RESUMO
The malaria parasite Plasmodium falciparum establishes in the host erythrocyte plasma membrane new permeability pathways that mediate nutrient uptake into the infected cell. These pathways simultaneously allow Na(+) influx, causing [Na(+)] in the infected erythrocyte cytosol to increase to high levels. The intraerythrocytic parasite itself maintains a low cytosolic [Na(+)] via unknown mechanisms. Here we present evidence that the intraerythrocytic parasite actively extrudes Na(+) against an inward gradient via PfATP4, a parasite plasma membrane protein with sequence similarities to Na(+)-ATPases of lower eukaryotes. Mutations in PfATP4 confer resistance to a potent class of antimalarials, the spiroindolones. Consistent with this, the spiroindolones cause a profound disruption in parasite Na(+) homeostasis, which is attenuated in parasites bearing resistance-conferring mutations in PfATP4. The mutant parasites also show some impairment of Na(+) regulation. Taken together, our results are consistent with PfATP4 being a Na(+) efflux ATPase and a target of the spiroindolones.
Assuntos
Adenosina Trifosfatases/metabolismo , Antimaláricos/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Plasmodium falciparum/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , Resistência a Medicamentos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Homeostase , Humanos , Indóis/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Ouabaína/farmacologia , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Compostos de Espiro/farmacologia , Trofozoítos/efeitos dos fármacos , Trofozoítos/metabolismoRESUMO
Early secretory and endoplasmic reticulum (ER)-localized proteins that are terminally misfolded or misassembled are degraded by a ubiquitin- and proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan pathogens, including the causative agents of malaria, toxoplasmosis, trypanosomiasis, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells, and, because of this, we observe that the malaria parasite Plasmodium falciparum is highly sensitive to the inhibition of components of this protein quality control system. Inhibitors that specifically target a putative protease component of ERAD, signal peptide peptidase (SPP), have high selectivity and potency for P. falciparum. By using a variety of methodologies, we validate that SPP inhibitors target P. falciparum SPP in parasites, disrupt the protein's ability to facilitate degradation of unstable proteins, and inhibit its proteolytic activity. These compounds also show low nanomolar activity against liver-stage malaria parasites and are also equipotent against a panel of pathogenic protozoan parasites. Collectively, these data suggest ER quality control as a vulnerability of protozoan parasites, and that SPP inhibition may represent a suitable transmission blocking antimalarial strategy and potential pan-protozoan drug target.
Assuntos
Antiparasitários/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Desenho de Fármacos , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Animais , Antiparasitários/química , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Sequência de Bases , Biologia Computacional , Resistência a Medicamentos/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Hep G2 , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/parasitologia , Dados de Sequência Molecular , Parasitos/efeitos dos fármacos , Parasitos/enzimologia , Parasitos/crescimento & desenvolvimento , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Inibidores de Proteases/química , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Proteoma/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologia , Toxoplasma/crescimento & desenvolvimento , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Recent reports of increased tolerance to artemisinin derivatives--the most recently adopted class of antimalarials--have prompted a need for new treatments. The spirotetrahydro-beta-carbolines, or spiroindolones, are potent drugs that kill the blood stages of Plasmodium falciparum and Plasmodium vivax clinical isolates at low nanomolar concentration. Spiroindolones rapidly inhibit protein synthesis in P. falciparum, an effect that is ablated in parasites bearing nonsynonymous mutations in the gene encoding the P-type cation-transporter ATPase4 (PfATP4). The optimized spiroindolone NITD609 shows pharmacokinetic properties compatible with once-daily oral dosing and has single-dose efficacy in a rodent malaria model.