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[This corrects the article DOI: 10.1371/journal.ppat.1011517.].
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Apicomplexans are widespread parasites of humans and other animals, and include the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). Existing anti-apicomplexan therapies are beset with issues around drug resistance and toxicity, and new treatment options are needed. The mitochondrial electron transport chain (ETC) is one of the few processes that has been validated as a drug target in apicomplexans. To identify new inhibitors of the apicomplexan ETC, we developed a Seahorse XFe96 flux analyzer approach to screen the 400 compounds contained within the Medicines for Malaria Venture 'Pathogen Box' for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC in T. gondii, at least four of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. MMV688853 belongs to a compound class, the aminopyrazole carboxamides, that were shown previously to target a kinase with a key role in parasite invasion of host cells. Our data therefore reveal that MMV688853 has dual targets in apicomplexans. We further developed our approach to pinpoint the molecular targets of these inhibitors, demonstrating that all target Complex III of the ETC, with MMV688853 targeting the ubiquinone reduction (Qi) site of the complex. Most of the compounds we identified remain effective inhibitors of parasites that are resistant to Complex III inhibitors that are in clinical use or development, indicating that they could be used in treating drug resistant parasites. In sum, we have developed a versatile, scalable approach to screen for compounds that target the ETC in apicomplexan parasites, and used this to identify and characterize novel inhibitors.
Assuntos
Parasitos , Toxoplasma , Toxoplasmose , Animais , Humanos , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons , Toxoplasmose/parasitologia , Plasmodium falciparumRESUMO
Neuropeptides play critical roles in cnidarian development. However, although they are known to play key roles in settlement and metamorphosis, their temporal and spatial developmental expression has not previously been characterized in any coral. We here describe Acropora millepora LWamide and RFamide and their developmental expression from the time of their first appearance, using in situ hybridization and FMRFamide immunohistochemistry. AmRFamide transcripts first appear in the ectoderm toward the oral end of the planula larva following blastopore closure. This oral bias becomes less apparent as the planula develops. The cell bodies of AmRFamide-expressing cells are centrally located in the ectoderm, with narrow projections to the mesoglea and to the cell surface. As the planula approaches settlement, AmRFamide expression disappears and is undetectable in the newly settled polyp. Expressing cells then gradually reappear as the polyp develops, becoming particularly abundant on the tentacles. AmLWamide transcripts first appear in ectodermal cells of the developing planula, with minimal expression at its two ends. The cell bodies of expressing cells lie just above the mesoglea, in a position distinct from those of AmRFamide-expressing cells, and have a narrow projection extending across the ectoderm to its surface. AmLWamide-expressing cells persist for most of the planula stage, disappearing shortly before settlement, but later than AmRFamide-expressing cells. As is the case with AmRFamide, expressing cells are absent from the polyp immediately after settlement, reappearing later on its oral side. AmLWamide expression lags that of AmRFamide in both its disappearance and reappearance. Antibodies to FMRFamide stain cells in a pattern similar to that of the transcripts, but also cells in areas where there is no expression revealed by in situ hybridization, most notably at the aboral end of the planula and in the adult polyp. Adult polyps have numerous staining cells on the tentacles and oral discs, as well as an immunoreactive nerve ring around the mouth. There are scattered staining cells in the coenosarc between polyps and staining cells are abundant in the mesenterial filaments. The above results are discussed in the context of our knowledge of the behavior of coral planulae at the time of their settlement and metamorphosis. Corals are facing multiple environmental threats, and these results both highlight the need for, and bring us a step closer to, a mechanistic understanding of a process that is critical to their survival.
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Antozoários/genética , Regulação da Expressão Gênica no Desenvolvimento , Metamorfose Biológica/genética , Neuropeptídeos/genética , Sequência de Aminoácidos , Animais , Antozoários/embriologia , Antozoários/metabolismo , Ectoderma/embriologia , Ectoderma/metabolismo , Hibridização In Situ , Neuropeptídeos/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismoRESUMO
Malaria continues to be a significant burden, particularly in Africa, which accounts for 95% of malaria deaths worldwide. Despite advances in malaria treatments, malaria eradication is hampered by insecticide and antimalarial drug resistance. Consequently, the need to discover new antimalarial lead compounds remains urgent. To help address this need, we evaluated the antiplasmodial activity of twenty-two amides and thioamides with pyridine cores and their non-pyridine analogues. Twelve of these compounds showed in vitro anti-proliferative activity against the intraerythrocytic stage of Plasmodium falciparum, the most virulent species of Plasmodium infecting humans. Thiopicolinamide 13i was found to possess submicromolar activity (IC50 = 142 nM) and was >88-fold less active against a human cell line. The compound was equally effective against chloroquine-sensitive and -resistant parasites and did not inhibit ß-hematin formation, pH regulation or PfATP4. Compound 13i may therefore possess a novel mechanism of action.
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Antimaláricos , Plasmodium falciparum , Piridinas , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/farmacologia , Antimaláricos/química , Humanos , Piridinas/farmacologia , Piridinas/química , Amidas/farmacologia , Linhagem Celular , Concentração Inibidora 50 , Resistência a Medicamentos , Descoberta de Drogas , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Tioamidas/farmacologia , Tioamidas/química , Testes de Sensibilidade ParasitáriaRESUMO
Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.
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Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Movimento Celular/fisiologia , Proteínas Contráteis , Células Eucarióticas/metabolismo , Pseudópodes/metabolismo , Citoesqueleto de Actina/ultraestrutura , Fatores de Despolimerização de Actina , Proteína 2 Relacionada a Actina , Actinas/antagonistas & inibidores , Actinas/deficiência , Actinas/genética , Animais , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Destrina , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células Eucarióticas/citologia , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Profilinas , Pseudópodes/ultraestrutura , Interferência de RNA , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Members of the Rho/Rac/Cdc42 superfamily of GTPases and their upstream activators, guanine nucleotide exchange factors (GEFs) , have emerged as key regulators of actin and microtubule dynamics. In their GTP bound form, these proteins interact with downstream effector molecules that alter actin and microtubule behavior. During Drosophila embryogenesis, a Galpha subunit (Concertina) and a Rho-type guanine nucleotide exchange factor (DRhoGEF2) have been implicated in the dramatic epithelial-cell shape changes that occur during gastrulation and morphogenesis . Using Drosophila S2 cells as a model system, we show that DRhoGEF2 induces contractile cell shape changes by stimulating myosin II via the Rho1 pathway. Unexpectedly, we found that DRhoGEF2 travels to the cell cortex on the tips of growing microtubules by interaction with the microtubule plus-end tracking protein EB1. The upstream activator Concertina, in its GTP but not GDP bound form, dissociates DRhoGEF2 from microtubule tips and also causes cellular contraction. We propose that DRhoGEF2 uses microtubule dynamics to search for cortical subdomains of receptor-mediated Galpha activation, which in turn causes localized actomyosin contraction associated with morphogenetic movements during development.
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Forma Celular/fisiologia , Proteínas de Drosophila/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Microtúbulos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Primers do DNA , Drosophila/embriologia , Epitélio/embriologia , Epitélio/metabolismo , Proteínas de Fluorescência Verde , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Miosina Tipo II/metabolismo , Plasmídeos/genética , Interferência de RNA , Transdução de Sinais/fisiologiaRESUMO
Three genome-wide RNA interference screens were performed in Drosophila S2 cells to dissect the contribution of host processes to Listeria monocytogenes entry, vacuolar escape, and intracellular growth. Among the 116 genes identified, several host pathways previously unrecognized as playing a role in listerial pathogenesis were identified: knockdowns affecting vacuolar trafficking to and from the multivesicular body bypassed the requirement for the essential pore-forming toxin listeriolysin O in mediating escape from phagocytic vacuoles and knockdowns affecting either subunit of serine palmitoyltransferase, a key enzyme in ceramide and sphingolipid biosynthesis, enhanced the toxicity of listeriolysin O expressed in the host cell cytosol, leading to lack of appropriate toxin activity compartmentalization and host cell death. Genome-wide RNA interference screens using Drosophila S2 cells proved to be a powerful approach to dissect host-pathogen interactions.
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Citoplasma/metabolismo , Listeria monocytogenes , Listeriose/metabolismo , Fagossomos/metabolismo , Interferência de RNA , Animais , Toxinas Bacterianas/metabolismo , Linhagem Celular , Citoplasma/microbiologia , Drosophila , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas , Listeria monocytogenes/metabolismo , Listeriose/genética , Fagossomos/genética , Fagossomos/microbiologiaRESUMO
The receptors for human interleukins 3 and 5 and granulocyte macrophage colony-stimulating factor are composed of ligand-specific alpha-subunits and a common beta-subunit (betac), the major signaling entity. The way in which betac interacts with ligands in the respective activation complexes has remained poorly understood. The recently determined crystal structure of the extracellular domain of betac revealed a possible ligand-binding interface composed of domain 1 of one chain of the betac dimer and the adjacent domain 4 of the symmetry-related chain. We have used site-directed mutagenesis, in conjunction with ligand binding and proliferation studies, to demonstrate the critical requirement of the domain 1 residues, Tyr(15) (A-B loop) and Phe(79) (E-F loop), in high affinity complex formation and receptor activation. The novel ligand-receptor interface formed between domains 1 and 4 represents the first example of a class I cytokine receptor interface to be composed of two noncontiguous fibronectin III domains.