receptor, were assigned as novel, potent anti-leishmanial drugs based on their binding affinity and energy. Of those, five ligands possessed cytotoxic and anti-cancer characteristics and showed good binding capacity to LPG receptor with ΔGbinding up to 8.5 kcal/mol more negative; while two compounds showed good binding capacity to glutamyl receptor with ΔGbinding up to 7.8 kcal/mol more negative. Conclusion: The latest software-based methods are powerful tools for scanning and predicting new peptide templates specific to biological targets in organisms for new drug discovery. However, the use of in vitro and in vivo techniques is a requirement for better evaluation of the potential of projected ligands with the help of in-silico approaches, identifying molecular mechanism of action of the more active compounds is possible. This can help in defining the most likely molecular target, so that the subsequent optimization using in vitro and in vivo techniques can be undertaken.


Assuntos
Antiprotozoários/farmacologia , Glutamato-Cisteína Ligase/antagonistas & inibidores , Glicoesfingolipídeos/antagonistas & inibidores , Leishmania infantum/efeitos dos fármacos , Sequência de Aminoácidos , Anfotericina B/farmacologia , Simulação por Computador , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Humanos , Leishmania infantum/química , Leishmania infantum/enzimologia , Ligantes , Antimoniato de Meglumina/farmacologia , Simulação de Acoplamento Molecular , Projetos de Pesquisa , Software
16.
PLoS Negl Trop Dis ; 13(12): e0007885, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790397

RESUMO

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity.


Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Testes de Sensibilidade Parasitária/métodos , Animais , Células Cultivadas , Feminino , Humanos , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/parasitologia , Camundongos , Testes de Sensibilidade Parasitária/normas
17.
Artigo em Inglês | MEDLINE | ID: mdl-31678841

RESUMO

Leishmania is the aethiological agent responsible for the visceral leishmaniasis, a serious parasite-borne disease widely spread all over the World. The emergence of resistant strains makes classical treatments less effective; therefore, new and better drugs are necessary. Naphthoquinones are interesting compounds for which many pharmacological properties have been described, including leishmanicidal activity. This work shows the antileishmanial effect of two series of terpenyl-1,4-naphthoquinones (NQ) and 1,4-anthraquinones (AQ) obtained from natural terpenoids, such as myrcene and myrceocommunic acid. They were evaluated both in vitro and ex vivo against the transgenic iRFP-Leishmania infantum strain and also tested on liver HepG2 cells to determine their selectivity indexes. The results indicated that NQ derivatives showed better antileishmanial activity than AQ analogues, and among them, compounds with a diacetylated hydroquinone moiety provided better results than their corresponding quinones. Regarding the terpenic precursor, compounds obtained from the monoterpenoid myrcene displayed good antiparasitic efficiency and low cytotoxicity for mammalian cells, whereas those derived from the diterpenoid showed better antileishmanial activity without selectivity. In order to explore their mechanism of action, all the compounds have been tested as potential inhibitors of Leishmania type IB DNA topoisomerases, but only some compounds that displayed the quinone ring were able to inhibit the recombinant enzyme in vitro. This fact together with the docking studies performed on LTopIB suggested the existence of another mechanism of action, alternative or complementary to LTopIB inhibition. In silico druglikeness and ADME evaluation of the best leishmanicidal compounds has shown good predictable druggability.


Assuntos
Antiprotozoários/farmacologia , DNA Topoisomerases/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/enzimologia , Quinonas/farmacologia , Animais , Antraquinonas/farmacologia , Camptotecina/química , Camptotecina/farmacologia , DNA Topoisomerases/química , DNA Topoisomerases/genética , Resistência a Medicamentos , Feminino , Células Hep G2/parasitologia , Humanos , Leishmaniose Visceral/tratamento farmacológico , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Naftoquinonas/farmacologia , Quinonas/química , Baço/citologia , Inibidores da Topoisomerase/química , Inibidores da Topoisomerase/farmacologia
18.
Exp Parasitol ; 207: 107789, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31669169

RESUMO

American visceral leishmaniasis is caused by the protozoan Leishmania infantum. The control of the disease depends on the magnitude of the Th1 cell response and IL-10 producing regulatory T cells. Administration of chemokine, such as CXCL10, has shown promising results in the leishmaniasis treatment. Previous studies from our group have shown that CXCL10 induces a reduction in parasite burden in the spleen and a decrease in IL-10 and TGF-ß production in L. infantum-infected BALB/c mice. This work investigated whether CXCL10-treatment reduces IL-10 + Treg cell populations (CD4+CD25+Foxp3+ and Tr1) and induces morphological changes in the spleen. BALB/c mice were infected and treated or not with CXCL10 on the 1st, 3rd and 7th days of infection. CXCL10-treatment was able to reduce the parasite load in the spleen in L. infantum-infected BALB/c mice and this decrease in the number of parasites correlated with the decrease in size of this organ in treated animals compared to untreated animals. 7, 23, and 45 days post-treatment (p.t.), the phenotype and frequency of IL-10 + Treg cells were evaluated by flow cytometry, and the morphological changes of the spleen were analyzed by optical microscopy. After 7 and 23 days p.t., CXCL10-treated animals showed a significant reduction of CD25-Foxp3-IL-10+ (Tr1) cells in the spleen when compared to untreated animals, whereas CD4+CD25+Foxp3+IL-10+ Treg cells reduced later at 23rd and 45th days p.t. Furthermore, while untreated animals showed a significant positive correlation between IL-10 production and Tr1 cells, in CXCL10-treated group this correlation was negative. Thus, these findings show that treatment with CXCL10 chemokine in L. infantum-infected BALB/c mice results in suppression of IL10+ Treg (Foxp3+ and Tr1) cells in the spleen, associated with a reduction in parasite load and splenomegaly.


Assuntos
Quimiocina CXCL10/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Baço/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Adjuvantes Imunológicos/uso terapêutico , Animais , Quimiocina CXCL10/administração & dosagem , Quimiocina CXCL10/farmacologia , Cricetinae , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Humanos , Injeções Intraperitoneais , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/imunologia , Leishmania infantum/patogenicidade , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/imunologia , Carga Parasitária , Baço/parasitologia , Baço/patologia , Linfócitos T Reguladores/imunologia , Virulência
19.
J Med Chem ; 62(23): 10664-10675, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31702921

RESUMO

We report the discovery of new 4-hydroxyphenyl phosphonium salt derivatives active in the submicromolar range (EC50 from 0.04 to 0.28 µM, SI > 10) against the protozoan parasite Leishmania donovani. The pharmacokinetics and in vivo oral efficacy of compound 1 [(16-(2,4-dihydroxyphenyl)-16-oxohexadecyl)triphenylphosphonium bromide] in a mouse model of visceral leishmaniasis were established. Compound 1 reduced the parasite load in spleen (98.9%) and liver (95.3%) of infected mice after an oral dosage of four daily doses of 1.5 mg/kg. Mode of action studies showed that compound 1 diffuses across the plasma membrane, as designed, and targets the mitochondrion of Leishmania parasites. Disruption of the energetic metabolism, with a decrease of intracellular ATP levels as well as mitochondrial depolarization together with a significant reactive oxygen species production, contributes to the leishmanicidal effect of 1. Importantly, this compound was equally effective against antimonials and miltefosine-resistant clinical isolates of Leishmania infantum, indicating its potential as antileishmanial lead.


Assuntos
Antiprotozoários/química , Antiprotozoários/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Animais , Antiprotozoários/síntese química , Fragmentação do DNA , Descoberta de Drogas , Resistência a Medicamentos , Feminino , Leishmania donovani/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Espécies Reativas de Oxigênio , Relação Estrutura-Atividade
20.
BMC Vet Res ; 15(1): 405, 2019 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-31706354

RESUMO

BACKGROUND: Canine leishmaniasis is a zoonotic disease caused by Leishmania infantum, being the dogs one of the major reservoirs of human visceral leishmaniasis. DNA topology is a consolidated target for drug discovery. In this regard, topoisomerase IB - one of the enzymes controlling DNA topology - has been poisoned by hundreds of compounds that increase DNA fragility and cell death. Aromathecins are novel molecules with a multiheterocyclic ring scaffold that have higher stability than camptothecins. RESULTS: Aromathecins showed strong activity against both forms of L. infantum parasites, free-living promastigotes and intra-macrophagic amastigotes harbored in ex vivo splenic explant cultures obtained from infected BALB/c mice. However, they prevented the relaxation activity of leishmanial topoisomerase IB weakly, which suggests that the inhibition of topoisomerase IB partially explains the antileishmanial effect of these compounds. The effect of aromathecins was also studied against a strain resistant to camptothecin, and results suggested that the trafficking of these compounds is not through the ABCG6 transporter. CONCLUSIONS: Aromathecins are promising novel compounds against canine leishmaniasis that can circumvent potential resistances based on drug efflux pumps.


Assuntos
Antiprotozoários/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Leishmania infantum/efeitos dos fármacos , Inibidores da Topoisomerase I/farmacologia , Animais , Técnicas de Cultura de Células , DNA Topoisomerases Tipo I/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Feminino , Leishmania infantum/enzimologia , Leishmania infantum/crescimento & desenvolvimento , Estágios do Ciclo de Vida/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/antagonistas & inibidores , Baço/parasitologia
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