RESUMO
Fragment-based screening is commonly used to identify compounds with relatively weak but efficient localized binding to protein surfaces. We used mass spectrometry to study fragment-sized three-dimensional natural products. We identified seven securinine-related compounds binding to Plasmodium falciparum 2'-deoxyuridine 5'-triphosphate nucleotidohydrolase (PfdUTPase). Securinine bound allosterically to PfdUTPase, enhancing enzyme activity and inhibiting viability of both P. falciparum gametocyte (sexual) and blood (asexual) stage parasites. Our results provide a new insight into mechanisms that may be applicable to transmission-blocking agents.
Assuntos
Antimaláricos/farmacologia , Produtos Biológicos/química , Estágios do Ciclo de Vida/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Alcaloides/química , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Antimaláricos/química , Antimaláricos/isolamento & purificação , Azepinas/química , Azepinas/isolamento & purificação , Azepinas/farmacologia , Nucleotídeos de Desoxiuracil/antagonistas & inibidores , Nucleotídeos de Desoxiuracil/química , Nucleotídeos de Desoxiuracil/metabolismo , Relação Dose-Resposta a Droga , Compostos Heterocíclicos de Anel em Ponte/química , Compostos Heterocíclicos de Anel em Ponte/isolamento & purificação , Compostos Heterocíclicos de Anel em Ponte/farmacologia , Cinética , Lactonas/química , Lactonas/isolamento & purificação , Lactonas/farmacologia , Estágios do Ciclo de Vida/fisiologia , Piperidinas/química , Piperidinas/isolamento & purificação , Piperidinas/farmacologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
Metallothionein-3 (MT-3), renamed as growth inhibitory factor (GIF), is a brain specific member of the metallothionein family. Human dUTPase is a recently found protein in brain that can interact with hMT-3. They have the growth inhibitory activity on neuron cell by interaction. To study the affection of hMT-3 to dUTPase's eliminating the cellular toxicity caused by dUTP, the pSVHA-dUTPase and pFLag-hMT-3 genes have been transfected into HEK293 cells. In addition, the dUTPase and hMT-3 proteins were expressed in BL21 to study the role of hMT-3 on the hydrolyzation of dUTP by dUTPase. The results demonstrate that the cells co-transfected with dUTPase and hMT-3 genes have more strong resistibility to dUTP than the cells transfected only with dUTPase gene. And that the hMT-3 protein can accelerate the hydrolyzation of dUTP by dUTPase. All these indicate that hMT-3 can cooperate with dUTPase to protect better the 293 cells from dUTP. This research offered the theoretic elements for the application of hMT-3 and dUTPase in chemic cure.