RESUMEN
Endemic Burkitt lymphoma (eBL) is characterized by an oncogenic IGH/c-MYC translocation and Epstein-Barr virus (EBV) positivity, and is epidemiologically linked to Plasmodium falciparum malaria. Both EBV and malaria are thought to contribute to eBL by inducing the expression of activation-induced cytidine deaminase (AID), an enzyme involved in the IGH/c-MYC translocation. AID/apolipoprotein B mRNA editing catalytic polypeptide-like (AID/APOBEC) family enzymes have recently emerged as potent mutagenic sources in a variety of cancers, but apart from AID, their involvement in eBL and their regulation by EBV and P. falciparum is unknown. Here, we show that upon inoculation with EBV, human B cells strongly upregulate the expression of enzymatically active APOBEC3B and APOBEC3G. In addition, we found significantly increased levels of APOBEC3A in B cells of malaria patients, which correlated with parasite load. Interestingly, despite the fact that APOBEC3A, APOBEC3B, and APOBEC3G caused c-MYC mutations when overexpressed in HEK293T cells, a mutational enrichment in eBL tumors was only detected in AID motifs. This suggests that even though the EBV- and P. falciparum-directed immune response triggers the expression and activity of several AID/APOBEC members, only the upregulation of AID has oncogenic consequences, while the induction of the APOBEC3 subfamily may primarily have immunoprotective functions.
Asunto(s)
Desaminasas APOBEC , Linfoma de Burkitt , Citidina Desaminasa , Infecciones por Virus de Epstein-Barr , Malaria Falciparum , Desaminasas APOBEC/genética , Desaminasa APOBEC-3G , Linfoma de Burkitt/enzimología , Linfoma de Burkitt/genética , Citidina Desaminasa/genética , Infecciones por Virus de Epstein-Barr/enzimología , Infecciones por Virus de Epstein-Barr/genética , Células HEK293 , Herpesvirus Humano 4 , Humanos , Malaria Falciparum/enzimología , Malaria Falciparum/genética , Antígenos de Histocompatibilidad Menor , MutágenosRESUMEN
Malaria is a life-threatening infectious disease primarily caused by the Plasmodium falciparum parasite. The increasing resistance to current antimalarial drugs and their side effects has led to an urgent need for novel malaria drug targets, such as the P. falciparum cGMP-dependent protein kinase (pfPKG). However, PKG plays an essential regulatory role also in the human host. Human cGMP-dependent protein kinase (hPKG) and pfPKG are controlled by structurally homologous cGMP-binding domains (CBDs). Here, we show that despite the structural similarities between the essential CBDs in pfPKG and hPKG, their respective allosteric networks differ significantly. Through comparative analyses of chemical shift covariance analyses, molecular dynamics simulations, and backbone internal dynamics measurements, we found that conserved allosteric elements within the essential CBDs are wired differently in pfPKG and hPKG to implement cGMP-dependent kinase activation. Such pfPKG versus hPKG rewiring of allosteric networks was unexpected because of the structural similarity between the two essential CBDs. Yet, such finding provides crucial information on which elements to target for selective inhibition of pfPKG versus hPKG, which may potentially reduce undesired side effects in malaria treatments.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico , Malaria Falciparum , Plasmodium falciparum , Regulación Alostérica , Proteínas Quinasas Dependientes de GMP Cíclico/química , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Humanos , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Simulación de Dinámica Molecular , Plasmodium falciparum/enzimología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.
Asunto(s)
Compuestos de Boro/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/farmacología , Administración Oral , Animales , Compuestos de Boro/administración & dosificación , Compuestos de Boro/química , Dominio Catalítico , Humanos , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Moleculares , Plasmodium falciparum/enzimología , Inhibidores de Proteasoma/administración & dosificación , Inhibidores de Proteasoma/químicaRESUMEN
Resistance to antimalarial drugs has spread rapidly over the past few decades. The WHO recommends artemisinin-based combination therapies for the treatment of uncomplicated malaria, but unfortunately these approaches are losing their efficacy in large areas of Southeast Asia. In 2016, artemisinin resistance was confirmed in 5 countries of the Greater Mekong subregion. We focused our study on Syk inhibitors as antimalarial drugs. The Syk protein is present in human erythrocytes, and the membrane of protein band 3 is its major target following activation by oxidant stress. Tyr phosphorylation of band 3 occurs during P. falciparum growth, leading to the release of microparticles containing hemicromes and structural weakening of the host cell membrane, simplifying merozoite reinfection. Syk inhibitors block these events by interacting with the Syk protein's catalytic site. We performed in vitro proteomics and in silico studies and compared the results. In vitro studies were based on treatment of the parasite's cellular cultures with different concentrations of Syk inhibitors, while proteomics studies were focused on the Tyr phosphorylation of band 3 by Syk protein with the same concentrations of drugs. In silico studies were based on different molecular modeling approaches in order to analyze and optimize the ligand-protein interactions and obtain the highest efficacy in vitro. In the presence of Syk inhibitors, we observed a marked decrease of band 3 Tyr phosphorylation according to the increase of the drug's concentration. Our studies could be useful for the structural optimization of these compounds and for the design of novel Syk inhibitors in the future.
Asunto(s)
Antimaláricos , Eritrocitos , Malaria Falciparum , Plasmodium falciparum/crecimiento & desarrollo , Inhibidores de Proteínas Quinasas , Quinasa Syk , Antimaláricos/química , Antimaláricos/farmacología , Relación Dosis-Respuesta a Droga , Eritrocitos/enzimología , Eritrocitos/parasitología , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinasa Syk/antagonistas & inhibidores , Quinasa Syk/química , Quinasa Syk/metabolismoRESUMEN
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.
Asunto(s)
Antimaláricos/farmacología , Ciclo Celular , Malaria Falciparum/enzimología , Plasmodium falciparum/enzimología , Transducción de Señal , Proteína Elk-1 con Dominio ets/antagonistas & inhibidores , Antimaláricos/química , Humanos , Malaria Falciparum/tratamiento farmacológico , Melatonina , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
The cytochrome bc1 complex is a key component of the mitochondrial respiratory chains of many eukaryotic microorganisms that are pathogenic for plants or humans, such as fungi responsible for crop diseases and Plasmodium falciparum, which causes human malaria. Cytochrome bc1 is an enzyme that contains two (ubi)quinone/quinol-binding sites, which can be exploited for the development of fungicidal and chemotherapeutic agents. Here, we review recent progress in determination of the structure and mechanism of action of cytochrome bc1 , and the associated development of antimicrobial agents (and associated resistance mechanisms) targeting its activity.
Asunto(s)
Antifúngicos/farmacología , Antimaláricos/uso terapéutico , Complejo III de Transporte de Electrones , Proteínas Fúngicas , Hongos/enzimología , Malaria Falciparum , Enfermedades de las Plantas/microbiología , Plasmodium falciparum/enzimología , Proteínas Protozoarias , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo III de Transporte de Electrones/metabolismo , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/metabolismo , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismoRESUMEN
Recently, we heterologously expressed, purified, and analyzed the function of the sole Plasmodium falciparum phosphatidylinositol 3-kinase (PI3K), found that the enzyme is a "class III" or "Vps34" PI3K, and found that it is irreversibly inhibited by Fe2+-mediated covalent, nonspecific interactions with the leading antimalarial drug, dihydroartemisinin [Hassett, M. R., et al. (2017) Biochemistry 56, 4335-4345]. One of several P. falciparum phosphatidylinositol 4-kinases [putative IIIß isoform (PfPI4KIIIß)] has generated similar interest as a druggable target; however, no validation of the mechanism of action for putative PfPI4K inhibitors has yet been possible due to the lack of purified PfPI4KIIIß. We therefore codon optimized the pfpi4kIIIß gene, successfully expressed the protein in yeast, and purified an N-lobe catalytic domain PfPI4KIIIß protein. Using an enzyme-linked immunosorbent assay strategy previously perfected for analysis of PfPI3K (PfVps34), we measured the apparent initial rate, Km,app(ATP), and other enzyme characteristics and found full activity for the construct and that PfPI4KIIIß activity is most consistent with the class IIIß designation. Because several novel antimalarial drug candidates with different chemical scaffolds have been proposed to target PfPI4KIIIß, we titrated enzyme inhibition for these candidates versus purified PfPI4KIIIß and PfVps34. We also analyzed the activity versus purified PfPI4KIIIß mutants previously expressed in P. falciparum selected for resistance to these drugs. Interestingly, we found that a putative PfPI4KIIIß inhibitor currently in advanced trials (MMV390048; MMV '0048) is a potent inhibitor of both PfVps34 and PfPI4KIIIß. These data are helpful for further preclinical optimization of an exciting new class of P. falciparum PI kinase inhibitor ("PfPIKi") antimalarial drugs.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Aminopiridinas/farmacología , Malaria Falciparum/parasitología , Plasmodium falciparum/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/metabolismo , Sulfonas/farmacología , 1-Fosfatidilinositol 4-Quinasa/aislamiento & purificación , Antimaláricos/farmacología , Técnicas de Cultivo de Célula , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Modelos Moleculares , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/aislamiento & purificación , Relación Estructura-Actividad , LevadurasRESUMEN
Subtilisin-like serine peptidases (subtilases) play important roles in the life cycle of many organisms, including the protozoan parasites that are the causative agent of malaria, Plasmodium spp. As with other peptidases, subtilase proteolytic activity has to be tightly regulated in order to prevent potentially deleterious uncontrolled protein degradation. Maturation of most subtilases requires the presence of an N-terminal propeptide that facilitates folding of the catalytic domain. Following its proteolytic cleavage, the propeptide acts as a transient, tightly bound inhibitor until its eventual complete removal to generate active protease. Here we report the identification of a stand-alone malaria parasite propeptide-like protein, called SUB1-ProM, encoded by a conserved gene that lies in a highly syntenic locus adjacent to three of the four subtilisin-like genes in the Plasmodium genome. Template-based modelling and ab initio structure prediction showed that the SUB1-ProM core structure is most similar to the X-ray crystal structure of the propeptide of SUB1, an essential parasite subtilase that is discharged into the parasitophorous vacuole (PV) to trigger parasite release (egress) from infected host cells. Recombinant Plasmodium falciparum SUB1-ProM was found to be a fast-binding, potent inhibitor of P. falciparum SUB1, but not of the only other essential blood-stage parasite subtilase, SUB2, or of other proteases examined. Mass-spectrometry and immunofluorescence showed that SUB1-ProM is expressed in the PV of blood stage P. falciparum, where it may act as an endogenous inhibitor to regulate SUB1 activity in the parasite.
Asunto(s)
Malaria Falciparum/genética , Plasmodium falciparum/genética , Serina Proteasas/química , Subtilisina/genética , Secuencia de Aminoácidos/genética , Animales , Eritrocitos/parasitología , Genoma/genética , Humanos , Estadios del Ciclo de Vida/genética , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Plasmodium falciparum/patogenicidad , Proteolisis , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Serina Proteasas/genética , Subtilisina/química , Vacuolas/parasitologíaRESUMEN
Carbon (C), hydrogen (H), nitrogen (N), oxygen (O), and sulfur (S) atoms intrigue as they are the foundation for amino acid (AA) composition and the folding and functions of proteins and thus define and control the survival of a cell, the smallest unit of life. Here, we calculated the proteomic atom distribution in >1500 randomly selected species across the entire current phylogenetic tree and identified uracil-5-methyltransferase (U5MTase) of the protozoan parasite Plasmodium falciparum (Pf, strain Pf3D7), with a distinct atom and AA distribution pattern. We determined its apicoplast location and in silico 3D protein structure to refocus attention exclusively on U5MTase with tremendous potential for therapeutic intervention in malaria. Around 300 million clinical cases of malaria occur each year in tropical and subtropical regions of the world, resulting in over one million deaths annually, placing malaria among the most serious infectious diseases. Genomic and proteomic research of the clades of parasites containing Pf is progressing slowly and the functions of most of the â¼5300 genes are still unknown. We applied a 'bottom-up' comparative proteomic atomics analysis across the phylogenetic tree to visualize a protein molecule on its actual basis - i. e., its atomic level. We identified a protruding Pf3D7-specific U5MTase, determined its 3D protein structure, and identified potential inhibitory drug molecules through in silico drug screening that might serve as possible remedies for the treatment of malaria. Besides, this atomic-based proteome map provides a unique approach for the identification of parasite-specific proteins that could be considered as novel therapeutic targets.
Asunto(s)
Biología Computacional/métodos , Malaria Falciparum/enzimología , Metiltransferasas/química , Plasmodium falciparum/enzimología , Proteoma/química , Proteoma/metabolismo , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Aminoácidos/química , Carbono/análisis , Simulación por Computador , Bases de Datos Genéticas , Humanos , Hidrógeno/análisis , Ligandos , Malaria Falciparum/tratamiento farmacológico , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Químicos , Nitrógeno/análisis , Oxígeno/análisis , Filogenia , Plasmodium falciparum/química , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Estructura Terciaria de Proteína , Proteómica , Azufre/análisis , Uracilo/metabolismoRESUMEN
FIKK kinases in the human malaria parasite Plasmodium falciparum are attractive targets for new anti-malaria drugs, as they have no orthologues in humans and have been linked to disease severity. Six FIKKs are known to be exported into red blood cells (RBCs) where they mediate dramatic structural and functional changes to RBCs that are central to pathogenesis. Eleven members of this family, which are predicted to be exported into infected RBCs (iRBCs), remain uncharacterised. Using a targeted gene-knockout approach, we have characterised these FIKKs and discovered that five are essential for parasite survival. Three of these five FIKKs (FIKK9.1, FIKK10.1, FIKK10.2) were exported from the parasite into iRBCs and for two of these (FIKK9.1 and FIKK10.1), export was via Maurer's clefts (parasite-derived structures involved in protein trafficking and pathognomonic of falciparum malaria). Of the remaining two essential kinases, FIKK3 was associated with rhoptries (specialised protein secretory organelles in the parasite) and FIKK9.5 was localised in the parasite nucleus. The diverse localisation and essentiality of these FIKKs demonstrate that they play different but essential roles in the survival of P. falciparum in RBCs and therefore are attractive new drug targets for the prevention or treatment of falciparum malaria.
Asunto(s)
Eritrocitos/enzimología , Malaria Falciparum/enzimología , Plasmodium falciparum/enzimología , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Eritrocitos/parasitología , Eritrocitos/patología , Humanos , Malaria Falciparum/genética , Malaria Falciparum/patología , Plasmodium falciparum/genética , Proteínas Quinasas/genética , Proteínas Protozoarias/genéticaRESUMEN
Plasmodium falciparum gametocytes infect mosquitoes and are responsible for malaria transmission. New interventions that block transmission could accelerate malaria elimination. Gametocytes develop within erythrocytes and activate protein export pathways that remodel the host cell. Plasmepsin V (PMV) is an aspartyl protease that is required for protein export in asexual parasites, but its function and essentiality in gametocytes has not been definitively proven, nor has PMV been assessed as a transmission-blocking drug target. Here, we show that PMV is expressed and can be inhibited specifically in P. falciparum stage I-II gametocytes. PMV inhibitors block processing and export of gametocyte effector proteins and inhibit development of stage II-V gametocytes. Gametocytogenesis in the presence of sublethal inhibitor concentrations results in stage V gametocytes that fail to infect mosquitoes. Therefore, PMV primes gametocyte effectors for export, which is essential for the development and fitness of gametocytes for transmission to mosquitoes.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Culicidae/crecimiento & desarrollo , Inhibidores Enzimáticos/farmacología , Gametogénesis/efectos de los fármacos , Malaria Falciparum/prevención & control , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Culicidae/efectos de los fármacos , Culicidae/parasitología , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Humanos , Estadios del Ciclo de Vida , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismoRESUMEN
The hemoglobin degradation process in Plasmodium parasites is vital for nutrient acquisition required for their growth and proliferation. In P. falciparum, falcipains (FP-2 and FP-3) are the major hemoglobinases, and remain attractive antimalarial drug targets. Other Plasmodium species also possess highly homologous proteins to FP-2 and FP-3. Although several inhibitors have been designed against these proteins, none has been commercialized due to associated toxicity on human cathepsins (Cat-K, Cat-L and Cat-S). Despite the two enzyme groups sharing a common structural fold and catalytic mechanism, distinct active site variations have been identified, and can be exploited for drug development. Here, we utilize in silico approaches to screen 628 compounds from the South African natural sources to identify potential hits that can selectively inhibit the plasmodial proteases. Using docking studies, seven abietane diterpenoids, binding strongly to the plasmodial proteases, and three additional analogs from PubChem were identified. Important residues involved in ligand stabilization were identified for all potential hits through binding pose analysis and their energetic contribution determined by binding free energy calculations. The identified compounds present important scaffolds that could be further developed as plasmodial protease inhibitors. Previous laboratory assays showed the effect of the seven diterpenoids as antimalarials. Here, for the first time, we demonstrate that their possible mechanism of action could be by interacting with falcipains and their plasmodial homologs. Dynamic residue network (DRN) analysis on the plasmodial proteases identified functionally important residues, including a region with high betweenness centrality, which had previously been proposed as a potential allosteric site in FP-2.
Asunto(s)
Abietanos/química , Antimaláricos/química , Simulación del Acoplamiento Molecular , Péptido Hidrolasas/química , Plasmodium falciparum/enzimología , Inhibidores de Proteasas/química , Proteínas Protozoarias , Animales , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , SudáfricaRESUMEN
Malaria is a highly inflammatory disease caused by Plasmodium infection of host erythrocytes. However, the parasite does not induce inflammatory cytokine responses in macrophages in vitro and the source of inflammation in patients remains unclear. Here, we identify oxidative stress, which is common in malaria, as an effective trigger of the inflammatory activation of macrophages. We observed that extracellular reactive oxygen species (ROS) produced by xanthine oxidase (XO), an enzyme upregulated during malaria, induce a strong inflammatory cytokine response in primary human monocyte-derived macrophages. In malaria patients, elevated plasma XO activity correlates with high levels of inflammatory cytokines and with the development of cerebral malaria. We found that incubation of macrophages with plasma from these patients can induce a XO-dependent inflammatory cytokine response, identifying a host factor as a trigger for inflammation in malaria. XO-produced ROS also increase the synthesis of pro-IL-1ß, while the parasite activates caspase-1, providing the two necessary signals for the activation of the NLRP3 inflammasome. We propose that XO-produced ROS are a key factor for the trigger of inflammation during malaria.
Asunto(s)
Inflamación/enzimología , Macrófagos/enzimología , Malaria Cerebral/enzimología , Malaria Falciparum/enzimología , Estrés Oxidativo , Plasmodium falciparum/patogenicidad , Especies Reactivas de Oxígeno/metabolismo , Xantina Oxidasa/metabolismo , Caspasa 1/metabolismo , Células Cultivadas , Citocinas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inflamación/sangre , Inflamación/parasitología , Mediadores de Inflamación/metabolismo , Activación de Macrófagos , Macrófagos/parasitología , Malaria Cerebral/sangre , Malaria Cerebral/parasitología , Malaria Falciparum/sangre , Malaria Falciparum/parasitología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de SeñalAsunto(s)
Malaria Falciparum , Modelos Biológicos , Plasmodium falciparum , Animales , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Malaria Falciparum/genética , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidadRESUMEN
Malaria is one of the three major global health threats. Drug development for malaria, especially for its most dangerous form caused by Plasmodium falciparum, remains an urgent task due to the emerging drug-resistant parasites. Exploration of novel antimalarial drug targets identified a trifunctional enzyme, malate quinone oxidoreductase (MQO), located in the mitochondrial inner membrane of P. falciparum (PfMQO). PfMQO is involved in the pathways of mitochondrial electron transport chain, tricarboxylic acid cycle, and fumarate cycle. Recent studies have shown that MQO is essential for P. falciparum survival in asexual stage and for the development of experiment cerebral malaria in the murine parasite P. berghei, providing genetic validation of MQO as a drug target. However, chemical validation of MQO, as a target, remains unexplored. In this study, we used active recombinant protein rPfMQO overexpressed in bacterial membrane fractions to screen a total of 400 compounds from the Pathogen Box, released by Medicines for Malaria Venture. The screening identified seven hit compounds targeting rPfMQO with an IC50 of under 5 µM. We tested the activity of hit compounds against the growth of 3D7 wildtype strain of P. falciparum, among which four compounds showed an IC50 from low to sub-micromolar concentrations, suggesting that PfMQO is indeed a potential antimalarial drug target.
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Inhibidores Enzimáticos/farmacología , Malaria Cerebral/tratamiento farmacológico , Malaria Falciparum/tratamiento farmacológico , Oxidorreductasas/antagonistas & inhibidores , Animales , Antimaláricos/metabolismo , Antimaláricos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Malaria Cerebral/enzimología , Malaria Cerebral/parasitología , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Malatos/metabolismo , Ratones , Mitocondrias/enzimología , Oxidorreductasas/genética , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/patogenicidad , Plasmodium falciparum/enzimología , Plasmodium falciparum/patogenicidad , Quinonas/metabolismoRESUMEN
BACKGROUND: Cytochrome P450 (CYP) enzymes are essential in the metabolism of most drugs used today. Single nucleotide polymorphism(s) occurring in CYP genes can adversely affect drug pharmacokinetics, efficacy, and safety. Individuals carrying the CYP2C8*2 c.805A > T (CYP2C8*2; rs11572103) allele have impaired amodiaquine metabolism, increased risk of amodiaquine-related adverse events, and may promote the selection of drug-resistant parasite strains. This study investigated the distribution of the CYP2C8*2 allele in Brazzaville, Republic of Congo, where artesunate + amodiaquine is used as the second-line treatment for uncomplicated Plasmodium falciparum malaria. METHODS: A total of 285 febrile children visiting the Marien Ngouabi paediatric hospital were genotyped for CYP2C8*2 using PCR-restriction fragment length polymorphism (PCR-RFLP). The allele frequencies and genotype distribution were determined. RESULTS: The CYP2C8*2 allele was successfully genotyped in 75% (213/285) of the study participants. The CYP2C8*2A allele had a frequency of 63%, whereas the CYP2C8*2T allele had a frequency of 37%. Genotypes CYP2C8*2AA (rapid metabolizer), CYP2C8*2AT (intermediate metabolizer), and CYP2C8*2TT (poor metabolizer) were observed in 44%, 38%, and 18% of the investigated participants, respectively. CONCLUSIONS: This study gives the first description of CYP2C8*2 allele distribution in the Republic of Congo and highlights the potential risk of amodiaquine-related adverse events. Information from this study will be beneficial during pharmacovigilance investigations.
Asunto(s)
Amodiaquina/uso terapéutico , Antimaláricos/uso terapéutico , Citocromo P-450 CYP2C8/genética , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/genética , Alelos , Artemisininas/uso terapéutico , Niño , Preescolar , Congo , Combinación de Medicamentos , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Lactante , Malaria Falciparum/enzimología , Masculino , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido SimpleRESUMEN
Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
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Criptosporidiosis , Cryptosporidium parvum/enzimología , Inhibidores Enzimáticos/farmacología , Lisina-ARNt Ligasa/antagonistas & inhibidores , Malaria Falciparum , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Criptosporidiosis/tratamiento farmacológico , Criptosporidiosis/enzimología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Humanos , Lisina-ARNt Ligasa/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Ratones SCID , Proteínas Protozoarias/metabolismoRESUMEN
Severe malaria due to Plasmodium falciparum remains a significant global health threat. DXR, the second enzyme in the MEP pathway, plays an important role to synthesize building blocks for isoprenoids. This enzyme is a promising drug target for malaria due to its essentiality as well as its absence in humans. In this study, we designed and synthesized a series of α,ß-unsaturated analogues of fosmidomycin, a natural product that inhibits DXR in P. falciparum. All compounds were evaluated as inhibitors of P. falciparum. The most promising compound, 18a, displays on-target, potent inhibition against the growth of P. falciparum (IC50 = 13 nM) without significant inhibition of HepG2 cells (IC50 > 50 µM). 18a was also tested in a luciferase-based Plasmodium berghei mouse model of malaria and showed exceptional in vivo efficacy. Together, the data support MEPicide 18a as a novel, potent, and promising drug candidate for the treatment of malaria.
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Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Antimaláricos/farmacología , Fosfomicina/análogos & derivados , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/crecimiento & desarrollo , Profármacos/farmacología , Animales , Antimaláricos/química , Femenino , Fosfomicina/química , Fosfomicina/farmacología , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Ratones , Plasmodium falciparum/efectos de los fármacos , Profármacos/química , Relación Estructura-ActividadRESUMEN
In erythrocytes, actively multiplying Plasmodium falciparum parasites exhibit a unique signature of virulence associated histone modifications, thereby epigenetically regulating the expression of the majority of genes. Histone acetylation is one such modification, effectuated and maintained by the dynamic interplay of two functionally antagonist enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Their inhibition leads to hypo/hyperacetylation and is known to be deleterious for P. falciparum, and hence they have become attractive molecular targets to design novel antimalarials. Many compounds, including four Food and Drug Administration (FDA) approved drugs, have been developed so far to inhibit HDAC activity but are not suitable to treat malaria as they lack selectivity and cause cytotoxicity in mammalian cells. In this study, we used comparative modeling and molecular docking to establish different binding modes of nonselective and selective compounds in the PfHDAC-1 (a class I HDAC protein in P. falciparum) active site and identified the involvement of active site nonidentical residues in binding of selective compounds. Further, we have applied virtual screening with precise selection criteria and molecular dynamics simulation to identify novel potential inhibitors against PfHDAC-1. We report 20 compounds (10 from ChEMBL and 10 from analogues compound library) bearing seven scaffolds having better affinity toward PfHDAC-1. Sixteen of these compounds are known antimalarials with 14 having activity in the nanomolar range against various drug resistant and sensitive strains of P. falciparum. The cytotoxicity of these compounds against various human cell lines are reported at relatively higher concentration and hence can be used as potential PfHDAC-1 inhibitors in P. falciparum. These findings indeed show great potential for using the above molecules as prospective antimalarials.
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Histona Desacetilasa 1 , Inhibidores de Histona Desacetilasas , Malaria Falciparum , Plasmodium falciparum/enzimología , Proteínas Protozoarias , Línea Celular , Histona Desacetilasa 1/química , Histona Desacetilasa 1/metabolismo , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
OBJECTIVE: We assessed the association of mutant allele frequencies of nitric oxide synthase 2 (NOS2) gene at two SNPs (-954 and -1173) with malaria disease severity in children from a malaria endemic area in Southern Ghana. METHOD: Using children recruited at the hospital, assigned into clinical subgroups of uncomplicated and severe malaria and matching with their "healthy control" counterparts, we designed a case control study. Genomic DNA was extracted and genotyping using Restriction Fragment Polymorphism was done. RESULT: A total of 123 malaria cases (91 uncomplicated, 32 severe) and 100 controls were sampled. Their corresponding mean Hbs were 9.6, 9.3 and 11.2g/dl and geometric mean parasite densities of 32097, 193252 and 0 parasites/ml respectively. Variant allele frequencies varied from 0.09 through 0.03 to 0.12 for G-954C and 0.06 through 0.03 to 0.07 for C-1173T in the uncomplicated, severe and healthy control groups respectively. There was a strong linkage disequilibrium between the two alleles (p<0.001). For the -954 position, the odds of developing severe malaria was found to be 2.5 times lower with the carriage of a C allele compared to those without severe malaria (χ2; p< 0.05) though this isn't the case with -1173. CONCLUSION: The carriage of a mutant allele in the -954 NOS2 gene may have a protective effect on malaria among Southern Ghanaian children.