RESUMEN
Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plants, some algae and protozoa. In Plasmodium falciparum, calcium-dependent protein kinase 1 (PfCDPK1) is expressed during schizogony in the erythrocytic stage as well as in the sporozoite stage. It is coexpressed with genes that encode the parasite motor complex, a cellular component required for parasite invasion of host cells, parasite motility and potentially cytokinesis. A targeted gene-disruption approach demonstrated that pfcdpk1 seems to be essential for parasite viability. An in vitro biochemical screen using recombinant PfCDPK1 against a library of 20,000 compounds resulted in the identification of a series of structurally related 2,6,9-trisubstituted purines. Compound treatment caused sudden developmental arrest at the late schizont stage in P. falciparum and a large reduction in intracellular parasites in Toxoplasma gondii, which suggests a possible role for PfCDPK1 in regulation of parasite motility during egress and invasion.
Asunto(s)
Adenina/análogos & derivados , Antimaláricos/farmacología , Ciclohexilaminas/farmacología , Regulación Enzimológica de la Expresión Génica/genética , Malaria/parasitología , Plasmodium falciparum/enzimología , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/genética , Proteínas Protozoarias/antagonistas & inhibidores , Adenina/química , Adenina/farmacología , Adenina/uso terapéutico , Animales , Antimaláricos/química , Antimaláricos/uso terapéutico , Células CHO , Línea Celular , Proliferación Celular/efectos de los fármacos , Cricetinae , Cricetulus , Ciclohexilaminas/química , Ciclohexilaminas/uso terapéutico , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Estadios del Ciclo de Vida/efectos de los fármacos , Malaria/tratamiento farmacológico , Malaria/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Estructura Molecular , Peso Molecular , Movimiento/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Quinasas/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Bibliotecas de Moléculas Pequeñas , Estereoisomerismo , Relación Estructura-Actividad , Distribución TisularRESUMEN
The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.