RESUMO
Plasmodium, a digenetic parasite, requires a host and a vector for its life cycle completion. Most Plasmodium species display circadian rhythmicity during their intraerythrocytic cycle within the host, aiding in immune evasion. This rhythmicity, however, diminishes in in vitro cultures, highlighting the importance of host-derived signals for synchronizing the parasite's asexual cycle. Studies indicate a species-specific internal clock in Plasmodium, dependent on these host signals. Melatonin, a hormone the pineal gland produces under circadian regulation, impacts various physiological functions and is extensively reviewed as the primary circadian marker affecting parasite rhythms. Research suggests that melatonin facilitates synchronization through the PLC-IP3 signaling pathway, activating phospholipase C, which triggers intracellular calcium release and gene expression modulation. This evidence strongly supports the role of melatonin as a key circadian marker for parasite synchronization, presenting new possibilities for targeting the melatonin pathway when developing novel therapeutic approaches.
Assuntos
Ritmo Circadiano , Melatonina , Plasmodium , Melatonina/metabolismo , Ritmo Circadiano/fisiologia , Animais , Humanos , Plasmodium/metabolismo , Plasmodium/fisiologia , Malária/parasitologia , Malária/metabolismo , Biomarcadores , Transdução de Sinais , Interações Hospedeiro-ParasitaRESUMO
During their life cycle, apicomplexan parasites pass through different microenvironments and encounter a range of ion concentrations. The discovery that the GPCR-like SR25 in Plasmodium falciparum is activated by a shift in potassium concentration indicates that the parasite can take advantage of its development by sensing different ionic concentrations in the external milieu. This pathway involves the activation of phospholipase C and an increase in cytosolic calcium. In the present report, we summarize the information available in the literature regarding the role of potassium ions during parasite development. A deeper understanding of the mechanisms that allow the parasite to cope with ionic potassium changes contributes to our knowledge about the cell cycle of Plasmodium spp.
Assuntos
Parasitos , Plasmodium , Toxoplasma , Animais , Toxoplasma/metabolismo , Parasitos/metabolismo , Plasmodium falciparum/metabolismo , Potássio/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Cytosolic Ca2+ regulates multiple steps in the host-cell invasion, growth, proliferation, and egress of blood-stage Plasmodium falciparum, yet our understanding of Ca2+ signaling in this endemic malaria parasite is incomplete. By using a newly generated transgenic line of P. falciparum (PfGCaMP3) that expresses constitutively the genetically encoded Ca2+ indicator GCaMP3, we have investigated the dynamics of Ca2+ release and influx elicited by inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pumps, cyclopiazonic acid (CPA), and thapsigargin (Thg). Here we show that in isolated trophozoite phase parasites: (i) both CPA and Thg release Ca2+ from intracellular stores in P. falciparum parasites; (ii) Thg is able to induce Ca2+ release from an intracellular compartment insensitive to CPA; (iii) only Thg is able to activate Ca2+ influx from extracellular media, through a mechanism resembling store-operated Ca2+ entry, typical of mammalian cells; and (iv) the Thg-sensitive Ca2+ pool is unaffected by collapsing the mitochondria membrane potential with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone or the release of acidic Ca2+ stores with nigericin. These data suggest the presence of two Ca2+ pools in P. falciparum with differential sensitivity to the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pump inhibitors, and only the release of the Thg-sensitive Ca2+ store induces Ca2+ influx. Activation of the store-operated Ca2+ entry-like Ca2+ influx may be relevant for controlling processes such as parasite invasion, egress, and development mediated by kinases, phosphatases, and proteases that rely on Ca2+ levels for their activation.
Assuntos
Cálcio/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio , Humanos , Transporte de Íons , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
Half of the world's population lives in countries at risk of malaria infection, which results in approximately 450,000 deaths annually. Malaria parasites infect erythrocytes in a coordinated manner, with cycle durations in multiples of 24 hours, which reflects a behavior consistent with the host's circadian cycle. Interference in cycle coordination can help the immune system to naturally fight infection. Consequently, there is a search for new drugs that interfere with the cycle duration for combined treatment with conventional antimalarials. Melatonin appears to be a key host hormone responsible for regulating circadian behavior in the parasite cycle. In addition to host factors, there are still unknown factors intrinsic to the parasite that control the cycle duration. In this review, we present a series of reports of indole compounds and melatonin derivatives with antimalarial activity that were tested on several species of Plasmodium to evaluate the cytotoxicity to parasites and human cells, in addition to the ability to interfere with the development of the erythrocytic cycle. Most of the reported compounds had an IC50 value in the low micromolar range, without any toxicity to human cells. Triptosil, an indole derivative of melatonin, was able to inhibit the effect of melatonin in vitro without causing changes to the parasitemia. The wide variety of tested compounds indicates that it is possible to develop a compound capable of safely eliminating parasites from the host and interfering with the life cycle, which is promising for the development of new combined therapies against malaria.
Assuntos
Antimaláricos/farmacologia , Descoberta de Drogas , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Falciparum/tratamento farmacológico , Melatonina/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Resistência a Múltiplos Medicamentos , Interações Hospedeiro-Patógeno , Humanos , Malária Falciparum/parasitologia , Melatonina/análogos & derivados , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
Throughout evolution, the need for single-celled organisms to associate and form a single cluster of cells has had several evolutionary advantages. In complex, multicellular organisms, each tissue or organ has a specialty and function that make life together possible, and the organism as a whole needs to act in balance and adapt to changes in the environment. Sensory organs are essential for connecting external stimuli into a biological response, through the senses: sight, smell, taste, hearing, and touch. The G-protein-coupled receptors (GPCRs) are responsible for many of these senses and therefore play a key role in the perception of the cells' external environment, enabling interaction and coordinated development between each cell of a multicellular organism. The malaria-causing protozoan parasite, Plasmodium falciparum, has a complex life cycle that is extremely dependent on a finely regulated cellular signaling machinery. In this review, we summarize strong evidence and the main candidates of GPCRs in protozoan parasites. Interestingly, one of these GPCRs is a sensor for K+ shift in Plasmodium falciparum, PfSR25. Studying this family of proteins in P. falciparum could have a significant impact, both on understanding the history of the evolution of GPCRs and on finding new targets for antimalarials.
Assuntos
Sinalização do Cálcio/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Malária Falciparum/metabolismo , Percepção/fisiologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Terapia de Alvo Molecular/métodos , Percepção/efeitos dos fármacos , Ligação Proteica , Receptores Acoplados a Proteínas G/antagonistas & inibidoresRESUMO
Ca2+ signaling has been involved in controling critical cellular functions such as activation of proteases, cell death, and cell cycle control. The endoplasmatic reticulum plays a significant role in Ca2+ storage inside the cell, but mitochondria have long been recognized as a fundamental Ca2+ pool. Protozoan parasites such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi display a Ca2+ signaling toolkit with similarities to higher eukaryotes, including the participation of mitochondria in Ca2+-dependent signaling events. This review summarizes the most recent knowledge in mitochondrial Ca2+ signaling in protozoan parasites, focusing on the mechanism involved in mitochondrial Ca2+ uptake by pathogenic protists.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Mitocôndrias/metabolismo , Parasitos/metabolismo , Animais , Eucariotos/metabolismo , Humanos , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Trypanosoma cruzi/metabolismoRESUMO
Melatonin and its indoles derivatives are central in the synchronization of malaria parasites. In this research, we discovered that melatonin is unable to increase the parasitemia in the human malaria Plasmodium falciparum that lacks the kinase PfeIK1. The PfeIK1 knockout strain is a valuable tool in the screening of indol-related compound that blocks the melatonin effect in wild-type (WT) parasite development. The assays were performed by using flow cytometry with simultaneous labeling for mitochondria viability with MitoTracker Deep Red and nucleus staining with SYBR Green. We found that Melatotosil leads to an increase in parasitemia in P. falciparum and blocks melatonin effect in the WT parasite. Using microscopy imaging system, we found that Melatotosil at 500 nM is able to induce cytosolic calcium rise in transgenic PfGCaMP3 parasites. On the contrary, the compound Triptiofen blocks P. falciparum cell cycle with IC50 9.76 µM ± 0.6, inhibits melatonin action, and does not lead to a cytosolic calcium rise in PfGCaMP3 parasites. We also found that the synthetic indol-related compounds arrested parasite cycle for PfeIK1 knockout and (WT) P. falciparum (3D7) in 72 hours culture assays with the IC50 values slighting lower for the WT strain. We concluded that the kinase PfeIK1 is central for melatonin downstream signaling pathways involved in parasite cell cycle progression. More importantly, the indol-related compounds block its cycle as an upstream essential mechanism for parasite survival. Our data clearly show that this class of compounds emerge as an alternative for the problem of resistance with the classical antimalarials.
Assuntos
Antimaláricos/farmacologia , Ciclo Celular , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Transdução de Sinais , Proteínas Elk-1 do Domínio ets/antagonistas & inibidores , Antimaláricos/química , Humanos , Malária Falciparum/tratamento farmacológico , Melatonina , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Porphyrins and analogous macrocycles exhibit interesting photochemical, catalytic, and luminescence properties demonstrating high potential in the treatment of several diseases. Among them can be highlighted the possibility of application in photodynamic therapy and antimicrobial/antiparasitic PDT, for example, of malaria parasite. However, the low efficiency generally associated with their low solubility in water and bioavailability have precluded biomedical applications. Nanotechnology can provide efficient strategies to enhance bioavailability and incorporate targeted delivery properties to conventional pharmaceuticals, enhancing the effectiveness and reducing the toxicity, thus improving the adhesion to the treatment. In this way, those limitations can be overcome by using two main strategies: (1) Incorporation of hydrophilic substituents into the macrocycle ring while controlling the interaction with biological systems and (2) by including them in nanocarriers and delivery nanosystems. This review will focus on antiparasitic drugs based on porphyrin derivatives developed according to these two strategies, considering their vast and increasing applications befitting the multiple roles of these compounds in nature.
Assuntos
Antiparasitários/química , Antiparasitários/farmacologia , Composição de Medicamentos , Porfirinas/química , Porfirinas/farmacologia , Antiparasitários/uso terapêutico , Técnicas Biossensoriais , Humanos , Indóis/química , Indóis/farmacologia , Isoindóis , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Malaria causes millions of deaths worldwide and is considered a huge burden to underdeveloped countries. The number of cases with resistance to all antimalarials is continuously increasing, making the identification of novel drugs a very urgent necessity. A potentially very interesting target for novel therapeutic intervention is the parasite mitochondrion. In this work, we studied in Plasmodium falciparum 3 genes coding for proteins homologues of the mammalian FIS1 (Mitochondrial Fission Protein 1) and DRP1 (Dynamin Related Protein 1) involved in mitochondrial fission. We studied the expression of P. falciparum genes that show ample sequence and structural homologies with the mammalian counterparts, namely FIS1, DYN1, and DYN2. The encoded proteins are characterized by a distinct pattern of expression throughout the erythrocytic cycle of P. falciparum, and their mRNAs are modulated by treating the parasite with the host hormone melatonin. We have previously reported that the knockout of the Plasmodium gene that codes for protein kinase 7 is essential for melatonin sensing. We here show that PfPk7 knockout results in major alterations of mitochondrial fission genes expression when compared to wild-type parasites, and no change in fission proteins expression upon treatment with the host hormone. Finally, we have compared the morphological characteristics (using MitoTracker Red CMX Ros) and oxygen consumption properties of P. falciparum mitochondria in wild-type parasites and PfPk7 Knockout strains. A novel GFP construct targeted to the mitochondrial matrix to wild-type parasites was also developed to visualize P. falciparum mitochondria. We here show that, the functional characteristics of P. falciparum are profoundly altered in cells lacking protein kinase 7, suggesting that this enzyme plays a major role in the control of mitochondrial morphogenesis and maturation during the intra-erythrocyte cell cycle progression.
Assuntos
Genes de Protozoários/efeitos dos fármacos , Melatonina/farmacologia , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/fisiologia , Plasmodium falciparum/metabolismo , Dinaminas/metabolismo , Eritrócitos/parasitologia , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Proteínas Quinases/metabolismoRESUMO
BACKGROUND: Plasmodium has a complex biology including the ability to interact with host signals modulating their function through cellular machinery. Tumor necrosis factor (TNF) elicits diverse cellular responses including effects in malarial pathology and increased infected erythrocyte cytoadherence. As TNF levels are raised during Plasmodium falciparum infection we have investigated whether it has an effect on the parasite asexual stage. METHODS: Flow cytometry, spectrofluorimetric determinations, confocal microscopy and PCR real time quantifications were employed for characterizing TNF induced effects and membrane integrity verified by wheat germ agglutinin staining. RESULTS: TNF is able to decrease intracellular parasitemia, involving calcium as a second messenger of the pathway. Parasites incubated for 48 h with TNF showed reduced erythrocyte invasion. Thus, TNF induced rises in intracellular calcium concentration, which were blocked by prior addition of the purinergic receptor agonists KN62 and A438079, or interfering with intra- or extracellular calcium release by thapsigargin or EGTA (ethylene glycol tetraacetic acid). Importantly, expression of PfPCNA1 which encodes the Plasmodium falciparum Proliferating-Cell Nuclear Antigen 1, decreased after P. falciparum treatment of TNF (tumor necrosis factor) or 6-Bnz cAMP (N(6)-benzoyladenosine-3',5'-cyclic monophosphate sodium salt). CONCLUSIONS: This is potentially interesting data showing the relevance of calcium in downregulating a gene involved in cellular proliferation, triggered by TNF. GENERAL SIGNIFICANCE: The data show that Plasmodium may subvert the immunological system and use TNF for the control of its proliferation within the vertebrate host.
Assuntos
Antimaláricos/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Eritrócitos/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/parasitologia , Interações Hospedeiro-Parasita , Humanos , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/imunologia , Plasmodium falciparum/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Protozoários/metabolismo , Fatores de TempoRESUMO
Plasmodium falciparum is the causative agent of the most dangerous form of malaria in humans. It has been reported that the P. falciparum genome encodes for a single ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), an enzyme that hydrolyzes extracellular tri- and di-phosphate nucleotides. The E-NTPDases are known for participating in invasion and as a virulence factor in many pathogenic protozoa. Despite its presence in the parasite genome, currently, no information exists about the activity of this predicted protein. Here, we show for the first time that P. falciparum E-NTPDase is relevant for parasite lifecycle as inhibition of this enzyme impairs the development of P. falciparum within red blood cells (RBCs). ATPase activity could be detected in rings, trophozoites, and schizonts, as well as qRT-PCR, confirming that E-NTPDase is expressed throughout the intraerythrocytic cycle. In addition, transfection of a construct which expresses approximately the first 500 bp of an E-NTPDase-GFP chimera shows that E-NTPDase co-localizes with the endoplasmic reticulum (ER) in the early stages and with the digestive vacuole (DV) in the late stages of P. falciparum intraerythrocytic cycle.
Assuntos
Apirase/metabolismo , Eritrócitos/parasitologia , Malária/parasitologia , Plasmodium falciparum/parasitologia , Animais , Células Cultivadas , Eritrócitos/metabolismo , Hidrólise , ParasitosRESUMO
Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca(2+) oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca(2+) enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca(2+) changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca(2+) oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca(2+) influx. This is the first study showing, in real time, Ca(2+) signals preceding egress and their direct link with motility, an essential virulence trait.
Assuntos
Sinalização do Cálcio , Toxoplasma/fisiologia , Animais , Ionóforos de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Linhagem Celular , Células HeLa , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Humanos , Ionomicina/farmacologia , Proteínas Luminescentes/genética , Proteínas Recombinantes/genética , Toxoplasma/genética , Toxoplasma/patogenicidade , Transfecção , VirulênciaRESUMO
BACKGROUND: A number of experiments have previously indicated that Plasmodium falciparum-infected erythrocytes (pRBC) were able to sense host environment. The basis of this ability to detect external cues is not known but in screening signalling molecules from pRBC using commercial antibodies, a 34 kDa phosphorylated molecule that possesses such ability was identified. METHODS: The pRBC were exposed to different culture conditions and proteins were extracted for 1D or 2D gel electrophoresis followed by Western blot. The localization of 34 kDa protein was examined by biochemical fractionation followed by Western blot. High-resolution mass spectrometric analysis of immune precipitants was used to identify this protein and real-time quantitative reverse transcriptase polymerase chain reaction was used for detecting mRNA expression level. RESULTS: The 34 kDa protein was called PfAB4 has immediate responses (dephosphorylation and rapid turnover) to host environmental stimuli such as serum depletion, osmolality change and cytokine addition. PfAB4 is expressed constitutively throughout the erythrocytic lifecycle with dominant expression in trophozoites 30 h post-infection. Tumour necrosis factor (TNF) treatment induced a transient detectable dephosphorylation of PfAB4 in the ItG strain (2 min after addition) and the level of expression and phosphorylation returned to normal within 1-2 h. PfAB4 localized dominantly in pRBC cytoplasm, with a transient shift to the nucleus under TNF stimulation as shown by biochemical fractionation. High-resolution mass spectrometric analysis of immune precipitants of AB4 antibodies revealed a 34 kDa PfAB4 component as a mixture of proliferating cellular nuclear antigen-1 (PCNA1) and exported protein-2 (EXP2), along with a small number of other inconsistently identified peptides. Different parasite strains have different PfAB4 expression levels, but no significant association between mRNA and PfAB4 levels was seen, indicating that the differences may be at the post-transcriptional, presumably phosphorylation, level. A triple serine phosphorylated PCNA1 peptide was identified from the PfAB4 high expression strain only, providing further evidence that the identity of PfAB4 is PCNA1 in P. falciparum. CONCLUSION: A protein element in the human malaria parasite that responds to external cues, including the pro-inflammatory cytokine TNF have been discovered. Treatment results in a transient change in phosphorylation status of the response element, which also migrates from the parasite cytoplasm to the nucleus. The response element has been identified as PfPCNA1. This sensing response could be regulated by a parasite checkpoint system and be analogous to bacterial two-component signal transduction systems.
Assuntos
Eritrócitos/metabolismo , Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/isolamento & purificação , Plasmodium falciparum/fisiologia , Transdução de Sinais/fisiologia , Dipeptídeos , Interações Hospedeiro-Parasita , Humanos , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/análise , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Fatores de Necrose Tumoral/farmacologia , XantonasRESUMO
OBJECTIVES: To semisynthesise piperazine derivatives of betulinic acid to evaluate antimalarial activity, cytotoxicity and action mechanism. METHODS: The new derivatives were evaluated against the CQ-sensitive Plasmodium falciparum 3D7 strain by flow cytometry (FC) using YOYO-1 as stain. Cytotoxicity of 4a and 4b was performed with HEK293T cells for 24 and 48 h by MTT assay. The capability of compound 4a to modulate Ca(2+) in the trophozoite stage was investigated. The trophozoites were stained with Fluo4-AM and analysed by spectrofluorimetry. Effect on mitochondrial membrane potential (ΔΨm) was tested for 4a by FC with DiOC6 (3) as stain. For ß-haematin assay, 4a was incubated for 24 h with reagents such as haemin, and the fluorescence was measured by FlexStation at an absorbance of 405 nm. RESULTS: Antimalarial activity of 4a and 4b was IC50 = 1 and 4 µm, respectively. Compound 4a displayed cytotoxicity with IC50 = 69 and 29 µm for 24 and 48 h, respectively, and 4b was not cytotoxic at the tested concentrations. Addition of 4a leads to an increase in cytosolic Ca(2+) . We have measured ΔΨm after treating parasites with the compound. Data on Figure 4a show that mitochondria were not affected. The action mechanism for 4a, inhibition of ß-haematin formation (17%), was lower than CQ treatment (83%; IC50 = 3 mm). CONCLUSION: Compound 4a showed excellent antimalarial activity, and its action mechanism is involved in Ca(2+) pathway(s).
Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Triterpenos/farmacologia , Antimaláricos/síntese química , Citometria de Fluxo , Células HEK293/efeitos dos fármacos , Hemeproteínas/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Triterpenos Pentacíclicos , Espectrometria de Fluorescência , Triterpenos/síntese química , Trofozoítos/efeitos dos fármacos , Ácido BetulínicoRESUMO
A new orbitide named ribifolin was isolated and characterized from Jatropha ribifolia using mass spectrometry, NMR spectroscopy, quantitative amino acid analysis, molecular dynamics/simulated annealing, and Raman optical activity measurements and calculations. Ribifolin (1) and its linear form (1a) were synthesized by solid-phase peptide synthesis, followed by evaluation of its antiplasmodial and cytotoxicity activities. Compound 1 was moderately effective (IC50 = 42 µM) against the Plasmodium falciparum strain 3D7.
Assuntos
Antimaláricos , Jatropha/química , Peptídeos Cíclicos , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/química , Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Testes de Sensibilidade Parasitária , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Extratos Vegetais/química , Técnicas de Síntese em Fase SólidaRESUMO
Several synthetic metallated protoporphyrins (M-PPIX) were tested for their ability to block the cell cycle of the lethal human malaria parasite Plasmodium falciparum. After encapsulating the porphyrin derivatives in micro- and nanocapsules of marine atelocollagen, their effects on cultures of red blood cells infected (RBC) with P. falciparum were verified. RBCs infected with synchronized P. falciparum incubated for 48 h showed a toxic effect over a micromolar range. Strikingly, the IC50 of encapsulated metalloporphyrins reached nanomolar concentrations, where Zn-PPIX showed the best antimalarial effect, with an IC50=330 nM. This value is an 80-fold increase in the antimalarial activity compared to the antimalarial effect of non-encapsulated Zn-PPIX. These findings reveal that the incubation of P. falciparum infected-RBCs with 20 µM Zn-PPIX reduced the size of hemozoin crystal by 34%, whereas a 28% reduction was noticed with chloroquine, confirming the importance of heme detoxification pathway in drug therapy. FROM THE CLINICAL EDITOR: In this study, synthetic metalloporphyrins were tested as therapeutics that target Plasmodium falciparum. The IC50 of encapsulated metalloporphyrins was found to be in the nanomolar concentration range, with encapsulated Zn-PPIX showing an 80-fold increase in its antimalarial activity compared to the non-encapsulated form.
Assuntos
Antimaláricos/administração & dosagem , Malária Falciparum/tratamento farmacológico , Metaloporfirinas/administração & dosagem , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/química , Colágeno/administração & dosagem , Colágeno/química , Humanos , Malária Falciparum/parasitologia , Nanocápsulas/administração & dosagem , Nanocápsulas/químicaRESUMO
During the last decade, a vast number of inhibitors, ligands and fluorescent probes have evolved for mammalian protein kinases; however, the suitability of these compounds for studies of evolutionarily divergent eukaryotes has mostly been left beyond the scope of research. Here, we examined whether adenosine analogue-oligoarginine conjugates that had been extensively characterized as efficient inhibitors of the human protein kinases are applicable for targeting Plasmodium protein kinases. We demonstrated that ARCs were not only able to bind to and inhibit a representative member of Plasmodium falciparum kinome (cGMP-dependent protein kinase) in biochemical assay, but also affected the general phosphorylation levels in parasites released from the infected red blood cells upon saponin treatment. These findings urge advantaging of already existing biochemical tools, whose initially generic, but intrinsically "tunable" selectivity profiles could be used for dissection of signaling pathways outside the initially defined group of biological targets.
Assuntos
Adenosina/análogos & derivados , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Corantes Fluorescentes/química , Regulação Enzimológica da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Malária Falciparum/tratamento farmacológico , Microscopia Confocal , Parasitemia/tratamento farmacológicoRESUMO
There is an increasing understanding that melatonin and the ubiquitin/ proteasome system (UPS) interact to regulate multiple cellular functions. Post-translational modifications such as ubiquitination are important modulators of signaling processes, cell cycle and many other cellular functions. Previously, we reported a melatonin-induced upregulation of gene expression related to ubiquitin/proteasome system (UPS) in Plasmodium falciparum, the human malaria parasite, and that P. falciparum protein kinase 7 influences this process. This implies a role of melatonin, an indolamine, in modulating intraerythrocytic development of the parasite. In this report we demonstrate by qPCR analysis, that melatonin induces gene upregulation in nine out of fourteen genes of the UPS, consisting of the same set of genes previously reported, between 4 to 5 h after melatonin treatment. We demonstrate that melatonin causes a temporally controlled gene expression of UPS members.
Assuntos
Malária/parasitologia , Melatonina/farmacologia , Parasitos/genética , Plasmodium falciparum/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Regulação para Cima/genética , Animais , Humanos , Parasitos/efeitos dos fármacos , Parasitos/enzimologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Most protocols used to study the dynamics of calcium (Ca2+) in the malaria parasite are based on dyes, which are invasive and do not allow discrimination between the signal from the host cell and the parasite. To avoid this pitfall, we have generated a parasite line expressing the genetically encoded calcium sensor GCaMP3. The PfGCaMP3 parasite line is an innovative tool for studying spontaneous intracellular Ca2+ oscillations without external markers. Using this parasite line, we demonstrate the occurrence of spontaneous Ca2+ oscillations in the ring, trophozoite, and schizont stages in Plasmodium falciparum. Using the Fourier transform to fluorescence intensity data extracted from different experiments, we observe cytosolic Ca2+ fluctuations. These spontaneous cytosolic Ca2+ oscillations occur in the three intraerythrocytic stages of the parasite, with most oscillations occurring in the ring and trophozoite stages. A control parasite line expressing only a GFP control did not reveal such fluctuations, demonstrating the specificity of the observations. Our results clearly show dynamic, spontaneous Ca2+ oscillations during the asexual stage in P. falciparum, independent from external stimuli.
RESUMO
BACKGROUND: The discovery and development of anti-malarial compounds of plant origin and semisynthetic derivatives thereof, such as quinine (QN) and chloroquine (CQ), has highlighted the importance of these compounds in the treatment of malaria. Ursolic acid analogues bearing an acetyl group at C-3 have demonstrated significant anti-malarial activity. With this in mind, two new series of betulinic acid (BA) and ursolic acid (UA) derivatives with ester groups at C-3 were synthesized in an attempt to improve anti-malarial activity, reduce cytotoxicity, and search for new targets. In vitro activity against CQ-sensitive Plasmodium falciparum 3D7 and an evaluation of cytotoxicity in a mammalian cell line (HEK293T) are reported. Furthermore, two possible mechanisms of action of anti-malarial compounds have been evaluated: effects on mitochondrial membrane potential (ΔΨm) and inhibition of ß-haematin formation. RESULTS: Among the 18 derivatives synthesized, those having shorter side chains were most effective against CQ-sensitive P. falciparum 3D7, and were non-cytotoxic. These derivatives were three to five times more active than BA and UA. A DiOC(6)(3) ΔΨm assay showed that mitochondria are not involved in their mechanism of action. Inhibition of ß-haematin formation by the active derivatives was weaker than with CQ. Compounds of the BA series were generally more active against P. falciparum 3D7 than those of the UA series. CONCLUSIONS: Three new anti-malarial prototypes were obtained from natural sources through an easy and relatively inexpensive synthesis. They represent an alternative for new lead compounds for anti-malarial chemotherapy.