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1.
Microbiol Res ; 263: 127134, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35905580

RESUMO

Streptococcus pneumoniae (Spn) kills Staphylococcus aureus (Sau) through a contact-dependent mechanism that is catalyzed by cations, including iron, to convert hydrogen peroxide (H2O2) to highly toxic hydroxyl radicals (•OH). There are two well-characterized ABC transporters that contribute to the pool of iron in Spn, named Pia and Piu. Some Spn strains have acquired genes mef(E)/mel encoding another ABC trasporter (Mega) that produces an inducible efflux pump for resistance to macrolides. In macrolide-resistant Spn clinical isolates the insertion of Mega class 1. IV and 2. IVc deleted the locus piaABCD and these strains were attenuated for intoxicating Sau. The goal of this study was to investigate if the disruption of iron acquisition, or the antimicrobial-resistance activity of Mega, contributed to inhibiting the killing mechanism. Neither depletion of iron with 2,2'-dipyridyl-d8 (DP) nor incubating with a double knockout mutant SpnΔpiaAΔpiuA, inhibited killing of Sau. Clinical Spn strains carrying Mega1. IV or Mega2. IVc showed a significant delay for killing Sau. An ex vivo recombination system was used to transfer Mega1. IV or Mega2. IVc to reference Spn strains, which was confirmed by whole genome sequencing, and recombinants TIGR4Mega2. IVc, D39Mega2. IVc, and D39Mega1. IV were delayed for killing Sau. We then compared Sau killing of selected Mega-carrying Spn strains when incubated with sub-inhibitory erythromycin (Mega-induced) or sub-inhibitory cefuroxime. Remarkably, killing of Sau was completely inhibited under the Mega-induced condition whereas incubation with cefuroxime did not interfere with killing. Both mef(E) and mel were upregulated > 400-fold, and spxB (encoding an enzyme responsible for production of most H2O2) was upregulated 14.2-fold, whereas transcription of the autolysin (lytA) gene was downregulated when incubated with erythromycin. We demonstrated that erythromycin induction of Mega inhibits the •OH-mediated intoxication of Sau and that the inhibition occurred at the post-translational level suggesting that an imbalance of ions in the membrane inhibits these reactions.


Assuntos
Infecções Estafilocócicas , Streptococcus pneumoniae , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cefuroxima , Farmacorresistência Bacteriana/genética , Eritromicina/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Ferro , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Staphylococcus aureus/genética , Streptococcus pneumoniae/genética
2.
ACS Infect Dis ; 5(10): 1738-1753, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31373203

RESUMO

Emerging resistance to current antimalarial medicines underscores the importance of identifying new drug targets and novel compounds. Malaria parasites are purine auxotrophic and import purines via the Plasmodium falciparum equilibrative nucleoside transporter type 1 (PfENT1). We previously showed that PfENT1 inhibitors block parasite proliferation in culture. Our goal was to identify additional, possibly more optimal chemical starting points for a drug discovery campaign. We performed a high throughput screen (HTS) of GlaxoSmithKline's 1.8 million compound library with a yeast-based assay to identify PfENT1 inhibitors. We used a parallel progression strategy for hit validation and expansion, with an emphasis on chemical properties in addition to potency. In one arm, the most active hits were tested for human cell toxicity; 201 had minimal toxicity. The second arm, hit expansion, used a scaffold-based substructure search with the HTS hits as templates to identify over 2000 compounds; 123 compounds had activity. Of these 324 compounds, 175 compounds inhibited proliferation of P. falciparum parasite strain 3D7 with IC50 values between 0.8 and ∼180 µM. One hundred forty-two compounds inhibited PfENT1 knockout (pfent1Δ) parasite growth, indicating they also hit secondary targets. Thirty-two hits inhibited growth of 3D7 but not pfent1Δ parasites. Thus, PfENT1 inhibition was sufficient to block parasite proliferation. Therefore, PfENT1 may be a viable target for antimalarial drug development. Six compounds with novel chemical scaffolds were extensively characterized in yeast-, parasite-, and human-erythrocyte-based assays. The inhibitors showed similar potencies against drug sensitive and resistant P. falciparum strains. They represent attractive starting points for development of novel antimalarial drugs.


Assuntos
Antimaláricos/farmacologia , Transporte Biológico/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Plasmodium falciparum/efeitos dos fármacos , Purinas/metabolismo , Antimaláricos/química , Eritrócitos/efeitos dos fármacos , Técnicas de Inativação de Genes , Células Hep G2/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Malária/parasitologia , Malária Falciparum/parasitologia , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/efeitos dos fármacos , Proteínas de Transporte de Nucleobases, Nucleosídeos, Nucleotídeos e Ácidos Nucleicos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/genética , Transcriptoma , Leveduras/efeitos dos fármacos
3.
Int J Parasitol Drugs Drug Resist ; 6(1): 1-11, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26862473

RESUMO

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/farmacologia
4.
ACS Chem Biol ; 10(3): 775-83, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25602169

RESUMO

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 64 560 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/farmacologia
5.
Ann N Y Acad Sci ; 1342: 19-28, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25424653

RESUMO

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 & inibidores
6.
Biochem J ; 446(2): 179-90, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22670848

RESUMO

Malaria, caused by Plasmodia parasites, affects hundreds of millions of people. As purine auxotrophs, Plasmodia use transporters to import host purines for subsequent metabolism by the purine salvage pathway. Thus purine transporters are attractive drug targets. All sequenced Plasmodia genomes encode four ENTs (equilibrative nucleoside transporters). During the pathogenic intraerythrocytic stages, ENT1 is a major route of purine nucleoside/nucleobase transport. Another plasma membrane purine transporter exists because Plasmodium falciparum ENT1-knockout parasites survive at supraphysiological purine concentrations. The other three ENTs have not been characterized functionally. Codon-optimized Pf- (P. falciparum) and Pv- (Plasmodium vivax) ENT4 were expressed in Xenopus laevis oocytes and substrate transport was determined with radiolabelled substrates. ENT4 transported adenine and 2'-deoxyadenosine at the highest rate, with millimolar-range apparent affinity. ENT4-expressing oocytes did not accumulate hypoxanthine, a key purine salvage pathway substrate, or AMP. Micromolar concentrations of the plant hormone cytokinin compounds inhibited both PfENT4 and PvENT4. In contrast with PfENT1, ENT4 interacted with the immucillin compounds in the millimolar range and was inhibited by 10 µM dipyridamole. Thus ENT4 is a purine transporter with unique substrate and inhibitor specificity. Its role in parasite physiology remains uncertain, but is likely to be significant because of the strong conservation of ENT4 homologues in Plasmodia genomes.


Assuntos
Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium vivax/metabolismo , Proteínas de Protozoários/metabolismo , Adenina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Citocininas/farmacologia , Desoxiadenosinas/metabolismo , Dipiridamol/farmacologia , Proteínas de Transporte de Nucleosídeo Equilibrativas/antagonistas & inibidores , Proteínas de Transporte de Nucleosídeo Equilibrativas/química , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Cinética , Moduladores de Transporte de Membrana/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Xenopus laevis
7.
J Biol Chem ; 285(22): 17001-10, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20335165

RESUMO

Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate K(m). We investigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methanethiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an alpha-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60 degrees arc suggesting that TM11 is largely alpha helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure.


Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Plasmodium falciparum/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Cisteína/química , Glicina/química , Hipoxantina/química , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Polimorfismo Genético , Estrutura Terciária de Proteína , Purinas/química , Homologia de Sequência de Aminoácidos
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