RESUMEN
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.
Asunto(s)
Antimaláricos/farmacología , Terapia Molecular Dirigida , Plasmodium falciparum/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antimaláricos/química , Antimaláricos/aislamiento & purificación , Antimaláricos/uso terapéutico , Gametogénesis/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Ratones , Ratones Endogámicos BALB C , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Inhibidores de Proteínas Quinasas/aislamiento & purificación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas Protozoarias/genética , Empalme del ARN/genética , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
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.