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1.
Science ; 365(6456)2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31467193

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/farmacologia
2.
Malar J ; 15(1): 535, 2016 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-27821169

RESUMO

BACKGROUND: Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite. RESULTS: Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2. CONCLUSIONS: Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2.


Assuntos
Inibidores Enzimáticos/metabolismo , Proteínas Mutantes/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Plasmodium falciparum/enzimologia , Purinas/metabolismo , Sulfonamidas/metabolismo , Espectrometria de Massas , Proteínas Mutantes/genética , Quinases Relacionadas a NIMA/genética
3.
Biochim Biophys Acta ; 1854(10 Pt B): 1650-6, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26143498

RESUMO

Until very recently there has been very little information about the phospho-signalling pathways in apicomplexan parasites including the most virulent species of human malaria parasite, Plasmodium falciparum. With the advancement of mass spectrometry-based phosphoproteomics and the development of chemical genetic approaches to target specific parasite protein kinases, the complexity of the essential role played by phosphorylation in maintaining the viability of apicomplexan parasites is now being revealed. This review will describe these recent advances and will discuss how these approaches can be used to validate parasite protein kinases as drug targets and to determine the on- and off-target action of protein kinase inhibitors. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.


Assuntos
Malária Falciparum/genética , Plasmodium falciparum/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/genética , Animais , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Espectrometria de Massas , Fosforilação , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade , Inibidores de Proteínas Quinases/química , Proteínas Quinases/biossíntese , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteômica , Transdução de Sinais/efeitos dos fármacos
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