RESUMO
The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the Plasmodium falciparum protein kinase PfCLK3, which we used in combination with chemogenetics to validate PfCLK3 as a drug target acting at multiple parasite life stages. Consistent with a role for PfCLK3 in RNA splicing, inhibition resulted in the down-regulation of more than 400 essential parasite genes. Inhibition of PfCLK3 mediated rapid killing of asexual liver- and blood-stage P. falciparum and blockade of gametocyte development, thereby preventing transmission, and also showed parasiticidal activity against P. berghei and P. knowlesi Hence, our data establish PfCLK3 as a target for drugs, with the potential to offer a cure-to be prophylactic and transmission blocking in malaria.
Assuntos
Antimaláricos/farmacologia , Terapia de Alvo Molecular , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antimaláricos/química , Antimaláricos/isolamento & purificação , Antimaláricos/uso terapêutico , Gametogênese/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Inibidores de Proteínas Quinases/isolamento & purificação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas de Protozoários/genética , Splicing de RNA/genética , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
We have successfully configured a new ultrasensitive fluorescent phosphate assay that detects free phosphate in solution through the formation of the fluorescent product resorufin. The phosphate assay relies on coupling phosphate generation to purine nucleoside phosphorylase, xanthine oxidase, and horseradish peroxidase. The response is excellent in the nanomolar range, being linear between 50 nM and 5 microM phosphate. This method is more sensitive (more than 10-fold) than other reported methods and is amenable to miniaturization. In particular, we have demonstrated the utility of this new method in a format suitable for ultra-high-throughput screening.