Estudo da internalização de macromoléculas em eritrócitos infectados pelo Plasmodium falciparum

The intraerythrocytic development of the malaria parasite Plasmodium falciparum is dependent on the uptake of essential nutrients from the host cell cytoplasm and blood plasma. It is widely recognized that it imports low molecular mass nutrients, such as polyols, amino acids, lipids, nucleosides, organic anions and cations from the plasma. However, although some studies show that P. falciparum is capable of importing macromolecules from the host, the route for internalization continues to be subject of debate between different groups, mainly due to the necessity of these macromolecules to pass through three membranes, namely the erythrocyte, parasitophorous vacuole and the parasite membrane itself. To better understand this process, this study provides new evidence to help elucidate the mechanism used by the parasite to internalize macromolecules with a focus on protein uptake. Using different experimentais approaches, we demonstrated that P. falciparum imports human plasminogen and the exogenous protein crotamine, at different stages of its intraeritrocitic development. In the infected erythrocyte, plasminogen is located in the parasite's cytosol, and is also present in the Maurer’s Cleft and tubular structures in the erythrocyte cytosol, which are described as being associated with the export of proteins. In addition, some interactions may be involved with the internalization of plasminogen, such as with Rabs proteins, kinesin, myosin, PfMC-2TM, Pf113 and heat shock proteins. There was a significant decrease in the uptake of plasminogen by the parasites after cytochalasin D treatment, suggesting a participation of the actin-myosin motor system in protein trafficking. Crotamine was selectively internalized by infected erythrocytes, and presented a colocalization with the parasite nucleus and part of the protein remains associated with the erythrocyte plasma membrane. The uptake was 2-deoxy-D-glucose sensitive, indicating that it is a mechanism dependent on energy via glycolysis. The results obtained here offer new elements involved in macromolecular uptake pathways in infected erythrocytes, which may constitute a potential target for the development of new drugs, in addition to identifying the permeability characteristics of the infected cell.O desenvolvimento intraeritrocítico do parasita da malária Plasmodium falciparum, é dependente da captação de uma série de nutrientes essenciais do citoplasma da célula hospedeira e do plasma sanguíneo. É amplamente reconhecido que o mesmo importa nutrientes de baixa massa molecular, tais como polióis, aminoácidos, lipídios, nucleosídeos, ânions orgânicos e cátions do plasma. No entanto, apesar de alguns estudos mostrarem que o P. falciparum é capaz de importar macromoléculas do hospedeiro, a rota para internalização continua sendo objeto de debate entre diferentes grupos, devido principalmente à necessidade destas macromoléculas passarem por três membranas, sendo elas a do eritrócito, vacúolo parasitóforo, e a do próprio parasita. Para melhor compreensão desse processo, este estudo fornece novas evidências que auxiliam na elucidação do mecanismo utilizado pelo parasita na internalização de macromoléculas, com um enfoque na captação de proteínas. Utilizando diferentes abordagens experimentais, demonstramos que o P. falciparum importa plasminogênio humano e a proteína exógena crotamina, em diferentes fases do seu desenvolvimento intraeritrócitico. No eritrócito infectado, o plasminogênio localiza-se no citosol do parasita, e também está presente nos Maurer’s Cleft e estruturas tubulares no citosol do eritrócito, as quais são descritas como associadas com a exportação de proteínas. Além disso, algumas interações podem estar envolvidas com a internalização do plasminogênio, tais como, com proteínas Rabs, cinesina, miosina, PfMC-2TM, Pf113 e heat shock proteins. Houve uma redução na captação do plasminogênio pelo parasita após o tratamento dos eritrócitos infectados com citocalasina D, o que sugere a participação do sistema motor actina-misiona no tráfego da proteína. Crotamina foi seletivamente internalizada por eritrócitos infectados, e apresentou uma colocalização com o núcleo do parasita e parte da proteína permaceu associada a membrana plasmática do eritrócito. Sua captação mostrou-se sensível ao tratamento com 2- deoxi-D-glicose, indicando ser um mecanismo dependente de energia via glicólise. Os resultados obtidos aqui oferecem novos elementos envolvidos nas vias de captação de macromoléculas em eritrócitos infectados, que podem constituir um potencial alvo para o desenvolvimento novos fármacos, além de identificar as características da permeabilidade da célula infectada.Dados abertos - Sucupira - Teses e dissertações (2019

Avaliação da atividade antimalárica de compostos telúricos e quimeras análogas à cloroquina

Malaria is a parasitic disease transmitted by the female Anopheles sp, infected by protozoa of Plasmodium. The cycle life of the protozoan involves invertebrate and vertebrate hosts, having an intraerytrocytic stage in vertebrates characterized by fever spikes and hemodynamic instability. The infection is a serious public health problem that infected 198 million people in 2013, causing 584 000 deaths, most on the African continent. The emergence of drug resistant strains of parasites determines the need for the development of new antimalarial drugs. In this sense, in the present study we tested two classes of .. compounds designed to act against Plasmodium falciparum. The first class comprises three derivatives of tellurium (RF05, RF07, RF19) and the second class comprises six chimeras based on the structure of chloroquine (GOS032, GOS033, GOS039 GOS042, GOS046 and GOS052). The damage caused by the compounds against the host was evaluated by cytotoxicity assays in HUVEC and hemolytic resistance experiments. For assessment of the antimalarial activity, P. falciparum strains 307 (not resistant to chloroquine) and W2 (chloroquine-resistant), were incubated for 72 hours with each compound. To elucidate the mechanism of action of the compounds as antimalarial, inhibition of proteases and cellular homeostasis assays in the parasites were performed. Except for the RF07, GOS032 and GOS046, the compounds have shown to be promising as antimalarial drugs, because they are not cytotoxic and hemolytic in concentrations that effectively inhibit the development of parasites in vitro of Plasmodium falciparum strains 307 and W2.A malária é uma doença parasitária transmitida pela fêmea do mosquito Anopheles sp, infectada pelo protozoário do gênero Plasmodium. O protozoário apresenta ciclo de vida envolvendo os hospedeiros invertebrado e vertebrado, possuindo um estágio intraeritrocítico no vertebrado caracterizado por picos febris e desequilíbrio hemodinâmico. A infecção é um sério problema de saúde pública que atingiu aproximadamente 198 milhões de pessoas em 2013, levando a 584 mil mortes, a maioria no continente africano. O crescente aparecimento de cepas resistentes aos fármacos disponíveis no mercado torna crítica a necessidade de se disponibilizar novas drogas antimaláricas. Neste sentido, o objetivo do nosso trabalho foi avaliar o potencial antimalárico de duas classes de compostos desenhados de modo a unir propriedades químicas que inibem o desenvolvimento do Plasmodium falciparum. A primeira compreende três derivados de telúrio nomeados RF05, RF07, RF19. O segundo grupo, engloba seis quimeras baseadas na estrutura da cloroquina, denominadas GOS032, GOS033, GOS039 GOS042, GOS046 e GOS052. Os danos causados pelos compostos no hospedeiro foram avaliados através de ensaios de citotoxicidade em HUVEC e resistência hemolítica. Para avaliação da ação antimalárica, foi determinada a parasitemia das cepas 307 (não resistente à cloroquina) e W2 (resistente à cloroquina) de P. falciparum, após incubação por 72 horas com cada composto. Para elucidar o mecanismo de ação antimalárica dos compostos, foram feitos testes de inibição das proteases e de homeostase celular dos parasitas. Com exceção do RF07, GOS032 e GOS046, os compostos mostraram-se promissores como potenciais antimaláricos, por não serem citotóxicos e nem hemolíticos em concentrações que inibem eficazmente o desenvolvimento dos parasitas in vitro das cepas 307 e W2 de P. falciparum.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Preparação, caracterização e estudo da eficiência na fotodegradação e adsorção de Rodamina B de heteroestruturas de TiO2/α-Fe2O3

A fotocatálise heterogênea, utilizando dióxido de titânio com estrutura do tipo anatase, tem se mostrado uma alternativa promissora para a remediação de sistemas aquáticos contaminados. Com o intuito de aumentar a eficiência do fotocatalisador, estudos científicos têm reportado a heterojunção deste óxido com estruturas do tipo hematita do óxido de ferro, α-Fe2O3. Neste estudo, foi investigada a síntese de nanoestruturas de TiO2 puro, α-Fe2O3 pura e heteroestruturas de TiO2/α-Fe2O3 em diferentes proporções dos óxidos a fim de testá-los como fotocatalisadores na degradação de um corante com elevada toxicidade, a Rodamina B. Os materiais foram sintetizados por rotas híbridas, utilizando o Metódo do Peróxido Oxidante com posterior tratamento hidrotérmico, totalizando a produção de 8 amostras. Foram empregadas três rotas de síntese: a rota (a) foi responsável pela síntese de TiO2 puro variando-se o pH do meio reacional na etapa de tratamento hidrotérmico, a rota (b) foi utilizada para a preparação de α-Fe2O3 pura e a rota (c) foi empregada na produção das heteroestruturas de TiO2/α-Fe2O3 nas concentrações de 0,1, 0,5, 1,0 e 5,0% p/p de hematita. As amostras foram caracterizadas quanto a sua estrutura, microestrutura e propriedades superficiais com o auxílio das técnicas de DRX, FT-Raman, método de BET, MEV/FEG, MET, TG e FT-IR. A análise dos resultados de caracterização estrutural mostrou que a rota (a) é eficiente para a síntese de TiO2 anatase quando realizada em meio básico, produzindo uma mistura de fases anatase/rutilo quando procedida em meio ácido. Sendo assim, na rota (c) foi utilizado o meio sintético básico para a síntese de TiO2 anatase e hematita. Em resumo, foi produzida pela rota (a) em meio básico a amostra HTTI1, que degradou a molécula de RhB em 95,0% e os materiais HTTI2 e HTTI3, produzidos em meio ácido, os quais degradaram a solução do corante em 70,9 e 24,4%, respectivamente, em 90 minutos de ensaio fotocatalítico, o que evidencia a importância em se obter nanomateriais com estrutura anatase do TiO2. Com o estudo da atividade fotocatalítica dos materiais produzidos, notou-se que alguns dos semicondutores apresentavam elevada taxa de adsorção no decorrer do processo. Devido a isso, a cinética de adsorção de RhB pelos materiais produzidos foi também estudada no período de 14 horas, cujos resultados mostraram que o óxido de ferro com estrutura hematita é um excelente adsorvente para o corante. Os resultados dos ensaios fotocatalíticos e de adsorção de RhB evidenciaram, principalmente, a elevada eficiência do TiO2 anatase como fotocatalisador e, em contrapartida, a elevada capacidade da hematita em adsorver as moléculas do corante. As heteroestruturas produzidas apresentaram o efeito de reduzir a eficiência na fotodegradação da RhB em relação a amostra HTTI1 com o aumento da concentração do óxido de ferro, porém as mesmas heterojunções se mostraram favoráveis para as propriedades adsortivas dos compostos quanto maior a concentração de α-Fe2O3.The heterogeneous photocatalysis using titanium dioxide with anatase type structure, has proved to be a promising alternative for the remediation of contaminated aquatic systems. In order to increase the efficiency of this photocatalyst, scientific studies have reported the heterojunction of this oxide with hematite structures of iron oxide, α-Fe2O3. In this study, it was investigated the synthesis of pure TiO2 nanostructures, pure α-Fe2O3 and TiO2/α-Fe2O3 heterostructures in different proportions in order to test them as photocatalysts in the degradation of a dye having high toxicity, the Rhodamine B. The materials were synthesized by hybrid routes, using the Oxidant Peroxo Method with subsequent hydrothermal treatment, producing a total of 8 samples. Three synthesis routes were employed: the route (a) was responsible for the pure TiO2 synthesis by varying the pH of the reaction medium in the hydrothermal treatment step, route (b) was used for the preparation of pure α-Fe2O3 and route (c) was employed in the production of TiO2/α-Fe2O3 heterostructures in concentrations of 0.1, 0.5, 1.0 and 5.0% w/w of hematite. The samples were characterized as their structure, microstructure and surface properties with XRD, FT-Raman, BET method, SEM/FEG, TEM, TG and FT-IR techniques. The results of the structural characterization showed that route (a) is effective for the synthesis of anatase TiO2 when carried out in basic medium, producing a mixture of phases anatase/rutile when performed in an acid medium. Thus, in route (c) it was used the basic synthetic medium for the synthesis of anatase TiO2 and hematite. In summary, it was produced by route (a) in a basic medium the HTTI1 sample, that degraded RhB molecule in 95.0%, HTTI2 and HTTI3 materials, produced in acid medium, which degraded dye solution in 70.9 and 24.4%, respectively, in 90 minutes of the photocatalytic test, which highlights the importance of obtaining nanomaterials with anatase TiO2 structure. With the study of the photocatalytic activity of the materials, it was noted that some of the semiconductor had high adsorption rate during the process. Because of this, the RhB adsorption kinetics by the materials produced was also studied within 14 hours. The results showed that the iron oxide with hematite structure is an excellent adsorbent for the dye. Especially, the results of the photocatalytic tests and RhB adsorption showed high efficiency of anatase TiO2 as a photocatalyst and, conversely, high capacity of hematite to adsorb dye molecules. The heterostructures produced had the effect of reducing the efficiency in photodegradation of RhB comparing with HTTI1 sample with increasing concentration of iron oxide, however the same heterojunctions demonstrated favorable for adsorptive properties of the compounds the greater the concentration of α-Fe2O3.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Theoretical insight into the mechanism for the inhibition of the cysteine protease cathepsin B by 1,2,4-thiadiazole derivatives

Several cellular disorders have been related to the overexpression of the cysteine protease cathepsin B (CatB), such as rheumatic arthritis, muscular dystrophy, osteoporosis, Alzheimer's disease, and tumor metastasis. Therefore, inhibiting CatB may be a way to control unregulated cellular functions and prevent tissue malformations. The inhibitory action of 1,2,4-thiadiazole (TDZ) derivatives has been associated in the literature with their ability to form disulfide bridges with the catalytic cysteine of CatB. In this work, we present molecular modeling and docking studies of a series of eight 1,2,4-thiadiazole compounds. Substitutions at two positions (3 and 5) on the 1,2,4-thiadiazole ring were analyzed, and the docking scores were correlated to experimental data. A correlation was found with the sequence of scores of four related compounds with different substituents at position 5. No correlation was observed for changes at position 3. In addition, quantum chemistry calculations were performed on smaller molecular models to study the mechanism of inhibition of TDZ at the active site of CatB. All possible protonation states of the ligand and the active site residues were assessed. The tautomeric form in which the proton is located on N2 was identified as the species that has the structural and energetic characteristics that would allow the ring opening of 1,2,4thiadiazole.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 mu M], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H+ homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles. (C) 2016 Elsevier Inc. All rights reserved.Fundacao de Amparo Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Univ Fed Sao Paulo UNIFESP, Dept Biofis, BR-04044020 Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Farmacol, Rua 3 de Maio 100,Ed INFAR,3rd Floor, BR-04044020 Sao Paulo, BrazilUniv Sao Paulo USP RP, Dept Bioquim & Imunol, Ribeirao Preto, BrazilUniv Fed Sao Paulo UNIFESP, Dept Biociencias, Rua Silva Jardim 136, BR-11015020 Santos, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dept Biofis, BR-04044020 Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Farmacol, Rua 3 de Maio 100,Ed INFAR,3rd Floor, BR-04044020 Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Biociencias, Rua Silva Jardim 136, BR-11015020 Santos, SP, BrazilWeb of Scienc

Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC50 values of 12 nM and 42 nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.Fundacao de Amparo a Pesquisa do Estado de Sao PauloConselho Nacional de Desenvolvimento Cientifico e TecnologicoUniv Fed Sao Paulo, Dept Biofis, Rua Pedro de Toledo 669, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Carlos, Dept Genet & Evolucao, Sao Carlos, SP, BrazilUniv Fed Sao Paulo, Dept Biociencias, Santos, BrazilUniv Fed Sao Paulo, Dept Biofis, Rua Pedro de Toledo 669, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biociencias, Santos, BrazilFAPESP: 13/12913-0FAPESP: 09/54598-9FAPESP: 09/53840-0FAPESP: 11/14403-4FAPESP: 12/50475-2FAPESP: 15/06861-3FAPESP: 15/19316-3FAPESP: 16/15298-3CNPq: 473.226/2010-3CNPq: 311745/2013-0Web of Scienc

Overexpression of Plasmodium falciparum M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development

Plasmodium falciparum, the most virulent of the human malaria parasite, is responsible for high mortality rates worldwide. We studied the M1 alanyl-aminopeptidase of this protozoan (PfA-M1), which is involved in the final stages of hemoglobin cleavage, an essential process for parasite survival. Aiming to help in the rational development of drugs against this target, we developed a new strain of P. falciparum overexpressing PfA-M1 without the signal peptide (overPfA-M1). The overPfA-M1 parasites showed a 2.5-fold increase in proteolytic activity toward the fluorogenic substrate alanyl-7-amido-4-methylcoumarin, in relation to the wild-type group. Inhibition studies showed that overPfA-M1 presented a lower sensitivity against the metalloaminopeptidase inhibitor bestatin and to other recombinant PfA-M1 inhibitors, in comparison with the wild-type strain, indicating that PfA-M1 is a target for the in vitro antimalarial activity of these compounds. Moreover, overPfA-M1 parasites present a decreased in vitro growth, showing a reduced number of merozoites per schizont, and also a decrease in the iRBC area occupied by the parasite in trophozoite and schizont forms when compared to the controls. Interestingly, the transgenic parasite displays an increase in the aminopeptidase activity toward Met-, Ala-, Leu- and Arg-7-amido-4-methylcoumarin. We also investigated the potential role of calmodulin and cysteine proteases in PfA-M1 activity. Taken together, our data show that the overexpression of PfA-M1 in the parasite cytosol can be a suitable tool for the screening of antimalarials in specific high-throughput assays and may be used for the identification of intracellular molecular partners that modulate their activity in P. falciparum

Synthesis, Structure–Activity Relationships, and Parasitological Profiling of Brussonol Derivatives as New Plasmodium falciparum Inhibitors

Malaria is a parasitic disease caused by protozoan parasites from the genus Plasmodium. Plasmodium falciparum is the most prevalent species worldwide and the causative agent of severe malaria. The spread of resistance to the currently available antimalarial therapy is a major concern. Therefore, it is imperative to discover and develop new antimalarial drugs, which not only treat the disease but also control the emerging resistance. Brussonol is an icetexane derivative and a member of a family of diterpenoids that have been isolated from several terrestrial plants. Here, the synthesis and antiplasmodial profiling of a series of brussonol derivatives are reported. The compounds showed inhibitory activities in the low micromolar range against a panel of sensitive and resistant P. falciparum strains (IC50s = 5–16 μM). Moreover, brussonol showed fast-acting in vitro inhibition and an additive inhibitory behavior when combined with the antimalarial artesunate (FICindex~1). The mode of action investigation indicated that brussonol increased the cytosolic calcium levels within the parasite. Hence, the discovery of brussonol as a new scaffold endowed with antiplasmodial activity will enable us to design derivatives with improved properties to deliver new lead candidates for malaria