RESUMO
Broadening of signals from atoms at interfaces can often be a limiting factor in applying solution NMR to the structure determination of complexes. Common contributors to such problems include exchange between free and bound states and the increased molecular weight of complexes relative to the free components, but another cause that can be more difficult to deal with occurs when conformational dynamics within the interface takes place at an intermediate rate on the chemical shift timescale. In this work we show how a carefully chosen mutation in the protein HMG-D rescued such a situation, making possible high-resolution structure determination of its complex with a dA2 bulge DNA ligand designed to mimic a natural DNA bend, and thereby revealing a new spatial organization of the complex.
RESUMO
An inherited gain-of-function variant (E756del) in the mechanosensitive cationic channel PIEZO1 was shown to confer a significant protection against severe malaria. Here, we demonstrate in vitro that human red blood cell (RBC) infection by Plasmodium falciparum is prevented by the pharmacological activation of PIEZO1. Yoda1 causes an increase in intracellular calcium associated with rapid echinocytosis that inhibits RBC invasion, without affecting parasite intraerythrocytic growth, division or egress. Notably, Yoda1 treatment significantly decreases merozoite attachment and subsequent RBC deformation. Intracellular Na+/K+ imbalance is unrelated to the mechanism of protection, although delayed RBC dehydration observed in the standard parasite culture medium RPMI/albumax further enhances the resistance to malaria conferred by Yoda1. The chemically unrelated Jedi2 PIEZO1 activator similarly causes echinocytosis and RBC dehydration associated with resistance to malaria invasion. Spiky outward membrane projections are anticipated to reduce the effective surface area required for both merozoite attachment and internalization upon pharmacological activation of PIEZO1. Globally, our findings indicate that the loss of the typical biconcave discoid shape of RBCs, together with an altered optimal surface to volume ratio, induced by PIEZO1 pharmacological activation prevent efficient P. falciparum invasion.
Assuntos
Malária , Parasitos , Animais , Humanos , Plasmodium falciparum , Desidratação/metabolismo , Eritrócitos/metabolismo , Malária/parasitologia , Parasitos/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismoRESUMO
The malaria parasite, Plasmodium falciparum, develops and multiplies in the human erythrocyte. It needs to synthesize considerable amounts of phospholipids (PLs), principally phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Several metabolic pathways coexist for their de novo biosynthesis, involving a dozen enzymes. Given the importance of these PLs for the survival of the parasite, we sought to determine their sources and to understand the connections and dependencies between the multiple pathways. We used three deuterated precursors (choline-d9, ethanolamine-d4, and serine-d3) to follow and quantify simultaneously their incorporations in the intermediate metabolites and the final PLs by LC/MS/MS. We show that PC is mainly derived from choline, itself provided by lysophosphatidylcholine contained in the serum. In the absence of choline, the parasite is able to use both other precursors, ethanolamine and serine. PE is almost equally synthesized from ethanolamine and serine, with both precursors being able to compensate for each other. Serine incorporated in PS is mainly derived from the degradation of host cell hemoglobin by the parasite. P. falciparum thus shows an unexpected adaptability of its PL synthesis pathways in response to different disturbances. These data provide new information by mapping the importance of the PL metabolic pathways of the malaria parasite and could be used to design future therapeutic approaches.
Assuntos
Malária Falciparum/parasitologia , Fosfolipídeos/metabolismo , Plasmodium falciparum/metabolismo , Redes e Vias Metabólicas , Fosfolipídeos/biossíntese , Plasmodium falciparum/fisiologiaRESUMO
Albitiazolium is the lead compound of bisthiazolium choline analogues and exerts powerful in vitro and in vivo antimalarial activities. Here we provide new insight into the fate of albitiazolium in vivo in mice and how it exerts its pharmacological activity. We show that the drug exhibits rapid and potent activity and has very favorable pharmacokinetic and pharmacodynamic properties. Pharmacokinetic studies in Plasmodium vinckei-infected mice indicated that albitiazolium rapidly and specifically accumulates to a great extent (cellular accumulation ratio, >150) in infected erythrocytes. Unexpectedly, plasma concentrations and the area under concentration-time curves increased by 15% and 69% when mice were infected at 0.9% and 8.9% parasitemia, respectively. Albitiazolium that had accumulated in infected erythrocytes and in the spleen was released into the plasma, where it was then available for another round of pharmacological activity. This recycling of the accumulated drug, after the rupture of the infected erythrocytes, likely extends its pharmacological effect. We also established a new viability assay in the P. vinckei-infected mouse model to discriminate between fast- and slow-acting antimalarials. We found that albitiazolium impaired parasite viability in less than 6 and 3 h at the ring and late stages, respectively, while parasite morphology was affected more belatedly. This highlights that viability and morphology are two parameters that can be differentially affected by a drug treatment, an element that should be taken into account when screening new antimalarial drugs.
Assuntos
Antimaláricos/farmacologia , Antimaláricos/farmacocinética , Eritrócitos/efeitos dos fármacos , Malária/tratamento farmacológico , Plasmodium/efeitos dos fármacos , Tiazóis/farmacologia , Tiazóis/farmacocinética , Animais , Eritrócitos/parasitologia , Feminino , Malária/parasitologia , Camundongos , Carga Parasitária , Testes de Sensibilidade Parasitária , Baço/efeitos dos fármacosRESUMO
The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis-Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.
Assuntos
Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , RNA Nucleotidiltransferases/química , Animais , Sítios de Ligação , Feminino , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismoRESUMO
Cation-π interactions to cognate ligands in enzymes have key roles in ligand binding and enzymatic catalysis. We have deciphered the key functional role of both charged and aromatic residues within the choline binding subsite of CTP:phosphocholine cytidylyltransferase and choline kinase from Plasmodium falciparum. Comparison of quaternary ammonium binding site structures revealed a general composite aromatic box pattern of enzyme recognition sites, well distinguished from the aromatic box recognition site of receptors.
Assuntos
Colina Quinase/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Plasmodium falciparum/enzimologia , Compostos de Amônio Quaternário/metabolismo , Sítios de Ligação , Colina Quinase/química , Colina-Fosfato Citidililtransferase/química , Malária Falciparum/parasitologia , Modelos Moleculares , Plasmodium falciparum/metabolismo , Ligação ProteicaRESUMO
The nucleotidase ISN1 is a potential therapeutic target of the purine salvage pathway of the malaria parasite Plasmodium falciparum. We identified PfISN1 ligands by in silico screening of a small library of nucleos(t)ide analogues and by thermal shift assays. Starting from a racemic cyclopentyl carbocyclic phosphonate scaffold, we explored the diversity on the nucleobase moiety and also proposed a convenient synthetic pathway to access the pure enantiomers of our initial hit (compound (±)-2). 2,6-Disubstituted purine containing derivatives such as compounds 1, (±)-7e and ß-L-(+)-2 showed the most potent inhibition of the parasite in vitro, with low micromolar IC50 values. These results are remarkable considering the anionic nature of nucleotide analogues, which are known to lack activity in cell culture experiments due to their scarce capacity to cross cell membranes. For the first time, we report the antimalarial activity of a carbocyclic methylphosphonate nucleoside with an L-like configuration.
Assuntos
Antimaláricos , Organofosfonatos , Plasmodium falciparum/metabolismo , Organofosfonatos/farmacologia , Antimaláricos/farmacologia , Antimaláricos/metabolismo , Nucleosídeos , Purinas/metabolismoRESUMO
In the human malaria parasite Plasmodium falciparum, the synthesis of the major and essential membrane phospholipid, phosphatidylcholine, occurs via the CDP-choline and the serine decarboxylase phosphoethanolamine methylation (SDPM) pathways, which are fueled by host choline, serine, and fatty acids. Both pathways share the final two steps catalyzed by two essential enzymes, P. falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) and choline-phosphate transferase (PfCEPT). We identified a novel class of phospholipid mimetics, which inhibit the growth of P. falciparum as well as Leishmania and Trypanosoma species. Metabolic analyses showed that one of these compounds, PG12, specifically blocks phosphatidylcholine biosynthesis from both the CDP-choline and SDPM pathways via inhibition of PfCCT. In vitro studies using recombinant PfCCT showed a dose-dependent inhibition of the enzyme by PG12. The potent antimalarial of this compound, its low cytotoxicity profile, and its established mode of action make it an excellent lead to advance for further drug development and efficacy in vivo.
Assuntos
Antimaláricos/farmacologia , Materiais Biomiméticos/farmacologia , Colina-Fosfato Citidililtransferase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Malária Falciparum/tratamento farmacológico , Fosfolipídeos/farmacologia , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Antimaláricos/química , Materiais Biomiméticos/química , Colina-Fosfato Citidililtransferase/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Malária Falciparum/enzimologia , Fosfolipídeos/química , Proteínas de Protozoários/metabolismoRESUMO
Phosphoinositide-specific phospholipase C (PI-PLC) is a major regulator of calcium-dependent signal transduction, which has been shown to be important in various processes of the malaria parasite Plasmodium. PI-PLC is generally implicated in calcium liberation from intracellular stores through the action of its product, inositol-(1,4,5)-trisphosphate, and is itself dependent on calcium for its activation. Here we describe the plc genes from Plasmodium species. The encoded proteins contain all domains typically found in PI-PLCs of the δ class but are almost twice as long as their orthologues in mammals. Transcriptional analysis by qRT-PCR of plc during the erythrocytic cycle of P. falciparum revealed steady expression levels that increased at the late schizont stages. Genetic analysis in the P. berghei model revealed that the plc locus was targetable but that plc gene knock-outs could not be obtained, thereby strongly indicating that the gene is essential during blood stage development. Alternatively, we attempted to modify plc expression through a promoter exchange approach but found the gene to be refractory to over-expression indicating that plc expression levels might additionally be tightly controlled.
Assuntos
Fosfoinositídeo Fosfolipase C/genética , Plasmodium berghei/enzimologia , Plasmodium falciparum/enzimologia , Sequência de Aminoácidos , Animais , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Fosfoinositídeo Fosfolipase C/química , Fosfoinositídeo Fosfolipase C/metabolismo , Plasmodium berghei/genética , Plasmodium falciparum/genética , Alinhamento de Sequência , Ativação TranscricionalRESUMO
The ROP2 protein and its paralogs are important virulence factors secreted into the host cell by the parasite Toxoplasma gondii. Here we describe the crystal structure of a large and soluble domain of mature ROP2, representative of the ROP2-like protein family. This is a structure of a protein-kinase fold that is devoid of catalytic residues and does not bind ATP. Various structural extensions constitute a signature of this protein family and act to maintain the protein kinase in an open conformation. Our ROP2 structure rules out a previous structural model of attachment of ROP2-like proteins to the parasitophorous vacuole membrane. We propose an alternative mode of membrane attachment implicating basic and amphiphatic helices present in the flexible N terminus of ROP2.
Assuntos
Proteínas de Membrana/química , Proteínas Quinases/química , Proteínas de Protozoários/química , Toxoplasma/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Dicroísmo Circular , Cricetinae , Primers do DNA , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Dobramento de Proteína , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Toxoplasma/patogenicidade , VirulênciaRESUMO
In vitro antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used in vitro antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.
Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Parasitária/métodos , Fatores de TempoRESUMO
The proliferation of the malaria-causing parasite Plasmodium falciparum within the erythrocyte is concomitant with massive phosphatidylcholine and phosphatidylethanolamine biosynthesis. Based on pharmacological and genetic data, de novo biosynthesis pathways of both phospholipids appear to be essential for parasite survival. The present study characterizes PfCK (P. falciparum choline kinase) and PfEK (P. falciparum ethanolamine kinase), which catalyse the first enzymatic steps of these essential metabolic pathways. Recombinant PfCK and PfEK were expressed as His6-tagged fusion proteins from overexpressing Escherichia coli strains, then purified to homogeneity and characterized. Using murine polyclonal antibodies against recombinant kinases, PfCK and PfEK were shown to be localized within the parasite cytoplasm. Protein expression levels increased during erythrocytic development. PfCK and PfEK appeared to be specific to their respective substrates and followed Michaelis-Menten kinetics. The Km value of PfCK for choline was 135.3+/-15.5 microM. PfCK was also able to phosphorylate ethanolamine with a very low affinity. PfEK was found to be an ethanolamine-specific kinase (Km=475.7+/-80.2 microM for ethanolamine). The quaternary ammonium compound hemicholinium-3 and an ethanolamine analogue, 2-amino-1-butanol, selectively inhibited PfCK or PfEK. In contrast, the bis-thiazolium compound T3, which was designed as a choline analogue and is currently in clinical trials for antimalarial treatment, affected PfCK and PfEK activities similarly. Inhibition exerted by T3 was competitive for both PfCK and PfEK and correlated with the impairment of cellular phosphatidylcholine biosynthesis. Comparative analyses of sequences and structures for both kinase types gave insights into their specific inhibition profiles and into the dual capacity of T3 to inhibit both PfCK and PfEK.
Assuntos
Colina Quinase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Amino Álcoois/farmacologia , Antimaláricos/farmacologia , Sítios de Ligação/genética , Western Blotting , Catálise/efeitos dos fármacos , Colina/metabolismo , Colina Quinase/química , Colina Quinase/genética , Dicroísmo Circular , Cristalografia por Raios X , Escherichia coli/genética , Etanolaminas/metabolismo , Hemicolínio 3/farmacologia , Cinética , Microscopia de Fluorescência , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plasmodium falciparum/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tiazóis/farmacologiaRESUMO
Malaria is an infectious disease caused by a parasite of the genus Plasmodium, and the emergence of parasites resistant to all current antimalarial drugs highlights the urgency of having new classes of molecules. We developed an effective method for the synthesis of a series of ß-modified acyclonucleoside phosphonate (ANP) derivatives, using commercially available and inexpensive materials (i.e., aspartic acid and purine heterocycles). Their biological evaluation in cell culture experiments and SAR revealed that the compounds' effectiveness depends on the presence of a hydroxyl group, the chain length (four carbons), and the nature of the nucleobase (guanine). The most active derivative inhibits the growth of Plasmodium falciparum in vitro in the nanomolar range (IC50 = 74 nM) with high selectivity index (SI > 1350). This compound also showed remarkable in vivo activity in P. berghei-infected mice (ED50 â¼ 0.5 mg/kg) when administered by the ip route and is, although less efficient, still active via the oral route. It is the first ANP derivative with such potent antimalarial activity and therefore has considerable potential for development as a new antimalarial drug.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/uso terapêutico , Feminino , Humanos , Células K562 , Camundongos , Nucleosídeos/química , Nucleosídeos/farmacologia , Nucleosídeos/uso terapêutico , Organofosfonatos/química , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Plasmodium falciparum/fisiologiaRESUMO
The development of the malaria parasite, Plasmodium falciparum, in the human erythrocyte, relies on phospholipid metabolism to fulfil the massive need for membrane biogenesis. Phosphatidylcholine (PC) is the most abundant phospholipid in Plasmodium membranes. PC biosynthesis is mainly ensured by the de novo Kennedy pathway that is considered as an antimalarial drug target. The CTP:phosphocholine cytidylyltransferase (CCT) catalyses the rate-limiting step of the Kennedy pathway. Here we report a series of structural snapshots of the PfCCT catalytic domain in its free, substrate- and product-complexed states that demonstrate the conformational changes during the catalytic mechanism. Structural data show the ligand-dependent conformational variations of a flexible lysine. Combined kinetic and ligand-binding analyses confirm the catalytic roles of this lysine and of two threonine residues of the helix αE. Finally, we assessed the variations in active site residues between Plasmodium and mammalian CCT which could be exploited for future antimalarial drug design.
Assuntos
Colina-Fosfato Citidililtransferase/química , Lipogênese/genética , Malária Falciparum/genética , Plasmodium falciparum/química , Sequência de Aminoácidos/genética , Animais , Antimaláricos/química , Antimaláricos/uso terapêutico , Catálise , Domínio Catalítico/genética , Colina-Fosfato Citidililtransferase/genética , Humanos , Cinética , Ligantes , Lipídeos/biossíntese , Lipídeos/química , Lipídeos/genética , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Ligação Proteica , Especificidade por SubstratoRESUMO
The histone-like nucleoid structuring (H-NS) protein is a global modulator of gene expression in Gram-negative bacteria. VicH, the H-NS protein of Vibrio cholerae, regulates the expression of certain major virulence determinants implicated in the pathogenesis of cholera. We present here the 2.5A crystal structure of the N-terminal oligomerisation domain of VicH (VicH_Nt). VicH_Nt adopts the same fold and dimeric assembly as the NMR structure of Escherichia coli H-NS_Nt, thus validating this fold against conflicting data. The structural similarity of V.cholerae VicH_Nt and E.coli H-NS_Nt, despite differences in origin, system of expression, experimental conditions and techniques used, indicates that the fold determined in our studies is robust to experimental conditions. Structural analysis and homology modelling were carried out to further elucidate the molecular basis of the functional polyvalence of the N-terminal domain. Our analysis of members of the H-NS superfamily supports the suggestion that the oligomerisation function of H-NS_Nt is conserved even in more distantly related proteins.
Assuntos
Cristalografia por Raios X , Vibrio cholerae/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
Phospholipid biosynthesis plays a key role in malarial infection and is regulated by CCT (CTP:phosphocholine cytidylyltransferase). This enzyme belongs to the group of amphitropic proteins which are regulated by reversible membrane interaction. To assess the role of the putative membrane-binding domain of Plasmodium falciparum CCT (PfCCT), we synthesized three peptides, K21, V20 and K54 corresponding to residues 274-294, 308-327 and 274-327 of PfCCT respectively. Conformational behaviour of the peptides, their ability to bind to liposomes and to destabilize lipid bilayers, and their insertion properties were investigated by different biophysical techniques. The intercalation mechanisms of the peptides were refined further by using surface-pressure measurements on various monolayers at the air/water interface. In the present study, we show that the three studied peptides are able to bind to anionic and neutral phospholipids, and that they present an alpha-helical conformation upon lipid binding. Peptides V20 and the full-length K54 intercalate their hydrophobic parts into an anionic bilayer and, to a lesser extent, a neutral one for V20. Peptide K21 interacts only superficially with both types of phospholipid vesicles. Adsorption experiments performed at the air/water interface revealed that peptide K54 is strongly surface-active in the absence of lipid. Peptide V20 presents an atypical behaviour in the presence of phosphatidylserine. Whatever the initial surface pressure of a phosphatidylserine film, peptide V20 and phosphatidylserine entities seem linked together in a special organization involving electrostatic and hydrophobic interactions. We showed that PfCCT presents different lipid-dependence properties from other studied CCTs. Although the lipid-binding domain seems to be located in the C-terminal region of the enzyme, as with the mammalian counterpart, the membrane anchorage, which plays a key role in the enzyme regulation, is driven by two alpha-helices, which behave differently from one another.
Assuntos
Colina-Fosfato Citidililtransferase/química , Metabolismo dos Lipídeos , Oligopeptídeos/química , Plasmodium falciparum/enzimologia , Adsorção , Animais , Sítios de Ligação , Colina-Fosfato Citidililtransferase/metabolismo , Dicroísmo Circular , Cinética , Lipossomos/química , Lipossomos/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência , Propriedades de Superfície , Água/químicaRESUMO
Phosphatidylcholine is the major lipid component of the malaria parasite membranes and is required for parasite multiplication in human erythrocytes. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) is the rate-limiting enzyme of the phosphatidylcholine biosynthesis pathway and thus considered as a potential antimalarial target. In contrast to its mammalian orthologs, PfCCT contains a duplicated catalytic domain. Here, we show that both domains are catalytically active with similar kinetic parameters. A virtual screening strategy allowed the identification of a drug-size molecule competitively inhibiting the enzyme. This compound also prevented phosphatidylcholine biosynthesis in parasites and exerted an antimalarial effect. This study constitutes the first step towards a rationalized design of future new antimalarial agents targeting PfCCT.
Assuntos
Domínio Catalítico , Colina-Fosfato Citidililtransferase/metabolismo , Citidina Difosfato Colina/análogos & derivados , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Antimaláricos/química , Antimaláricos/farmacologia , Vias Biossintéticas/genética , Colina-Fosfato Citidililtransferase/antagonistas & inibidores , Colina-Fosfato Citidililtransferase/genética , Citidina Difosfato Colina/química , Citidina Difosfato Colina/farmacologia , Humanos , Immunoblotting , Cinética , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/química , Plasmodium falciparum/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Homologia de Sequência de AminoácidosRESUMO
The enzyme CTP:phosphocholine cytidylyltransferase (CCT) is essential in the lipid biosynthesis of Plasmodia (Haemosporida), presenting a promising antimalarial target. Here, we identified two independent gene duplication events of CCT within Apicomplexa and characterized a truncated construct of Plasmodium falciparum CCT that forms a dimer resembling the molecular architecture of CCT enzymes from other sources. Based on biophysical and enzyme kinetics methods, our data show that the CDP-choline product of the CCT enzymatic reaction binds to the enzyme considerably stronger than either substrate (CTP or choline phosphate). Interestingly, in the presence of Mg²âº , considered to be a cofactor of the enzyme, the binding of the CTP substrate is attenuated by a factor of 5. The weaker binding of CTP:Mg²âº , similarly to the related enzyme family of aminoacyl tRNA synthetases, suggests that, with lack of Mg²âº , positively charged side chain(s) of CCT may contribute to CTP accommodation. Thermodynamic investigations by isothermal titration calorimetry and fluorescent spectroscopy studies indicate that accommodation of the choline phosphate moiety in the CCT active site is different when it appears on its own as one of the substrates or when it is linked to the CDP-choline product. A tryptophan residue within the active site is identified as a useful internal fluorescence sensor of enzyme-ligand binding. Results indicate that the catalytic mechanism of Plasmodium falciparum CCT may involve conformational changes affecting the choline subsite of the enzyme.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Evolução Molecular , Modelos Moleculares , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Apicomplexa/enzimologia , Apicomplexa/genética , Apicomplexa/metabolismo , Biocatálise , Domínio Catalítico , Colina-Fosfato Citidililtransferase/química , Colina-Fosfato Citidililtransferase/genética , Citidina Difosfato Colina/química , Citidina Difosfato Colina/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , Dimerização , Estabilidade Enzimática , Deleção de Genes , Duplicação Gênica , Magnésio/metabolismo , Dados de Sequência Molecular , Fosforilcolina/química , Fosforilcolina/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Triptofano/químicaRESUMO
Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the main membrane phospholipids (PLs) of Plasmodium parasites and can be generated by the de novo (Kennedy) CDP-choline and CDP-ethanolamine pathways and by the CDP-diacylglycerol dependent pathway. The Kennedy pathways initiate from exogenous choline and ethanolamine involving choline kinase (CK) and ethanolamine kinase (EK), followed by the choline-phosphate cytidylyltransferase (CCT) and ethanolamine-phosphate cytidylyltransferase (ECT) that catalyse the formation of CDP-choline and CDP-ethanolamine. Finally, in Plasmodium, PC and PE are apparently synthesized by a common choline/ethanolamine-phosphotransferase (CEPT). Here, we have studied the essential nature of the Kennedy pathways in Plasmodium berghei, a rodent malaria parasite. Sequence analysis of the P. berghei CEPT, CCT, ECT and CK enzymes revealed the presence of all catalytic domains and essential residues and motifs necessary for enzymatic activities. Constructs were designed for the generation of gene knockout and GFP-fusions of the cept, cct, ect and ck genes in P. berghei. We found that all four genes were consistently refractory to knockout attempts. At the same time, successful tagging of these proteins with GFP demonstrated that the loci were targetable and indicated that these genes are essential in P. berghei blood stage parasites. GFP-fusions of CCT, ECT and CK were found in the cytosol whereas the GFP-CEPT mainly localised in the endoplasmic reticulum. These results indicate that both CDP-choline and CDP-ethanolamine de novo pathways are essential for asexual P. berghei development and are non-redundant with other possible sources of PC and PE.