Computer-aided drug design approaches applied to screen natural product’s structural analogs targeting arginase in Leishmania spp [version 3; peer review: 1 approved, 2 approved with reservations]

Introduction: Leishmaniasis is a disease with high mortality rates and approximately 1.5 million new cases each year. Despite the new approaches and advances to fight the disease, there are no effective therapies. Methods: Hence, this study aims to screen for natural products' structural analogs as new drug candidates against leishmaniasis. We applied Computer-aided drug design (CADD) approaches, such as virtual screening, molecular docking, molecular dynamics simulation, molecular mechanics–generalized Born surface area (MM–GBSA) binding free estimation, and free energy perturbation (FEP) aiming to select structural analogs from natural products that have shown anti-leishmanial and anti-arginase activities and that could bind selectively against the Leishmania arginase enzyme. Results: The compounds 2H-1-benzopyran, 3,4-dihydro-2-(2-methylphenyl)-(9CI), echioidinin, and malvidin showed good results against arginase targets from three parasite species and negative results for potential toxicities. The echioidinin and malvidin ligands generated interactions in the active center at pH 2.0 conditions by MM-GBSA and FEP methods. Conclusions: This work suggests the potential anti-leishmanial activity of the compounds and thus can be further in vitro and in vivo experimentally validated

In vivo antileishmanial efficacy of a naphthoquinone derivate incorporated into a Pluronic? F127-based polymeric micelle system against Leishmania amazonensis infection.

New therapeutic strategies against leishmaniasis are desirable, since the treatment against disease presents problems, such as the toxicity, high cost and/or parasite resistance. As consequence, new antileishmanial compounds are necessary to be identified, as presenting high activity against Leishmania parasites, but low toxicity in mammalian hosts. Flau-A is a naphthoquinone derivative recently showed to presents an in vitro effective action against Leishmania amazonensis and L. infantum species. In the present work, the in vivo efficacy of Flau-A, which was incorporated into a Poloxamer 407-based micelle system, was evaluated in a murine model against L. amazonensis infection. Amphotericin B (AmB) and Ambisome? were used as controls. The animals were infected and later treated with the compounds. Thirty days after the treatment, parasitological and immunological parameters were evaluated. Results showed that AmB, Ambisome? , Flau-A or Flau-A/M-treated animals presented significantly lower average lesion diameter and parasite burden in tissue and organs evaluated, when compared to the control (saline and micelle) groups. Flau-A or Flau-A/M-treated mice were those presenting the most significant reductions in the parasite burden, when compared to the others. These animals developed also a more polarized antileishmanial Th1 immune response, which was based on significantly higher levels of IFN-?, IL-12, TNF-?, GM-CSF, and parasite-specific IgG2a isotype; associated with low levels of IL-4, IL10, and IgG1 antibody. The absence of toxicity was found in these animals, although mice receiving AmB have showed high levels of renal and hepatic damage markers. In conclusion, results suggested that the Flau-A/M compound may be considered as a possible therapeutic target to be evaluated against human leishmaniasis

Recent updates and perspectives on approaches for the development of vaccines against visceral leishmaniasis

All rights reserved. Visceral leishmaniasis (VL) is one of the most important tropical diseases worldwide. Although chemotherapy has been widely used to treat this disease, problems related to the development of parasite resistance and side effects associated with the compounds used have been noted. Hence, alternative approaches for VL control are desirable. Some methods, such as vector control and culling of infected dogs, are insufficiently effective, with the latter not ethically recommended. The development of vaccines to prevent VL is a feasible and desirable measure for disease control, for example, some vaccines designed to protect dogs against VL have recently been brought to market. These vaccines are based on the combination of parasite fractions or recombinant proteins with adjuvants that are able to induce cellular immune responses, however, their partial efficacy and the absence of a vaccine to protect against human leishmaniasis underline the need for characterization of new vaccine candidates. This review presents recent advances in control measures for VL based on vaccine development, describing extensively studied antigens, as well as new antigenic proteins recently identified using immuno-proteomic techniquesThis work was supported by grants from Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica, Rede Nanobiotec/Brasil-Universidade Federal de Uberlândia/CAPES, PRONEX-FAPEMIG (APQ-01019-09), FAPEMIG (CBB-APQ-00819-12 and CBB-APQ-01778-2014), and CNPq (APQ-482976/2012-8, APQ-488237/2013-0, and APQ-467640/2014-9). EAFC and LRG are recipients of the grant from CNPq. MACF is the recipient of grants from FAPEMIG/CAPE

Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis.

Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons-? (IFN-?), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-? (TNF-?), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-? and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-?, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-? production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease

Avaliação de uma proteína hipotética específica de Leishmania no sorodiagnóstico e desenvolvimento de uma vacina contra as leishmanioses

Exportado OPUSMade available in DSpace on 2019-08-13T16:26:18Z (GMT). No. of bitstreams: 3 disserta__o_final_daniela_pdf.pdf: 8938758 bytes, checksum: aa8881c65b17387d4d115d7bf6a92d21 (MD5) daniela_pagliara_lage___imt_ata_de_defesa.pdf: 308676 bytes, checksum: 69426377a1107cde87a4f6438e6c05a3 (MD5) ficha_catalogr_fica_daniela_pagliara.pdf: 9157 bytes, checksum: d67fe336613c0b82086b3b9344a8319e (MD5) Previous issue date: 6A leishmaniose é um complexo de doenças incidente no Brasil e no mundo, apresentando elevada morbidade e mortalidade. Nosso país responde por aproximadamente 95% dos casos de leishmaniose visceral (LV) nas Américas, sendo o cão o principal reservatório doméstico da doença. O sorodiagnóstico da LV canina (LVC) apresenta problemas relacionados à sua sensibilidade e/ou especificidade. No presente estudo, uma proteína hipotética específica de Leishmania, LiHyD, sob sua forma recombinante (rLiHyD), foi avaliada em experimentos de ELISA para o sorodiagnóstico da LVC. Três epitopos de linfócitos B da proteína foram sintetizados (Peptídeo-1, Peptídeo-2 e Peptídeo3) e também avaliados como marcadores diagnósticos. A proteína recombinante e o Peptídeo-3 mostraram os melhores resultados, tendo sido reconhecidos por anticorpos presentes em soros de cães com VL sintomática e assintomática, e não apresentaram reatividade cruzada com anticorpos presentes em soros de cães com doença de Chagas, ehrlichiose, babesiose ou de cães sem leishmaniose e/ou vacinados com a vacina Leish-Tec®. Na busca por se selecionar um antígeno candidato a compor uma vacina contra as leishmanioses, uma vacina baseada na combinação da proteína rLiHyD com saponina foi testada em camundongos BALB/c contra a infecção causada pelas espécies Leishmania infantum, Leishmania major e Leishmania braziliensis. A imunogenicidade da vacina foi avaliada e os resultados mostraram que os animais imunizados produziram níveis elevados de IFN-, IL-12 e GMCSF após o estímulo in vitro de esplenócitos com a proteína ou usando os extratos proteicos de L. infantum, L. major ou L. braziliensis. Após o desafio, os animais vacinados mostraram reduções significativas na carga parasitária em todos os órgãos e tecidos avaliados, quando comparados com os animais que receberam salina ou que foram imunizados apenas com saponina ou com a proteína isolada. A proteção obtida com a vacina rLiHyD/saponina foi associada com a produção de IFN- específica contra o parasito e dependente de IL-12, que foi produzida principalmente por linfócitos T CD4+. Nesses animais, uma redução na produção de IL-4 e IL-10, associada à presença de níveis elevados de anticorpos do isotipo IgG2a específicos à proteína e aos parasitos, foram também encontrados. O presente estudo mostrou que a proteína hipotética LiHyD, inicialmente identificada em L. infantum, pode ser utilizada como um antígeno para o sorodiagnóstico da LVC e, quando associada a um adjuvante Th1, pode também compor uma vacina para proteção contra as leishmanioses visceral e tegumentar.Leishmaniasis is a disease complex with a large incidence in Brazil and in the world, presenting high morbidity and mortality. Our country accounts for approximately 95% of the VL cases in Americas, being the dog the main domestic reservoir of the parasites. The serodiagnosis of canine visceral leishmaniasis (CVL) presents problems related to its sensitivity and/or specificity. In the present study, a Leishmania-specific hypothetical protein, rLiHyD, in a recombinant form (rLiHyD), was evaluated in ELISA experiments for the CVL serodiagnosis. Three B cell epitopes of LiHyD were synthesized (Peptide-1, Peptide-2 and Peptide-3) and also evaluated as diagnostic markers. The recombinant protein and the Peptide-3 showed the best results, being recognized by antibodies in sera from dogs with asymptomatic and symptomatic VL, and did not show cross-reactivity with antibodies in dog sera of dogs with Chagas disease, ehrlichiosis, babesiosis or animals without leishmaniasis and/or vaccinated with the Leish-Tec® vaccine. In the search to also select a candidate antigen for composing a vaccine against leishmaniasis, a combination between rLiHyD and saponin was tested in BALB/c mice against infection by Leishmania infantum, Leishmania major and Leishmania braziliensis. The immunogenicity of rLiHyD/saponin vaccine was evaluated, and the results showed that immunized mice produced higher levels of IFN-, IL-12 and GM-CSF after splenocytes in vitro stimulation with rLiHyD or L. infantum, L. major or L. braziliensis protein extracts. After challenge, vaccinated animals showed significant reductions in the parasite burden in all evaluated organs and tissues, when compared to those that were inoculated with saline or immunized with saponin or the protein alone. The protection obtained with the rLiHyD/saponin was associated with a parasite-specific IL-12dependent IFN- production, which was produced mainly by CD4+T cells. In these animals, a decrease in the parasite-mediated IL-4 and IL-10 responses, associated with the presence of high levels of LiHyD and parasite-specific IgG2a isotype antibodies, were also found. The present study showed that the hypothetical protein LiHyD, which was firstly identified in L. infantum, can be used for the CVL serodiagnosis and, when combined with a Th1 adjuvant, can compose a vaccine and confer protection against visceral and tegumentary leishmaniasis

Treatment of murine visceral leishmaniasis using an 8-hydroxyquinoline-containing polymeric micelle system.

Newtherapeutics are urgently needed to treat visceral leishmaniasis (VL). Due to the fact that drug discovery is a long and expensive process, the development of delivery systems to carry old and toxic drugs could be considered, as well as the evaluation of new molecules that have already shown to present biological activity. In this context, the present study evaluated the in vitro and in vivo antileishmanial activity of an 8-hydroxyquinoline (8-HQN)-containing polymeric micelle (8-HQN/M) system against Leishmania infantum, the main causative agent of VL in the Americas. The experimental strategy used was based on the evaluation of the parasite load by a limiting-dilution technique in the spleen, liver, bone marrow and draining lymph nodes of the infected and treated animals, as well as by a quantitative PCR (qPCR) technique to also assess the splenic parasite load. The immune response developed was evaluated by the production of IFN-γ, IL-4, IL-10, IL-12 and GM-CSF cytokines, as well as by antileishmanial nitrite dosage and antibodies production. Hepatic and renal enzymes were also investigated to verify cellular injury as a result of treatments toxicity. In the results, 8-HQN/M-treated mice, when compared to the other groups: saline, free amphotericin B (AmpB, as a drug control), 8-HQN and B-8-HQN/M (as a micelle control) showed more significant reductions in their parasite burden in all evaluated organs. These animals also showed an antileishmanial Th1 immunity, which was represented by high levels of IFN-γ, IL-12, GM-CSF and nitrite, associated with a low production of IL-4 and IL-10 and anti-Leishmania IgG1 isotype antibodies. In addition, any hepatic or renal damage was found in these treated animals. In conclusion, 8-HQN/M was effective in treating L. infantum-infected BALB/c mice, and can be considered alone, or combined with other drugs, as an alternative treatment for VL

Poloxamer 407 (Pluronic® F127)-based polymeric micelles for amphotericin B : in vitro biological activity, toxicity and in vivo therapeutic efficacy against murine tegumentary leishmaniasis.

In the present study, a Poloxamer 407-based amphotericin B (AmpB)-containing polymeric micelles system (AmpB/M) was employed in the treatment of Leishmania amazonensis-infected BALB/c mice. Initially, the in vitro antileishmanial activity (IC50 value) of AmpB/M and B-AmpB/M (empty micelles) against stationary promastigotes and amastigotes-like forms of the parasites was determined, and results were of 1.83 ± 0.4 and 22.1 ± 0.7 mM, respectively, for the promastigotes, and of 2.27 ± 0.5 and 33.98 ± 2.6 mM, respectively, for the amastigotes-like. The cytotoxic concentration (CC50) values of these products were also evaluated, and we found the results of 119.5 ± 9.6 and 134.7 ± 10.3 mM, respectively. With these values, the selectivity index (SI) was calculated and results were of 65.3 and 5.4, respectively, for the promastigotes, and of 59.3 and 3.96, respectively, for the amastigotes-like of the parasites. Free AmpB showed IC50 values of 1.2 ± 0.3 and 2.5 ± 0.5 mM for the promastigotes and amastigotes-like, respectively, whereas the CC50 value was of 9.5 ± 0.4 mM. The SI values of this drug were of 7.9 and 3.8, respectively, for the promastigote and amastigote-like stages of the parasites. After, animals were infected and received saline or were treated subcutaneously with free AmpB, AmpB/M or B-AmpB/M. In the results, free AmpB-treated and infected mice showed reductions in their body weight, which were associated with hepatic and renal damage; however, no organic alteration was observed in the AmpB/Mtreated animals. In addition, these animals showed significant reductions in their lesion average size and in the parasite burden in all evaluated infected tissue and organs, when compared to the other groups; as well as significantly higher levels of antileishmanial IFN-g, IL-12, GM-CSF and nitrite, which were associated with low production of IL-4, IL-10 and IgG1 isotype antibodies. In conclusion, this AmpB/M system could be considered as an alternative for future studies in the treatment of tegumentary leishmaniasis

Recent updates and perspectives on approaches for the development of vaccines against visceral leishmaniasis

Abstract: Visceral leishmaniasis (VL) is one of the most important tropical diseases worldwide. Although chemotherapy has been widely used to treat this disease, problems related to the development of parasite resistance and side effects associated with the compounds used have been noted. Hence, alternative approaches for VL control are desirable. Some methods, such as vector control and culling of infected dogs, are insufficiently effective, with the latter not ethically recommended. The development of vaccines to prevent VL is a feasible and desirable measure for disease control; for example, some vaccines designed to protect dogs against VL have recently been brought to market. These vaccines are based on the combination of parasite fractions or recombinant proteins with adjuvants that are able to induce cellular immune responses; however, their partial efficacy and the absence of a vaccine to protect against human leishmaniasis underline the need for characterization of new vaccine candidates. This review presents recent advances in control measures for VL based on vaccine development, describing extensively studied antigens, as well as new antigenic proteins recently identified using immuno-proteomic techniques

Strychnos pseudoquina

The development of new and cost-effective alternative therapeutic strategies to treat leishmaniasis has become a high priority. In the present study, the antileishmanial activity of Strychnos pseudoquina St. Hil. was investigated and pure compounds that presented this biological effect were isolated. An ethyl acetate extract was prepared, and it proved to be effective against Leishmania amazonensis. A bioactivity-guided fractionation was performed, and two flavonoids were identified, quercetin 3-O-methyl ether and strychnobiflavone, which presented an effective antileishmanial activity against L. amazonensis, and studies were extended to establish their minimum inhibitory concentrations (IC50), their leishmanicidal effects on the intra-macrophage Leishmania stage, as well as their cytotoxic effects on murine macrophages (CC50), and in O+ human red blood cells. The data presented in this study showed the potential of an ethyl acetate extract of S. pseudoquina, as well as two flavonoids purified from it, which can be used as a therapeutic alternative on its own, or in association with other drugs, to treat disease evoked by L. amazonensis