RESUMO
Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amoebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 = 0.14 ± 0.04 µM) that was toxic to trophozoites (EC50 = 0.21 ± 0.02 µM) while the reported CC50 was >300 µM. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the end of the one-week observation, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. However, intranasal HEX delivery was not curative as brains of six of the eight survivors were positive for amoebae. These findings suggest that HEX requires further evaluation to develop as a lead for treatment of PAM.
Assuntos
Infecções Protozoárias do Sistema Nervoso Central , Naegleria fowleri , Fosfopiruvato Hidratase , Animais , Naegleria fowleri/efeitos dos fármacos , Infecções Protozoárias do Sistema Nervoso Central/tratamento farmacológico , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Fosfopiruvato Hidratase/metabolismo , Fosfopiruvato Hidratase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Camundongos , Ratos , Humanos , Simulação de Acoplamento MolecularRESUMO
Heme, an iron-containing organic ring, is essential for virtually all living organisms by serving as a prosthetic group in proteins that function in diverse cellular activities ranging from diatomic gas transport and sensing, to mitochondrial respiration, to detoxification. Cellular heme levels in microbial pathogens can be a composite of endogenous de novo synthesis or exogenous uptake of heme or heme synthesis intermediates. Intracellular pathogenic microbes switch routes for heme supply when heme availability fluctuates in their replicative environment throughout infection. Here, we show that Toxoplasma gondii, an obligate intracellular human pathogen, encodes a functional heme biosynthesis pathway. A chloroplast-derived organelle, termed apicoplast, is involved in heme production. Genetic and chemical manipulation revealed that de novo heme production is essential for T. gondii intracellular growth and pathogenesis. Surprisingly, the herbicide oxadiazon significantly impaired Toxoplasma growth, consistent with phylogenetic analyses that show T. gondii protoporphyrinogen oxidase is more closely related to plants than mammals. This inhibition can be enhanced by 15- to 25-fold with two oxadiazon derivatives, lending therapeutic proof that Toxoplasma heme biosynthesis is a druggable target. As T. gondii has been used to model other apicomplexan parasites, our study underscores the utility of targeting heme biosynthesis in other pathogenic apicomplexans, such as Plasmodium spp., Cystoisospora, Eimeria, Neospora, and Sarcocystis.
Assuntos
Heme/genética , Filogenia , Protoporfirinogênio Oxidase/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasmose/genética , Heme/biossíntese , Humanos , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Plantas/genética , Protoporfirinogênio Oxidase/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Toxoplasmose/enzimologiaRESUMO
Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate that these agents are potent inhibitors of N. fowleri ENO ( Nf ENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC 50 value of 0.14 ± 0.04 µM) that was toxic to trophozoites (EC 50 value of 0.21 ± 0.02 µM) while the reported CC 50 was >300 µM. Molecular docking simulation revealed that HEX binds strongly to the active site of Nf ENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of Nf ENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the conclusion of the experiment, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. Brains of six of the eight survivors were positive for amoebae, suggesting the agent at the tested dose suppressed, but did not eliminate, infection. These findings suggest that HEX is a promising lead for the treatment of PAM.
RESUMO
Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, serving as the lone source of ATP production for the bloodstream form (BSF) parasite in the glucose-rich environment of the host blood. Recently, phosphonate inhibitors of human enolase (ENO), the enzyme responsible for the interconversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) in glycolysis or PEP to 2-PG in gluconeogenesis, have been developed for the treatment of glioblastoma multiforme (GBM). Here, we have tested these agents against T. brucei ENO (TbENO) and found the compounds to be potent enzyme inhibitors and trypanocides. For example, (1-hydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (deoxy-SF2312) was a potent enzyme inhibitor (IC50 value of 0.60 ± 0.23 µM), while a six-membered ring-bearing phosphonate, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX), was less potent (IC50 value of 2.1 ± 1.1 µM). An analog with a larger seven-membered ring, (1-hydroxy-2-oxoazepan-3-yl) phosphonic acid (HEPTA), was not active. Molecular docking simulations revealed that deoxy-SF2312 and HEX had binding affinities of -6.8 and -7.5 kcal/mol, respectively, while the larger HEPTA did not bind as well, with a binding of affinity of -4.8 kcal/mol. None of these compounds were toxic to BSF parasites; however, modification of enzyme-active phosphonates through the addition of pivaloyloxymethyl (POM) groups improved activity against T. brucei, with POM-modified (1,5-dihydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (POMSF) and POMHEX having EC50 values of 0.45 ± 0.10 and 0.61 ± 0.08 µM, respectively. These findings suggest that HEX is a promising lead against T. brucei and that further development of prodrug HEX analogs is warranted.
RESUMO
Infections of Toxoplasma gondii can cause severe and sometimes fatal diseases in immunocompromised individuals. The de novo heme biosynthesis pathway is required for intracellular growth and pathogenesis, making it an appealing therapeutic target. We synthesized a small library of derivatives of the herbicide oxadiazon, a known inhibitor of the penultimate reaction within the heme biosynthesis pathway in plants, catalyzed by protoporphyrinogen oxidase (PPO). Seven of the 18 analogs exhibit potent intracellular growth inhibition of wild-type T. gondii (IC50 = 1 to 2.4 µM). An assay of the compounds against Toxoplasma PPO knockout and complementation strains confirmed the mode of action to be due to the potent inhibition of PPO. The most potent compounds have no detectable cytotoxicity against human foreskin fibroblast cells up to 100 µM. This study suggests that oxadiazon derivatives may represent a new molecular scaffold for the effective treatment of T. gondii infections.