RESUMEN
Malaria causes worldwide morbidity and mortality, and while chemotherapy remains an excellent means of malaria control, drug-resistant parasites necessitate the discovery of new antimalarials. Peptidases are a promising class of drug targets and perform several important roles during the Plasmodium falciparum erythrocytic life cycle. Herein, we report a multidisciplinary effort combining activity-based protein profiling, biochemical, and peptidomic approaches to functionally analyze two genetically essential P. falciparum metallo-aminopeptidases (MAPs), PfA-M1 and Pf-LAP. Through the synthesis of a suite of activity-based probes (ABPs) based on the general MAP inhibitor scaffold, bestatin, we generated specific ABPs for these two enzymes. Specific inhibition of PfA-M1 caused swelling of the parasite digestive vacuole and prevented proteolysis of hemoglobin (Hb)-derived oligopeptides, likely starving the parasite resulting in death. In contrast, inhibition of Pf-LAP was lethal to parasites early in the life cycle, prior to the onset of Hb degradation suggesting that Pf-LAP has an essential role outside of Hb digestion.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Leucina/análogos & derivados , Malaria/parasitología , Técnicas de Sonda Molecular , Sondas Moleculares/metabolismo , Familia de Multigenes , Secuencia de Aminoácidos , Aminopeptidasas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hemoglobinas/metabolismo , Leucina/química , Leucina/farmacología , Leucil Aminopeptidasa/antagonistas & inhibidores , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Péptidos/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Análisis por Matrices de Proteínas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacosRESUMEN
A novel set of activity-based probes (ABPs) for functionally profiling metallo-aminopeptidases was synthesized based on the bestatin inhibitor scaffold, the first synthesis of bestatin analogues using solid-phase techniques. These ABPs were shown to label metallo-aminopeptidases, using both a biotin and a fluorophore reporter, in an activity-dependent manner. This probe class was also shown to be amenable to 'click' chemistry labeling for possible use in live cells. Finally, we demonstrate that the ABPs are able to label an aminopeptidase in a complex proteome. Thus, these bestatin-based probes should have wide utility to functionally profile aminopeptidases in many biological systems.
