RESUMO
Plasmodium falciparum causes the most severe form of malaria and causes approximately 500â¯000 deaths per year. P. falciparum parasites resistant to current antimalarial treatments are spreading. Therefore, it is imperative to develop new antimalarial drugs. Malaria parasites are purine auxotrophic. They rely on purine import from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Recently, inhibitors of the P. falciparum ENT1 (PfENT1) that inhibit proliferation of malaria parasites in culture have been identified as promising starting points for antimalarial drug development. Genome sequencing of P. falciparum field isolates has identified nonsynonymous single nucleotide polymorphisms (SNPs) in the gene encoding PfENT1. Here we evaluate the impact of these PfENT1 SNPs on purine substrate affinity and inhibitor efficacy. We expressed each PfENT1-SNP in Saccharomyces cerevisiae. Using PfENT1-SNP-expressing yeast, we characterized the PfENT1 purine substrate affinity using radiolabeled substrate uptake inhibition experiments. Four of the 13 SNPs altered affinity for one or more purines by up to 7-fold. Three of the SNPs reduced the potency of a subset of the inhibitors by up to 7-fold. One SNP, Q284E, reduced the potency of all six inhibitor chemotypes. We tested drug efficacy in available parasite strains containing PfENT1 SNPs. While PfENT1-SNP-expressing yeast had decreased sensitivity to PfENT1 inhibitors, parasite strains containing SNPs showed similar or more potent inhibition of proliferation with all PfENT1 inhibitors. Thus, parasite strains bearing PfENT1 SNPs are not resistant to these PfENT1 inhibitors. This supports PfENT1 as a promising target for further development of novel antimalarial drugs.
Assuntos
Antimaláricos/farmacologia , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/antagonistas & inibidores , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Purinas/metabolismo , Transporte Biológico , Desenvolvimento de Medicamentos , Resistência a Medicamentos , Genoma de Protozoário , Concentração Inibidora 50 , Polimorfismo de Nucleotídeo Único , Saccharomyces cerevisiae/genéticaRESUMO
Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophic. They import purines via an equilibrative nucleoside transporter (ENT). In P. falciparum, the most virulent species, the equilibrative nucleoside transporter 1 (PfENT1) represents the primary purine uptake pathway. This transporter is a potential target for the development of antimalarial drugs. In the absence of a high-resolution structure for either PfENT1 or a homologous ENT, we used the substituted cysteine accessibility method (SCAM) to investigate the membrane-spanning domain structure of PfENT1 to identify potential inhibitor-binding sites. We previously used SCAM to identify water-accessible residues that line the permeation pathway in transmembrane segment 11 (TM11). TM2 and TM10 lie adjacent to TM11 in an ab initio model of a homologous Leishmania donovani nucleoside transporter. To identify TM2 and TM10 residues in PfENT1 that are at least transiently on the water-accessible transporter surface, we assayed the reactivity of single cysteine-substitution mutants with three methanethiosulfonate (MTS) derivatives. Cysteines substituted for 12 of 14 TM2 segment residues reacted with MTS-ethyl-ammonium-biotin (MTSEA-biotin). At eight positions, MTSEA-biotin inhibited transport, and at four positions substrate transport was potentiated. On an α helical wheel projection of TM2, the four positions where potentiation occurred were located in a cluster on one side of the helix. In contrast, although MTSEA-biotin inhibited 9 of 10 TM10 cysteine-substituted mutants, the reactive residues did not form a pattern consistent with either an α helix or ß sheet. These results may help identify the binding site(s) of PfENT1 inhibitors.
Assuntos
Substituição de Aminoácidos/genética , Permeabilidade da Membrana Celular/genética , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/antagonistas & inibidores , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/química , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Antimaláricos , Sítios de Ligação , Transporte Biológico , Cisteína , Desenho de Fármacos , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/genética , Plasmodium falciparum , Proteínas de Protozoários/genética , Purinas/metabolismo , Solubilidade , Água/químicaRESUMO
Infection with Plasmodium falciparum and vivax cause most cases of malaria. Emerging resistance to current antimalarial medications makes new drug development imperative. Ideally a new antimalarial drug should treat both falciparum and vivax malaria. Because malaria parasites are purine auxotrophic, they rely on purines imported from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Thus, the purine import transporters represent a potential target for antimalarial drug development. For falciparum parasites the primary purine transporter is the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Recently we identified potent PfENT1 inhibitors with nanomolar IC50 values using a robust, yeast-based high throughput screening assay. In the current work we characterized the Plasmodium vivax ENT1 (PvENT1) homologue and its sensitivity to the PfENT1 inhibitors. We expressed a yeast codon-optimized PvENT1 gene in Saccharomyces cerevisiae. PvENT1-expressing yeast imported both purines ([(3)H]adenosine) and pyrimidines ([(3)H]uridine), whereas wild type (fui1Δ) yeast did not. Based on radiolabel substrate uptake inhibition experiments, inosine had the lowest IC50 (3.8 µM), compared to guanosine (14.9 µM) and adenosine (142 µM). For pyrimidines, thymidine had an IC50 of 183 µM (vs. cytidine and uridine; mM range). IC50 values were higher for nucleobases compared to the corresponding nucleosides; hypoxanthine had a 25-fold higher IC50 than inosine. The archetypal human ENT1 inhibitor 4-nitrobenzylthioinosine (NBMPR) had no effect on PvENT1, whereas dipyridamole inhibited PvENT1, albeit with a 40 µM IC50, a 1000-fold less sensitive than human ENT1 (hENT1). The PfENT1 inhibitors blocked transport activity of PvENT1 and the five known naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) with similar IC50 values. Thus, the PfENT1 inhibitors also target PvENT1. This implies that development of novel antimalarial drugs that target both falciparum and vivax ENT1 may be feasible.
Assuntos
Antimaláricos/farmacologia , Descoberta de Drogas , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/antagonistas & inibidores , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium vivax/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Adenosina/farmacologia , Dipiridamol/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/genética , Guanosina/farmacologia , Humanos , Concentração Inibidora 50 , Inosina/farmacologia , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Malária Vivax/prevenção & controle , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/genética , Plasmodium falciparum/metabolismo , Plasmodium vivax/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Purinas/metabolismo , Purinas/farmacologia , Pirimidinas/metabolismo , Saccharomyces cerevisiae/genética , Uridina/farmacologiaRESUMO
Equilibrative transporters are potential drug targets; however, most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64â¯560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2-2 µM). These nine compounds completely blocked [(3)H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5-50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5-50 µM). Wild-type (WT) parasite IC50 values were up to 4-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that, in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development.
Assuntos
Antimaláricos/farmacologia , Ensaios de Triagem em Larga Escala , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Trofozoítos/efeitos dos fármacos , Adenosina/metabolismo , Antimaláricos/química , Cultura Axênica , Transporte Biológico/efeitos dos fármacos , Deleção de Genes , Expressão Gênica , Teste de Complementação Genética , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/genética , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/metabolismo , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismo , Uridina/análogos & derivados , Uridina/farmacologiaRESUMO
Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophs. In all life cycle stages, they require purines for RNA and DNA synthesis and other cellular metabolic processes. Purines are imported from the host erythrocyte by equilibrative nucleoside transporters (ENTs). They are processed via purine salvage pathway enzymes to form the required purine nucleotides. The Plasmodium falciparum genome encodes four putative ENTs (PfENT1-4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary purine transporter supplying the purine salvage pathway. Protein mass spectrometry shows that PfENT1 is expressed in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine concentrations found in human blood (<10 µM). Thus, PfENT1 is a potential target for novel antimalarial drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target.
Assuntos
Antimaláricos/metabolismo , Sistemas de Liberação de Medicamentos/tendências , Descoberta de Drogas/tendências , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Purinas/metabolismo , Animais , Antimaláricos/administração & dosagem , Humanos , Malária/tratamento farmacológico , Malária/metabolismo , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/antagonistas & inibidores , Parasitos/efeitos dos fármacos , Parasitos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidoresRESUMO
Nucleoside transporters play important physiological roles by regulating intra- and extra-cellular concentrations of purine and pyrimidine (deoxy)nucleosides. This review describes the biological function and activity of the two major families of membrane nucleoside transporters that exist in mammalian cells. These include equilibrative nucleoside transporters that transport nucleosides in a gradient-dependent fashion and concentrative nucleoside transporters that import nucleosides against a gradient by coupling movement with sodium transport. Particular emphasis is placed on describing the roles of nucleoside transport in normal physiological processes, including inflammation, cardiovascular function and nutrient transport across the blood-brain barrier. In addition, the role of nucleoside transport in pathological conditions such as cardiovascular disease and cancer are discussed. The potential therapeutic applications of manipulating nucleoside transport activities are discussed, focusing on nucleoside analogs as anti-neoplastic agents. Finally, we discuss future directions for the development of novel chemical entities to measure nucleoside transport activity at the cellular and organismal level.
Assuntos
Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/metabolismo , Nucleosídeos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Doenças Cardiovasculares/tratamento farmacológico , Humanos , Neoplasias/tratamento farmacológico , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/antagonistas & inibidores , Nucleosídeos/farmacologia , Nucleosídeos/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the K(m) and/or K(i) for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the K(i) for PfENT1 transport of hypoxanthine and adenosine is in the 300-700microM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport K(m). PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [(3)H]hypoxanthine or [(3)H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.