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1.
SLAS Discov ; 24(1): 38-46, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30142014

RESUMO

Malaria remains a major cause of morbidity and mortality worldwide with ~3.3 billion people at risk of contracting malaria and an estimated 450,000 deaths each year. While tools to reduce the infection prevalence to low levels are currently under development, additional efforts will be required to interrupt transmission. Transmission between human host and vector by the malaria parasite involves gametogenesis in the host and uptake of gametocytes by the mosquito vector. This stage is a bottleneck for reproduction of the parasite, making it a target for small-molecule drug discovery. Targeting this stage, we used whole Plasmodium falciparum gametocytes from in vitro culture and implemented them into 1536-well plates to create a live/dead phenotypic antigametocyte assay. Using specialized equipment and upon further validation, we screened ~150,000 compounds from the NIH repository currently housed at Scripps Florida. We identified 100 primary screening hits that were tested for concentration response. Additional follow-up studies to determine specificity, potency, and increased efficacy of the antigametocyte candidate compounds resulted in a starting point for initial medicinal chemistry intervention. From this, 13 chemical analogs were subsequently tested as de novo powders, which confirmed original activity from the initial analysis and now provide a point of future engagement.


Assuntos
Antimaláricos/farmacologia , Gametogênese/efeitos dos fármacos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Florida , Humanos , Células Jurkat , Malária Falciparum/parasitologia , Fenótipo
2.
Parasit Vectors ; 10(1): 216, 2017 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-28464929

RESUMO

BACKGROUND: Blocking malaria gametocyte development in RBCs or their fertilization in the mosquito gut can prevent infection of the mosquito vector and passage of disease to the human host. A 'transmission blocking' strategy is a component of future malaria control. However, the lack of robust culture systems for producing large amounts of Plasmodium falciparum gametocytes has limited our understanding of sexual-stage malaria biology and made vaccine or chemotherapeutic discoveries more difficult. METHODS: The Wave BioreactorTM 20/50 EHT culture system was used to develop a convenient and low-maintenance protocol for inducing commitment of P. falciparum parasites to gametocytogenesis. Culture conditions were optimised to obtain mature stage V gametocytes within 2 weeks in a large-scale culture of up to a 1 l. RESULTS: We report a simple method for the induction of gametocytogenesis with N-acetylglucosamine (10 mM) within a Wave Bioreactor. By maintaining the culture for 14-16 days as many as 100 million gametocytes (stage V) were produced in a 1 l culture. Gametocytes isolated using magnetic activated cell sorting (MACS) columns were frozen in aliquots for storage. These were revitalised by thawing and shown to retain their ability to exflagellate and infect mosquitoes (Anopheles stephansi). CONCLUSIONS: The production of gametocytes in the Wave Bioreactor under GMP-compliant conditions will not only facilitate cellular, developmental and molecular studies of gametocytes, but also the high-throughput screening for new anti-malarial drugs and, possibly, the development of whole-cell gametocyte or sporozoite-based vaccines.


Assuntos
Reatores Biológicos , Plasmodium falciparum/crescimento & desenvolvimento , Esporozoítos/crescimento & desenvolvimento , Animais , Anopheles/parasitologia , Técnicas de Cultura , Descoberta de Drogas , Vacinas Antimaláricas , Mosquitos Vetores/parasitologia , Plasmodium falciparum/fisiologia , Esporozoítos/fisiologia
3.
Exp Parasitol ; 169: 13-21, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27373432

RESUMO

Infection with the apicomplexan parasite Plasmodium falciparum is a major cause of morbidity and mortality worldwide. One of the striking features of this parasite is its ability to remodel and decrease the deformability of host red blood cells, a process that contributes to disease. To further understand the virulence of Pf we investigated the biochemistry and function of a putative Pf S33 proline aminopeptidase (PfPAP). Unlike other P. falciparum aminopeptidases, PfPAP contains a predicted protein export element that is non-syntenic with other human infecting Plasmodium species. Characterization of PfPAP demonstrated that it is exported into the host red blood cell and that it is a prolyl aminopeptidase with a preference for N-terminal proline substrates. In addition genetic deletion of this exopeptidase was shown to lead to an increase in the deformability of parasite-infected red cells and in reduced adherence to the endothelial cell receptor CD36 under flow conditions. Our studies suggest that PfPAP plays a role in the rigidification and adhesion of infected red blood cells to endothelial surface receptors, a role that may make this protein a novel target for anti-disease interventions strategies.


Assuntos
Aminopeptidases/metabolismo , Deformação Eritrocítica/fisiologia , Plasmodium falciparum/enzimologia , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/genética , Aminopeptidases/imunologia , Anticorpos Antiprotozoários/imunologia , Northern Blotting , Western Blotting , Adesão Celular/fisiologia , Elasticidade , Membrana Eritrocítica/genética , Membrana Eritrocítica/fisiologia , Eritrócitos/parasitologia , Técnicas de Inativação de Genes , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Plasmodium falciparum/genética , RNA de Protozoário/química , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transfecção
4.
Mol Microbiol ; 98(6): 1101-14, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26304012

RESUMO

The malaria parasite Plasmodium falciparum dramatically remodels its host red blood cell to enhance its own survival, using a secretory membrane system that it establishes outside its own cell. Cisternal organelles, called Maurer's clefts, act as a staging point for the forward trafficking of virulence proteins to the red blood cell (RBC) membrane. The Ring-EXported Protein-1 (REX1) is a Maurer's cleft resident protein. We show that inducible knockdown of REX1 causes stacking of Maurer's cleft cisternae without disrupting the organization of the knob-associated histidine-rich protein at the RBC membrane. Genetic dissection of the REX1 sequence shows that loss of a repeat sequence domain results in the formation of giant Maurer's cleft stacks. The stacked Maurer's clefts are decorated with tether-like structures and retain the ability to dock onto the RBC membrane skeleton. The REX1 mutant parasites show deficient export of the major virulence protein, PfEMP1, to the red blood cell surface and markedly reduced binding to the endothelial cell receptor, CD36. REX1 is predicted to form a largely α-helical structure, with a repetitive charge pattern in the repeat sequence domain, providing potential insights into the role of REX1 in Maurer's cleft sculpting.


Assuntos
Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Antígenos CD36/metabolismo , DNA de Protozoário , Membrana Eritrocítica/metabolismo , Eritrócitos/parasitologia , Técnicas de Silenciamento de Genes , Humanos , Mutação , Plasmodium falciparum/genética , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas/química , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequências Repetitivas de Ácido Nucleico , Fatores de Virulência/genética
6.
J Biomol Screen ; 19(7): 1107-15, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24619116

RESUMO

The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP, suggesting that it is a valid target for new antimalaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository). A cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counter screen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from high-throughput screening. Two structurally related compounds, CID 6852389 and CID 23724194, yielded micromolar potency and were inactive in CTSL1 titration experiments (IC50>59.6 µM). As measured by the K(i) assay, both compounds demonstrated micromolar noncompetitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrated potency in malaria growth assays (IC504 µM and 1.3 µM, respectively).


Assuntos
Aminopeptidases/antagonistas & inibidores , Antimaláricos/química , Glutamil Aminopeptidase/antagonistas & inibidores , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/farmacologia , Catepsina L/química , Análise por Conglomerados , Desenho de Fármacos , Eritrócitos/parasitologia , Fasciola hepatica/enzimologia , Glutamil Aminopeptidase/química , Humanos , Concentração Inibidora 50 , Cinética , Peptídeos/metabolismo , Plasmodium falciparum/enzimologia , Proteínas Recombinantes/química , Bibliotecas de Moléculas Pequenas/química , Software , Espectrometria de Fluorescência , Especificidade por Substrato
7.
Org Lett ; 16(6): 1716-9, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24611932

RESUMO

A marine-derived Streptomyces sp. (CMB-M0244) isolated from a sediment collected off South Molle Island, Queensland, produced mollemycin A (1) as a new first in class glyco-hexadepsipeptide-polyketide. The structure of 1 was assigned by detailed spectroscopic analysis, supported by chemical derivatization and degradation, and C3 Marfey's analysis. Mollemycin A (1) exhibits exceptionally potent and selective growth inhibitory activity against Gram-positive and Gram-negative bacteria (IC50 10-50 nM) and drug-sensitive (3D7; IC50 7 nM) and multidrug-resistant (Dd2; IC50 9 nM) clones of the malaria parasite Plasmodium falciparum.


Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Policetídeos/isolamento & purificação , Policetídeos/farmacologia , Streptomyces/química , Antibacterianos/química , Antimaláricos/metabolismo , Austrália , Depsipeptídeos/química , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Concentração Inibidora 50 , Biologia Marinha , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Policetídeos/química , Streptomyces/genética , Relação Estrutura-Atividade
8.
ACS Chem Biol ; 8(12): 2654-9, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-24079418

RESUMO

Fragment-based screening is commonly used to identify compounds with relatively weak but efficient localized binding to protein surfaces. We used mass spectrometry to study fragment-sized three-dimensional natural products. We identified seven securinine-related compounds binding to Plasmodium falciparum 2'-deoxyuridine 5'-triphosphate nucleotidohydrolase (PfdUTPase). Securinine bound allosterically to PfdUTPase, enhancing enzyme activity and inhibiting viability of both P. falciparum gametocyte (sexual) and blood (asexual) stage parasites. Our results provide a new insight into mechanisms that may be applicable to transmission-blocking agents.


Assuntos
Antimaláricos/farmacologia , Produtos Biológicos/química , Estágios do Ciclo de Vida/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Alcaloides/química , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Antimaláricos/química , Antimaláricos/isolamento & purificação , Azepinas/química , Azepinas/isolamento & purificação , Azepinas/farmacologia , Nucleotídeos de Desoxiuracil/antagonistas & inibidores , Nucleotídeos de Desoxiuracil/química , Nucleotídeos de Desoxiuracil/metabolismo , Relação Dose-Resposta a Droga , Compostos Heterocíclicos de Anel em Ponte/química , Compostos Heterocíclicos de Anel em Ponte/isolamento & purificação , Compostos Heterocíclicos de Anel em Ponte/farmacologia , Cinética , Lactonas/química , Lactonas/isolamento & purificação , Lactonas/farmacologia , Estágios do Ciclo de Vida/fisiologia , Piperidinas/química , Piperidinas/isolamento & purificação , Piperidinas/farmacologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade
9.
J Infect Dis ; 208(7): 1170-4, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23847056

RESUMO

Gametocytogenesis by Plasmodium falciparum is essential for transmission of the parasite from human to mosquito, yet developing gametocytes lack expression of surface proteins required for cytoadherence. Therefore, elimination from the circulation should occur unless they are sequestered in regions of low blood flow such as the extracellular spaces of the bone marrow. Our data indicate that gametocytogenesis is enhanced in the presence of erythroid progenitors found within the bone marrow. Furthermore, atomic force microscopy indicates that developing gametocytes undergo remarkable shifts in their erythrocyte membrane elasticity, which may allow them to be retained within the bone marrow until maturation.


Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Células-Tronco/parasitologia , Membrana Celular/fisiologia , Elasticidade , Humanos , Microscopia de Força Atômica
10.
Malar J ; 12: 134, 2013 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-23607486

RESUMO

BACKGROUND: The production of gametocytes is essential for transmission of malaria parasites from the mammalian host to the mosquito vector. However the process by which the asexual blood-stage parasite undergoes commitment to sexual development is not well understood. This process is known to be sensitive to environmental stimuli and it has been suggested that a G protein dependent system may mediate the switch, but there is little evidence that the Plasmodium falciparum genome encodes heterotrimeric G proteins. Previous studies have indicated that the malaria parasite can interact with endogenous erythrocyte G proteins, and other components of the cyclic nucleotide pathway have been identified in P. falciparum. Also, the polypeptide cholera toxin, which induces commitment to gametocytogenesis is known to catalyze the ADP-ribosylation of the α(s) class of heterotrimeric G protein α subunits in mammalian systems has been reported to detect a number of G(α) subunits in P. falciparum-infected red cells. METHODS: Cholera toxin and Mas 7 (a structural analogue of Mastoparan) were used to assess the role played by putative G protein signalling in the commitment process, both are reported to interact with different components of classical Gas and Gai/o signalling pathways. Their ability to induce gametocyte production in the transgenic P. falciparum line Pfs16-GFP was determined and downstream effects on the secondary messenger cAMP measured. RESULTS: Treatment of parasite cultures with either cholera toxin or MAS 7 resulted in increased gametocyte production, but only treatment with MAS 7 resulted in a significant increase in cAMP levels. This indicates that MAS 7 acts either directly or indirectly on the P. falciparum adenylyl cyclase. CONCLUSION: The observation that cholera toxin treatment did not affect cAMP levels indicates that while addition of cholera toxin does increase gametocytogenesis the method by which it induces increased commitment is not immediately obvious, except that is unlikely to be via heterotrimeric G proteins.


Assuntos
Plasmodium falciparum/citologia , Plasmodium falciparum/crescimento & desenvolvimento , Transdução de Sinais , Toxina da Cólera/metabolismo , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/metabolismo , Fatores de Tempo
11.
J Mol Biol ; 422(4): 495-507, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22709581

RESUMO

The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.


Assuntos
Aminopeptidases/química , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Aminoácidos/química , Aminoácidos/metabolismo , Aminopeptidases/metabolismo , Animais , Domínio Catalítico , Cristalografia por Raios X/métodos , Eritrócitos/metabolismo , Humanos , Malária Falciparum/parasitologia , Peptídeos/química , Peptídeos/metabolismo , Proteólise , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
12.
Antimicrob Agents Chemother ; 56(6): 3244-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22450967

RESUMO

Malaria remains a significant risk in many areas of the world, with resistance to the current antimalarial pharmacopeia an ever-increasing problem. The M1 alanine aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) are believed to play a role in the terminal stages of digestion of host hemoglobin and thereby generate a pool of free amino acids that are essential for parasite growth and development. Here, we show that an orally bioavailable aminopeptidase inhibitor, CHR-2863, is efficacious against murine malaria.


Assuntos
Aminopeptidases/antagonistas & inibidores , Antimaláricos/farmacologia , Inibidores Enzimáticos/farmacologia , Animais , Antimaláricos/química , Inibidores Enzimáticos/química , Feminino , Malária/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade
13.
PLoS One ; 7(2): e31938, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22359643

RESUMO

BACKGROUND: Plasmodium falciparum, the causative agent of human malaria, expresses two aminopeptidases, PfM1AAP and PfM17LAP, critical to generating a free amino acid pool used by the intraerythrocytic stage of the parasite for proteins synthesis, growth and development. These exopeptidases are potential targets for the development of a new class of anti-malaria drugs. METHODOLOGY/PRINCIPAL FINDINGS: To define the substrate specificity of recombinant forms of these two malaria aminopeptidases we used a new library consisting of 61 fluorogenic substrates derived both from natural and unnatural amino acids. We obtained a detailed substrate fingerprint for recombinant forms of the enzymes revealing that PfM1AAP exhibits a very broad substrate tolerance, capable of efficiently hydrolyzing neutral and basic amino acids, while PfM17LAP has narrower substrate specificity and preferentially cleaves bulky, hydrophobic amino acids. The substrate library was also exploited to profile the activity of the native aminopeptidases in soluble cell lysates of P. falciparum malaria. CONCLUSIONS/SIGNIFICANCE: This data showed that PfM1AAP and PfM17LAP are responsible for majority of the aminopeptidase activity in these extracts. These studies provide specific substrate and mechanistic information important for understanding the function of these aminopeptidases and could be exploited in the design of new inhibitors to specifically target these for anti-malaria treatment.


Assuntos
Aminopeptidases/química , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Aminoácidos/química , Desenho de Fármacos , Humanos , Malária Falciparum/tratamento farmacológico , Terapia de Alvo Molecular , Bibliotecas de Moléculas Pequenas , Especificidade por Substrato
14.
Antimicrob Agents Chemother ; 56(5): 2283-9, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22354298

RESUMO

The antiretroviral protease inhibitors (APIs) ritonavir, saquinavir, and lopinavir, used to treat HIV infection, inhibit the growth of Plasmodium falciparum at clinically relevant concentrations. Moreover, it has been reported that these APIs potentiate the activity of chloroquine (CQ) against this parasite in vitro. The mechanism underlying this effect is not understood, but the degree of chemosensitization varies between the different APIs and, with the exception of ritonavir, appears to be dependent on the parasite exhibiting a CQ-resistant phenotype. Here we report a study of the role of the P. falciparum chloroquine resistance transporter (PfCRT) in the interaction between CQ and APIs, using transgenic parasites expressing different PfCRT alleles and using the Xenopus laevis oocyte system for the heterologous expression of PfCRT. Our data demonstrate that saquinavir behaves as a CQ resistance reverser and that this explains, at least in part, its ability to enhance the effects of CQ in CQ-resistant P. falciparum parasites.


Assuntos
Cloroquina/farmacologia , Malária Falciparum/tratamento farmacológico , Oócitos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antimaláricos/farmacologia , Transporte Biológico/efeitos dos fármacos , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Inibidores da Protease de HIV/farmacologia , Humanos , Lopinavir/farmacologia , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Oócitos/citologia , Oócitos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Ritonavir/farmacologia , Saquinavir/farmacologia , Trítio , Xenopus laevis
15.
Malar J ; 11: 34, 2012 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-22305482

RESUMO

BACKGROUND: Recent renewed emphasis on the eradication of malaria has highlighted the need for more tools with which to achieve this ambitious goal. One high priority area is the need to determine the gametocytocidal activity of both currently used anti-malarial drugs and those in the development pipeline. However, testing the activity of compounds against Plasmodium falciparum gametocytes is technically challenging both in vivo and in vitro. METHODS: Here the use of a simple robust assay to screen a panel of currently used and experimental anti-malarial drugs against mature P. falciparum gametocytes is described. RESULTS: Eight of 44 compounds tested reduced gametocyte viability by at least 50% and three showed IC50 values in nM range. CONCLUSIONS: There is a need to identify new compounds with activity against late stage gametocytes and the information provided by this in vitro assay is a valuable first step, which can guide future clinical studies.


Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Concentração Inibidora 50 , Testes de Sensibilidade Parasitária/métodos
16.
Mol Biochem Parasitol ; 180(2): 127-31, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21939693

RESUMO

Recent success in the global reduction campaign against malaria has resulted in the possibility that it may be feasible to drastically reduce or even eradicate malaria even without the introduction of a vaccine. However, while there has been significant effort to design the next generation of antimalarial drugs, one area that is underrepresented in the current antimalarial pharmacopeia is that of transmission blocking drugs directed at late-stage gametocytes. Here we describe the development of a robust and simple assay that is amenable to a high throughput format for the discovery of new antigametocyte drugs.


Assuntos
Antimaláricos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Malária Falciparum/fisiopatologia , Plasmodium falciparum/efeitos dos fármacos , Humanos , Malária Falciparum/tratamento farmacológico , Testes de Sensibilidade Parasitária , Plasmodium falciparum/crescimento & desenvolvimento
17.
Mol Microbiol ; 81(4): 982-93, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21696460

RESUMO

The malaria parasite Plasmodium falciparum assembles knob structures underneath the erythrocyte membrane that help present the major virulence protein, P. falciparum erythrocyte membrane protein-1 (PfEMP1). Membranous structures called Maurer's clefts are established in the erythrocyte cytoplasm and function as sorting compartments for proteins en route to the RBC membrane, including the knob-associated histidine-rich protein (KAHRP), and PfEMP1. We have generated mutants in which the Maurer's cleft protein, the ring exported protein-1 (REX1) is truncated or deleted. Removal of the C-terminal domain of REX1 compromises Maurer's cleft architecture and PfEMP1-mediated cytoadherance but permits some trafficking of PfEMP1 to the erythrocyte surface. Deletion of the coiled-coil region of REX1 ablates PfEMP1 surface display, trapping PfEMP1 at the Maurer's clefts. Complementation of mutants with REX1 partly restores PfEMP1-mediated binding to the endothelial cell ligand, CD36. Deletion of the coiled-coil region or complete deletion of REX1 is tightly associated with the loss of a subtelomeric region of chromosome 2, encoding KAHRP and other proteins. A KAHRP-green fluorescent protein (GFP) fusion expressed in the REX1-deletion parasites shows defective trafficking. Thus, loss of functional REX1 directly or indirectly ablates the assembly of the P. falciparum virulence complex at the surface of host erythrocytes.


Assuntos
Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fatores de Virulência/metabolismo , Antígenos CD36/metabolismo , Adesão Celular , Células Endoteliais/metabolismo , Eritrócitos/parasitologia , Teste de Complementação Genética , Humanos , Transporte Proteico , Deleção de Sequência
18.
Proc Natl Acad Sci U S A ; 107(38): 16643-8, 2010 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-20823248

RESUMO

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate the adherence of parasite-infected red blood cells (IRBCs) to various host receptors. A previous study has shown that the parasite protein, cytoadherence-linked asexual gene 9 (CLAG9), is also essential for IRBC adherence. However, how CLAG9 influences this process remains unknown. In this study, we show that CLAG9 interacts with VAR2CSA, a PfEMP1 that mediates IRBC adherence to chondroitin 4-sulfate in the placenta. Importantly, our results show that the adherent parasites synthesize CLAG9 at two stages--the early ring and late trophozoite stages. Localization studies revealed that a substantial level of CLAG9 is located mainly at or in close proximity of the IRBC membrane in association with VAR2CSA. Upon treatment of IRBCs with trypsin, a significant amount of CLAG9 (≈150 kDa) was converted into ≈142-kDa polypeptide. Together these data demonstrate that a considerable amount of CLAG9 is embedded in the IRBC membrane such that at least a portion of the polypeptide at either N or C terminus is exposed on the cell surface. In parasites lacking CLAG9, VAR2CSA failed to express on the IRBC surface and was located within the parasite. Based on these findings, we propose that CLAG9 plays a critical role in the trafficking of PfEMP1s onto the IRBC surface. These results have important implications for the development of therapeutics for cerebral, placental, and other cytoadherence-associated malaria illnesses.


Assuntos
Antígenos de Protozoários/fisiologia , Moléculas de Adesão Celular/fisiologia , Plasmodium falciparum/fisiologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/fisiologia , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Sequência de Bases , Adesão Celular/fisiologia , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Sulfatos de Condroitina/fisiologia , DNA de Protozoário/genética , Membrana Eritrocítica/parasitologia , Membrana Eritrocítica/fisiologia , Membrana Eritrocítica/ultraestrutura , Eritrócitos/parasitologia , Feminino , Técnicas de Inativação de Genes , Genes de Protozoários , Interações Hospedeiro-Parasita/fisiologia , Humanos , Técnicas In Vitro , Microscopia Imunoeletrônica , Complexos Multiproteicos , Placenta/parasitologia , Placenta/fisiologia , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Gravidez , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética
19.
Infect Disord Drug Targets ; 10(3): 217-25, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20334618

RESUMO

Novel targets for new drug development are urgently required to combat malaria, a disease that puts half of the world's population at risk. One group of enzymes identified within the genome of the most lethal of the causative agents of malaria, Plasmodium falciparum, that may have the potential to become new targets for antimalarial drug development are the aminopeptidases. These enzymes catalyse the cleavage of the N-terminal amino acids from proteins and peptides. P. falciparum appears to encode for at least nine aminopeptidases, two neutral aminopeptidases, one aspartyl aminopeptidase, one aminopeptidase P, one prolyl aminopeptidase and four methionine aminopeptidases. Recent advances in our understanding of these genes and their protein products are outlined in this review, including their potential for antimalarial drug development.


Assuntos
Aminopeptidases/antagonistas & inibidores , Aminopeptidases/metabolismo , Antimaláricos/farmacologia , Plasmodium/enzimologia , Inibidores de Proteases/farmacologia , Animais , Glutamil Aminopeptidase/metabolismo , Humanos , Malária/tratamento farmacológico , Malária/parasitologia , Metionil Aminopeptidases , Plasmodium/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia
20.
Proc Natl Acad Sci U S A ; 107(6): 2449-54, 2010 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-20133789

RESUMO

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the X-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the X-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.


Assuntos
Aminopeptidases/química , Desenho de Fármacos , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/metabolismo , Antimaláricos/química , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Catálise , Domínio Catalítico , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Metais/química , Metais/metabolismo , Modelos Moleculares , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Especificidade por Substrato
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