A docking-based structural analysis of geldanamycin-derived inhibitor binding to human or Leishmania Hsp90.

Leishmaniasis is a neglected disease that affects millions of individuals around the world. Regardless of clinical form, treatment is based primarily on the use of pentavalent antimonials. However, such treatments are prolonged and present intense side effects, which lead to patient abandonment in many cases. The search for chemotherapeutic alternatives has become a priority. Heat Shock Protein 90 (Hsp90) inhibitors have recently come under investigation due to antiparasitic activity in Plasmodium sp., Trypanosoma sp. and Leishmania sp. Some of these inhibitors, such as geldanamycin and its analogs, 17-AAG and 17-DMAG, bind directly to Hsp90, thereby inhibiting its activity. Previous studies have demonstrated that different parasite species are more susceptible to some of these inhibitors than host cells. We hypothesized that this increased susceptibility may be due to differences in binding of Hsp90 inhibitors to Leishmania protein compared to host protein. Based on the results of the in silico approach used in the present study, we propose that geldanamycin, 17-AAG and 17-DMAG present an increased tendency to bind to the N-terminal domain of Leishmania amazonensis Hsp83 in comparison to human Hsp90. This could be partially explained by differences in intermolecular interactions between each of these inhibitors and Hsp83 or Hsp90. The present findings demonstrate potential for the use of these inhibitors in the context of anti-Leishmania therapy.

Proteomic Analysis Reveals a Predominant NFE2L2 (NRF2) Signature in Canonical Pathway and Upstream Regulator Analysis of Leishmania-Infected Macrophages.

CBA mice macrophages (MØ) control infection by Leishmania major and are susceptive to Leishmania amazonensis, suggesting that both parasite species induce distinct responses that play important roles in infection outcome. To evaluate the MØ responses to infection arising from these two Leishmania species, a proteomic study using a Multidimensional Protein Identification Technology (MudPIT) approach with liquid chromatography tandem mass spectrometry (LC-MS/MS) was carried out on CBA mice bone-marrow MØ (BMMØ). Following SEQUEST analysis, which revealed 2,838 proteins detected in BMMØ, data mining approach found six proteins significantly associated with the tested conditions. To investigate their biological significance, enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). A three steps IPA approach revealed 4 Canonical Pathways (CP) and 7 Upstream Transcriptional Factors (UTFs) strongly associated with the infection process. NRF2 signatures were present in both CPs and UTFs pathways. Proteins involved in iron metabolism, such as heme oxigenase 1 (HO-1) and ferritin besides sequestosome (SQSMT1 or p62) were found in the NRF2 CPs and the NRF2 UTFs. Differences in the involvement of iron metabolism pathway in Leishmania infection was revealed by the presence of HO-1 and ferritin. Noteworty, HO-1 was strongly associated with L. amazonensis infection, while ferritin was regulated by both species. As expected, higher HO-1 and p62 expressions were validated in L. amazonensis-infected BMMØ, in addition to decreased expression of ferritin and nitric oxide production. Moreover, BMMØ incubated with L. amazonensis LPG also expressed higher levels of HO-1 in comparison to those stimulated with L. major LPG. In addition, L. amazonensis-induced uptake of holoTf was higher than that induced by L. major in BMMØ, and holoTf was also detected at higher levels in vacuoles induced by L. amazonensis. Taken together, these findings indicate that NRF2 pathway activation and increased HO-1 production, together with higher levels of holoTf uptake, may promote permissiveness to L. amazonensis infection. In this context, differences in protein signatures triggered in the host by L. amazonensis and L. major infection could drive the outcomes in distinct clinical forms of leishmaniasis.

Encapsulation of the HSP-90 Chaperone Inhibitor 17-AAG in Stable Liposome Allow Increasing the Therapeutic Index as Assessed, in vitro, on Leishmania (L) amazonensis Amastigotes-Hosted in Mouse CBA Macrophages.

The current long-term treatment for leishmaniasis causes severe side effects and resistance in some cases. An evaluation of the anti-leishmanial potential of an HSP90-inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), demonstrated its potent effect against Leishmania spp. in vitro and in vivo. We have previously shown that 17-AAG can kill L. (L) amazonensis promastigotes with an IC50 of 65 nM and intracellular amastigote at concentrations as low as 125 nM. As this compound presents low solubility and high toxicity in human clinical trials, we prepared an inclusion complex containing hydroxypropyl-ß-cyclodextrin and 17-AAG (17-AAG:HPßCD) to improve its solubility. This complex was characterized by scanning electron microscopy, and X-ray diffraction. Liposomes-containing 17-AAG:HPßCD was prepared and evaluated for encapsulation efficiency (EE%), particle size, polydispersity index (PDI), pH, and zeta potential, before and after accelerated and long-term stability testing. An evaluation of leishmanicidal activity against promastigotes and intracellular amastigotes of L. (L) amazonensis was also performed. The characterization techniques utilized confirmed the formation of the inclusion complex, HPßCD:17-AAG, with a resulting 33-fold-enhancement in compound water solubility. Stability studies revealed that 17-AAG:HPßCD-loaded liposomes were smaller than 200 nm, with 99% EE. Stability testing detected no alterations in PDI that was 0.295, pH 7.63, and zeta potential +22.6, suggesting liposome stability, and suitability for evaluating leishmanicidal activity. Treatment of infected macrophages with 0.006 nM of 17-AAG:HPßCD or 17-AAG:HPßCD-loaded liposomes resulted in almost complete amastigote clearance inside macrophages after 48 h. This reduction is similar to the one observed in infected macrophages treated with 2 µM amphotericin B. Our results showed that nanotechnology and drug delivery systems could be used to increase the antileishmanial efficacy and potency of 17-AAG in vitro, while also resulting in reduced toxicity that indicates these formulations may represent a potential therapeutic strategy against leishmaniasis.

In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195.

BACKGROUND: Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE: To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS: The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS: Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS: PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.

In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195

BACKGROUND Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.

Advances in Development of New Treatment for Leishmaniasis.

Leishmaniasis is a neglected infectious disease caused by several different species of protozoan parasites of the genus Leishmania. Current strategies to control this disease are mainly based on chemotherapy. Despite being available for the last 70 years, leishmanial chemotherapy has lack of efficiency, since its route of administration is difficult and it can cause serious side effects, which results in the emergence of resistant cases. The medical-scientific community is facing difficulties to overcome these problems with new suitable and efficient drugs, as well as the identification of new drug targets. The availability of the complete genome sequence of Leishmania has given the scientific community the possibility of large-scale analysis, which may lead to better understanding of parasite biology and consequent identification of novel drug targets. In this review we focus on how high-throughput analysis is helping us and other groups to identify novel targets for chemotherapeutic interventions. We further discuss recent data produced by our group regarding the use of the high-throughput techniques and how this helped us to identify and assess the potential of new identified targets.

Preparation and characterization of a bacterial cellulose/silk fibroin sponge scaffold for tissue regeneration.

Bacterial cellulose (BC) and silk fibroin (SF) are natural biopolymers successfully applied in tissue engineering and biomedical fields. In this work nanocomposites based on BC and SF were prepared and characterized by scanning electron microscopy (SEM), infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA). In addition, the investigation of cytocompatibility was done by MTT, XTT and Trypan Blue dye technique. Cellular adhesion and proliferation were detected additionally. The evaluation of genotoxicity was realized by micronucleus assay. In vitro tests showed that the material is non-cytotoxic or genotoxic. SEM images revealed a greater number of cells attached at the BC/SF:50% scaffold surface than the pure BC one, suggesting that the presence of fibroin improved cell attachment. This could be related to the SF amino acid sequence that acts as cell receptors facilitating cell adhesion and growth. Consequently, BC/SF:50% scaffolds configured an excellent option in bioengineering depicting its potential for tissue regeneration and cultivation of cells on nanocomposites.

Avaliação do 17-AAG como agente leishmanicida e seu mecanismo de ação na indução da morte de parasitos do gênero leishmania spp / 17-AAG assessment as leishmanicidal agent and its mechanism of action for induction of parasites of the Leishmania genus death spp

A leishmaniose é uma doença endêmica no Brasil causada por parasitos protozoários do gênero Leishmania. A quimioterapia continua sendo a forma mais efetiva de tratamento com os antimoniais pentavalentes sendo usados há mais de 70 anos como a primeira linha de tratamento. O uso deste e de outros fármacos apresenta efeitos adversos graves, os esquemas terapêuticos empregados são desconfortáveis, além de relatos do aumento de casos de resistência. A proteína de choque térmico 90 (HSP90) é um membro da família das chaperonas presente em células eucarióticas e bactérias. Essa proteína é fundamental para o dobramento e estabilização de diferentes proteínas, chamadas genericamente de proteínas cliente. Essa chaperona vem sendo considerada um importante alvo molecular para o tratamento de diferentes doenças parasitárias. Nessa tese, o inibidor específico da atividade ATPásica da HSP90, o 17-allilamino-17-demethoxigeldanamicina (17- AAG) foi testado em parasitos do gênero Leishmania. Inicialmente, avaliamos o efeito em cultura axênica e observamos que o 17-AAG causa a morte desses parasitos em concentrações inferiores às necessárias para causar a morte de macrófagos. Observamos também que o tratamento com 17-AAG promove a morte intracelular dos parasitos em concentrações que variam de 25 a 500 nM nos tempos de 24 e 48 h...

Leishmaniases are endemic disease in Brazil caused by protozoan parasites from the genus Leishmania. Chemotherapy remains the most effective way of treatment and pentavalent antimonials, used for more than 70 years, remaining as first choice drugs for leishmaniasis treatment. The use of this and other drugs causes severe side effects, therapeutic regimens employed for leishmaniasis treatment are unpleasant, besides an increase number of resistance cases. The Heat Shock Protein 90 (HSP90) is a member of the chaperone family present in bacteria and eukaryotic cells. This protein is essential for the folding and stabilization of different proteins, known as client proteins. This chaperone has been considered an important molecular target for the treatment of different parasitic diseases. In this thesis, the specific inhibitors of the ATPase activity from the HSP90, 17-allylamino- 17-demethoxygeldanamycin (17-AAG), were tested against parasites from the genus Leishmania. First we evaluated its effect on axenic culture and observed that 17- AAG induces parasite cell death in lowerconcentrations than those needed to induce macrophage cell death. We also observed that 17-AAG intracellular parasite death in concentrations ranging from 25 to 500 nM after 24 or 48 h...

17-AAG kills intracellular Leishmania amazonensis while reducing inflammatory responses in infected macrophages.

BACKGROUND: Leishmaniasis is a neglected endemic disease with a broad spectrum of clinical manifestations. Pentavalent antimonials have been the treatment of choice for the past 70 years and, due to the emergence of resistant cases, the efficacy of these drugs has come under scrutiny. Second-line drugs are less efficacious, cause a range of side effects and can be costly. The formulation of new generations of drugs, especially in developing countries, has become mandatory. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the anti-leishmanial effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an HSP90 inhibitor, in vitro. This inhibitor is currently in clinical trials for cancer treatment; however, its effects against intracellular Leishmania remain untested. Macrophages infected with L. amazonensis were treated with 17-AAG (25-500 nM) and parasite load was quantified using optical microscopy. Parasite load declined in 17-AAG-treated macrophages in a dose- and time-dependent manner. Intracellular parasite death became irreversible after 4 h of treatment with 17-AAG, and occurred independent of nitric oxide (NO) and superoxide (O(2) (-)) production. Additionally, intracellular parasite viability was severely reduced after 48 h of treatment. Interestingly, treatment with 17-AAG reduced pro-inflammatory mediator production, including TNF-α, IL-6 and MCP-1, yet IL-12 remained unaffected. Electron microscopy revealed morphological alterations, such as double-membrane vacuoles and myelin figures at 24 and 48 h after 17-AAG treatment. CONCLUSIONS/SIGNIFICANCE: The HSP90 inhibitor, 17-AAG, possesses high potency under low dosage and reduces both pro-inflammatory and oxidative molecule production. Therefore, further studies are warranted to investigate this inhibitor's potential in the development of new generations of anti-leishmanials.

Control of Mycobacterium fortuitum and Mycobacterium intracellulare infections with respect to distinct granuloma formations in livers of BALB/c mice.

Mycobacterium fortuitum is a rapidly growing nontuberculous Mycobacterium that can cause a range of diseases in humans. Complications from M. fortuitum infection have been associated with numerous surgical procedures. A protective immune response against pathogenic mycobacterial infections is dependent on the granuloma formation. Within the granuloma, the macrophage effector response can inhibit bacterial replication and mediate the intracellular killing of bacteria. The granulomatous responses of BALB/c mice to rapidly and slowly growing mycobacteria were assessed in vivo and the bacterial loads in spleens and livers from M. fortuitum and Mycobacterium intracellulare-infected mice, as well as the number and size of granulomas in liver sections, were quantified. Bacterial loads were found to be approximately two times lower in M. fortuitum-infected mice than in M. intracellulare-infected mice and M. fortuitum-infected mice presented fewer granulomas compared to M. intracellulare-infected mice. These granulomas were characterized by the presence of Mac-1+ and CD4+ cells. Additionally, IFN-γmRNA expression was higher in the livers of M. fortuitum-infected mice than in those of M. intracellulare-infected mice. These data clearly show that mice are more capable of controlling an infection with M. fortuitum than M. intracellulare. This capacity is likely related to distinct granuloma formations in mice infected with M. fortuitum but not with M. intracellulare.

Control of Mycobacterium fortuitum and Mycobacterium intracellulare infections with respect to distinct granuloma formations in livers of BALB/c mice

Mycobacterium fortuitum is a rapidly growing nontuberculous Mycobacterium that can cause a range of diseases in humans. Complications from M. fortuitum infection have been associated with numerous surgical procedures. A protective immune response against pathogenic mycobacterial infections is dependent on the granuloma formation. Within the granuloma, the macrophage effector response can inhibit bacterial replication and mediate the intracellular killing of bacteria. The granulomatous responses of BALB/c mice to rapidly and slowly growing mycobacteria were assessed in vivo and the bacterial loads in spleens and livers from M. fortuitum and Mycobacterium intracellulare-infected mice, as well as the number and size of granulomas in liver sections, were quantified. Bacterial loads were found to be approximately two times lower in M. fortuitum-infected mice than in M. intracellulare-infected mice and M. fortuitum-infected mice presented fewer granulomas compared to M. intracellulare-infected mice. These granulomas were characterized by the presence of Mac-1+ and CD4+ cells. Additionally, IFN-γmRNA expression was higher in the livers of M. fortuitum-infected mice than in those of M. intracellulare-infected mice. These data clearly show that mice are more capable of controlling an infection with M. fortuitum than M. intracellulare. This capacity is likely related to distinct granuloma formations in mice infected with M. fortuitum but not with M. intracellulare.