RESUMO
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.
Assuntos
Adenina/análogos & derivados , Antimaláricos/farmacologia , Cicloexilaminas/farmacologia , Regulação Enzimológica da Expressão Gênica/genética , Malária/parasitologia , Plasmodium falciparum/enzimologia , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas de Protozoários/antagonistas & inibidores , Adenina/química , Adenina/farmacologia , Adenina/uso terapêutico , Animais , Antimaláricos/química , Antimaláricos/uso terapêutico , Células CHO , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Cicloexilaminas/química , Cicloexilaminas/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária/tratamento farmacológico , Malária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Movimento/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Testes de Sensibilidade Parasitária , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas Quinases/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
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.
Assuntos
Cisteína/metabolismo , Dissulfetos/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Noxas/farmacologia , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/metabolismo , Acroleína/análogos & derivados , Acroleína/química , Acroleína/metabolismo , Acroleína/farmacologia , Animais , Cisteína/química , Dissulfetos/química , Ditiotreitol/farmacologia , Condutividade Elétrica , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/química , Metanossulfonato de Etila/metabolismo , Metanossulfonato de Etila/farmacologia , Humanos , Camundongos , Mostardeira/química , Mostardeira/metabolismo , Noxas/química , Noxas/metabolismo , Dor/induzido quimicamente , Dor/fisiopatologia , Óleos de Plantas/química , Óleos de Plantas/metabolismo , Óleos de Plantas/farmacologia , Canais de Potencial de Receptor Transitório/químicaRESUMO
[reaction: see text] (S)-S-(2-Cyclohexenyl) N,N-diisopropylmonothiocarbamate [(-)-(S)-8] was deprotonated by sec-butyllithium/TMEDA to form a configurationally stable lithium compound (S)-9, which is the first example of a new class of alpha-thio-substituted organolithium compounds with improved properties. It is regioselectively alkylated by alkyl halides with complete stereoinversion to form the monothiocarbamates (+)-10 which afford highly enantioenriched tertiary 2-cyclohexene-1-thiols (+)-6 on reductive cleavage.