RESUMO
The lack of disease models adequately resembling human tissue has hindered our understanding of amoebic brain infection. Three-dimensional structured organoids provide a microenvironment similar to human tissue. This study demonstrates the use of cerebral organoids to model a rare brain infection caused by the highly lethal amoeba Balamuthia mandrillaris. Cerebral organoids were generated from human pluripotent stem cells and infected with clinically isolated B. mandrillaris trophozoites. Histological examination showed amoebic invasion and neuron damage following coculture with the trophozoites. The transcript profile suggested an alteration in neuron growth and a proinflammatory response. The release of intracellular proteins specific to neuronal bodies and astrocytes was detected at higher levels postinfection. The amoebicidal effect of the repurposed drug nitroxoline was examined using the human cerebral organoids. Overall, the use of human cerebral organoids was important for understanding the mechanism of amoeba pathogenicity, identify biomarkers for brain injury, and in the testing of a potential amoebicidal drug in a context similar to the human brain.
Assuntos
Amebíase , Balamuthia mandrillaris , Encéfalo , Organoides , Humanos , Organoides/parasitologia , Balamuthia mandrillaris/efeitos dos fármacos , Encéfalo/parasitologia , Encéfalo/patologia , Amebíase/parasitologia , Amebíase/tratamento farmacológico , Trofozoítos/efeitos dos fármacos , Neurônios/parasitologia , Células-Tronco PluripotentesRESUMO
Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a 'brain-eating' disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections.
Assuntos
Balamuthia mandrillaris/genética , Desenho de Fármacos/métodos , Trofozoítos/genética , Acanthamoeba/genética , Amebíase/tratamento farmacológico , Amoeba/genética , Balamuthia mandrillaris/efeitos dos fármacos , Balamuthia mandrillaris/crescimento & desenvolvimento , Sequência de Bases , Encéfalo/patologia , Descoberta de Drogas/métodos , Encefalite/patologia , Expressão Gênica/genética , Naegleria fowleri/genética , Transcriptoma/genética , Trofozoítos/efeitos dos fármacosRESUMO
Diseases caused by pathogenic free-living amoebae include primary amoebic meningoencephalitis (Naegleria fowleri), granulomatous amoebic encephalitis (Acanthamoeba spp.), Acanthamoeba keratitis, and Balamuthia amoebic encephalitis (Balamuthia mandrillaris). Each of these are difficult to treat and have high morbidity and mortality rates due to lack of effective therapeutics. Since repurposing drugs is an ideal strategy for orphan diseases, we conducted a high throughput phenotypic screen of 12,000 compounds from the Calibr ReFRAME library. We discovered a total of 58 potent inhibitors (IC50 <1 µM) against N. fowleri (n = 19), A. castellanii (n = 12), and B. mandrillaris (n = 27) plus an additional 90 micromolar inhibitors. Of these, 113 inhibitors have never been reported to have activity against Naegleria, Acanthamoeba or Balamuthia. Rapid onset of action is important for new anti-amoeba drugs and we identified 19 compounds that inhibit N. fowleri in vitro within 24 hours (halofuginone, NVP-HSP990, fumagillin, bardoxolone, belaronib, and BPH-942, solithromycin, nitracrine, quisinostat, pabinostat, pracinostat, dacinostat, fimepinostat, sanguinarium, radicicol, acriflavine, REP3132, BC-3205 and PF-4287881). These compounds inhibit N. fowleri in vitro faster than any of the drugs currently used for chemotherapy. The results of these studies demonstrate the utility of phenotypic screens for discovery of new drugs for pathogenic free-living amoebae, including Acanthamoeba for the first time. Given that many of the repurposed drugs have known mechanisms of action, these compounds can be used to validate new targets for structure-based drug design.
Assuntos
Amebíase/tratamento farmacológico , Amebicidas/farmacologia , Reposicionamento de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Acanthamoeba/efeitos dos fármacos , Balamuthia mandrillaris/efeitos dos fármacos , Bases de Dados de Produtos Farmacêuticos , Naegleria fowleri/efeitos dos fármacos , Doenças Negligenciadas/tratamento farmacológico , Bibliotecas de Moléculas PequenasRESUMO
Balamuthia mandrillaris and Naegleria fowleri are free-living amoebae that can cause life-threatening infections involving the central nervous system. The high mortality rates of these infections demonstrate an urgent need for novel treatment options against the amoebae. Considering that indole and thiazole compounds possess wide range of antiparasitic properties, novel bisindole and thiazole derivatives were synthesized and evaluated against the amoebae. The antiamoebic properties of four synthetic compounds i.e., two new bisindoles (2-Bromo-4-(di (1H-indol-3-yl)methyl)phenol (denoted as A1) and 2-Bromo-4-(di (1H-indol-3-yl)methyl)-6-methoxyphenol (A2)) and two known thiazole (4-(3-Nitrophenyl)-2-(2-(pyridin-3-ylmethylene)hydrazinyl)thiazole (A3) and 4-(Biphenyl-4-yl)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (A4)) were evaluated against B. mandrillaris and N. fowleri. The ability of silver nanoparticle (AgNPs) conjugation to enrich antiamoebic activities of the compounds was also investigated. The synthetic heterocyclic compounds demonstrated up to 53% and 69% antiamoebic activities against B. mandrillaris and N. fowleri respectively, while resulting in up to 57% and 68% amoebistatic activities, respectively. Antiamoebic activities of the compounds were enhanced by up to 71% and 51% against B. mandrillaris and N. fowleri respectively, after conjugation with AgNPs. These compounds exhibited potential antiamoebic effects against B. mandrillaris and N. fowleri and conjugation of synthetic heterocyclic compounds with AgNPs enhanced their activity against the amoebae.
Assuntos
Amebíase/tratamento farmacológico , Balamuthia mandrillaris/efeitos dos fármacos , Infecções Protozoárias do Sistema Nervoso Central/tratamento farmacológico , Indóis/administração & dosagem , Naegleria fowleri/efeitos dos fármacos , Tiazóis/administração & dosagem , Amebíase/parasitologia , Amebicidas/administração & dosagem , Amebicidas/química , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Células HeLa , Humanos , Indóis/química , Concentração Inibidora 50 , Nanopartículas Metálicas , Tiazóis/químicaRESUMO
Balamuthia mandrillaris is an under-reported, pathogenic free-living amoeba that causes Balamuthia amoebic encephalitis (BAE) and cutaneous skin infections. Although cutaneous infections are not typically lethal, BAE with or without cutaneous involvement is usually fatal. This is due to the lack of drugs that are both efficacious and can cross the blood-brain barrier. We aimed to discover new leads for drug discovery by screening the open-source Medicines for Malaria Venture (MMV) Malaria Box and MMV Pathogen Box, with 800 compounds total. From an initial single point screen at 1 and 10 µM, we identified 54 hits that significantly inhibited the growth of B. mandrillarisin vitro Hits were reconfirmed in quantitative dose-response assays and 23 compounds (42.6%) were confirmed with activity greater than miltefosine, the current standard of care.
Assuntos
Amebicidas/farmacologia , Antimaláricos/farmacologia , Balamuthia mandrillaris/efeitos dos fármacos , Amebíase/parasitologia , Animais , Balamuthia mandrillaris/crescimento & desenvolvimento , Simulação por Computador , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Feminino , Papio , GravidezRESUMO
Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris-a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions-have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.IMPORTANCEBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections.
Assuntos
Amebicidas/farmacologia , Balamuthia mandrillaris/efeitos dos fármacos , Nitroquinolinas/farmacologia , Amebíase/tratamento farmacológico , Amebíase/parasitologia , Amebíase/patologia , Balamuthia mandrillaris/crescimento & desenvolvimento , Encéfalo/parasitologia , Encéfalo/patologia , Linhagem Celular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Fibroblastos/parasitologia , Fibroblastos/patologia , Humanos , Modelos Biológicos , Testes de Sensibilidade ParasitáriaRESUMO
Brain-eating amoebae (Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri) can cause opportunistic infections involving the central nervous system. It is troubling that the mortality rate is more than 90% despite advances in antimicrobial chemotherapy over the last few decades. Here, we describe urgent key priorities for improving outcomes from infections due to brain-eating amoebae.
Assuntos
Infecções Protozoárias do Sistema Nervoso Central , Prioridades em Saúde , Pesquisa , Acanthamoeba/efeitos dos fármacos , Acanthamoeba/genética , Animais , Antiprotozoários/provisão & distribuição , Antiprotozoários/uso terapêutico , Balamuthia mandrillaris/efeitos dos fármacos , Balamuthia mandrillaris/genética , Infecções Protozoárias do Sistema Nervoso Central/tratamento farmacológico , Infecções Protozoárias do Sistema Nervoso Central/mortalidade , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Infecções Protozoárias do Sistema Nervoso Central/prevenção & controle , Descoberta de Drogas , Previsões , Acessibilidade aos Serviços de Saúde , Humanos , Naegleria fowleri/efeitos dos fármacos , Naegleria fowleri/genética , Infecções Oportunistas/parasitologia , Infecções Oportunistas/prevenção & controle , Testes ImediatosRESUMO
Balamuthia mandrillaris, a free-living ameba, causes rare but frequently fatal granulomatous amebic encephalitis (GAE). Few patients have survived after receiving experimental drug combinations, with or without brain lesion excisions. Some GAE survivors have been treated with a multi-drug regimen including miltefosine, an investigational anti-leishmanial agent with in vitro amebacidal activity. Miltefosine dosing for GAE has been based on leishmaniasis dosing because no data exist in humans concerning its pharmacologic distribution in the central nervous system. We describe results of limited cerebrospinal fluid (CSF) and serum drug level testing performed during clinical management of a child with fatal GAE who was treated with a multiple drug regimen including miltefosine. Brain biopsy specimens, CSF, and sera were tested for B. mandrillaris using multiple techniques, including culture, real-time polymerase chain reaction, immunohistochemical techniques, and serology. CSF and serum miltefosine levels were determined using a liquid chromatography method coupled to tandem mass spectrometry. The CSF miltefosine concentration on hospital admission day 12 was 0.4 µg/mL. The serum miltefosine concentration on day 37, about 80 h post-miltefosine treatment, was 15.3 µg/mL. These are the first results confirming some blood-brain barrier penetration by miltefosine in a human, although with low-level CSF accumulation. Further evaluation of brain parenchyma penetration is required to determine optimal miltefosine dosing for Balamuthia GAE, balanced with the drug's toxicity profile. Additionally, the Balamuthia isolate was evaluated by real-time polymerase chain reaction (PCR), demonstrating genetic variability in 18S ribosomal RNA (18S rRNA) sequences and possibly signaling the first identification of multiple Balamuthia strains with varying pathogenicities.
Assuntos
Amebíase/tratamento farmacológico , Amebicidas/farmacocinética , Balamuthia mandrillaris/efeitos dos fármacos , Barreira Hematoencefálica/parasitologia , Encefalite/tratamento farmacológico , Fosforilcolina/análogos & derivados , Amebíase/parasitologia , Amebicidas/administração & dosagem , Balamuthia mandrillaris/isolamento & purificação , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/parasitologia , Encéfalo/patologia , Criança , Encefalite/parasitologia , Evolução Fatal , Humanos , Masculino , Fosforilcolina/administração & dosagem , Fosforilcolina/farmacocinéticaRESUMO
Balamuthia mandrillaris is a free-living protist pathogen that can cause life-threatening granulomatous amoebic encephalitis. Given the lack of effective available drugs against B. mandrillaris encephalitis with a mortality rate of more than 90%, here we screened drugs, targeting vital cellular receptors and biochemical pathways, that are already in approved clinical use for their potential clinical usefulness. Amoebicidal assays were performed by incubating B. mandrillaris with drugs (3 × 10(5) cells/0.5 mL/well) in phosphate buffered saline for 24 h and viability was determined using Trypan blue exclusion staining. For controls, amoebae were incubated with the solvent alone. To determine whether effects are reversible, B. mandrillaris were pre-exposed to drugs for 24 h, washed twice, and incubated with human brain microvascular endothelial cells, which constitute the blood-brain barrier as food source, for up to 48 h. Of the ten drugs tested, amlodipine, apomorphine, demethoxycurcumin, haloperidol, loperamide, prochlorperazine, procyclidine, and resveratrol showed potent amoebicidal effects, while amiodarone and digoxin exhibited minimal effectiveness. When pre-treated with these drugs, no viable trophozoites re-emerged, suggesting that drugs destroyed parasite irreversibly. Based on the in vitro assay, amlodipine, apomorphine, demethoxycurcumin, haloperidol, loperamide, prochlorperazine, procyclidine, and resveratrol are potential antimicrobials for further testing against B. mandrillaris encephalitis. These findings may provide novel strategies for therapy but further research is needed to determine clinical usefulness of aforementioned drugs against granulomatous amoebic encephalitis caused by B. mandrillaris, and other free-living amoebae, such as Acanthamoeba spp., and Naegleria fowleri.
Assuntos
Antiprotozoários/isolamento & purificação , Antiprotozoários/farmacologia , Balamuthia mandrillaris/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos , Balamuthia mandrillaris/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Humanos , Testes de Sensibilidade ParasitáriaRESUMO
Granulomatous amoebic encephalitis (GAE) is caused by two protist pathogens, Acanthamoeba spp., and Balamuthia mandrillaris. Although rare, it almost always results in death. In the present study, amoebae were treated with various combinations of clinically-approved drugs, targeting vital cellular receptors and biochemical pathways. The results revealed that among the seven different combinations tested, three proved highly effective against both Acanthamoeba castellanii as well as B. mandrillaris at a concentration of 100µM. These combinations included (i) prochlorperazine plus loperamide; (ii) prochlorperazine plus apomorphine; and (iii) procyclidine plus loperamide. In viability assays, none of the drug-treated amoebae emerged as viable trophozoites, suggesting irreversible amoebicidal effects. Four combinations of drugs tested showed varied potency against A. castellanii and B. mandrillaris at 100µM. The combination of haloperidol and loperamide was highly effective against A. castellanii at 100µM, but potent effects against B. mandrillaris were observed only at 250µM. Digoxin and amlodipine were effective against A. castellanii and B. mandrillaris at 100µM and 250µM, respectively. In contrast, the combination of apomorphine and haloperidol was effective against B. mandrillaris and A. castellanii at 100µM and 250µM, respectively. At 100µM, the combination of procyclidine and amiodarone was effective against neither A. castellanii nor B. mandrillaris. In this case, amoebicidal properties were observed at 750µM for A. castellanii, and 950µM for B. mandrillaris. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic encephalitis caused by A. castellanii and B. mandrillaris.
Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Amebíase/tratamento farmacológico , Amebicidas/farmacologia , Balamuthia mandrillaris/efeitos dos fármacos , Encefalite/tratamento farmacológico , Amebíase/parasitologia , Amiodarona/farmacologia , Anlodipino/farmacologia , Apomorfina/farmacologia , Encéfalo/irrigação sanguínea , Células Cultivadas , Digoxina/farmacologia , Sinergismo Farmacológico , Quimioterapia Combinada , Encefalite/parasitologia , Células Endoteliais/citologia , Haloperidol/farmacologia , Humanos , Loperamida/farmacologia , Microvasos/citologia , Proclorperazina/farmacologia , Prociclidina/farmacologiaRESUMO
Balamuthia amoebic encephalitis (BAE) is a life threatening human disease which, always lead to death. Amoebae invasion of the bloodstream is considered an important step in BAE followed by their haematogenous spread. It is more likely that Balamuthia mandrillaris enters into the central nervous system through blood-brain barrier (BBB) sites. The objective of the present study was to determine the impact of cytokines on biological properties of Balamuthia in vitro. Human brain microvascular endothelial cells (HBMEC), which constitutes the BBB were used in vitro test model for the present investigation. It was observed that Balamuthia exhibited >90 % binding and >70% cytotoxicity to HBMEC. However, cytokines did not affect amoebic binding and cytotoxicity except lipopolysaccharide (LPS) which reduced Balamuthia-mediated HBMEC cytotoxicity. It is also important to note that amoebic numbers were reduced in the presence of LPS within 24 h. We have shown previously the bacterial uptake by Balamuthia is very limited which is further investigated in the presence of cytokines and observed a slight reduction of bacterial uptake during phagocytosis assay. Zymography assays revealed there is no effect of cytokines on proteolytic activity of Balamuthia. Overall we described for the first time that cytokines has no inhibitory effects on biological properties of Balamuthia in vitro.
Assuntos
Balamuthia mandrillaris/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Citocinas/farmacologia , Células Endoteliais/parasitologia , Balamuthia mandrillaris/metabolismo , Barreira Hematoencefálica , Células Cultivadas , Humanos , Lipopolissacarídeos/farmacologia , FagocitoseRESUMO
Infections caused by free-living amebae (FLA) are severe and life-threatening. These infections include primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri and granulomatous amebic encephalitis caused by Balamuthia mandrillaris and Acanthamoeba species. Although several drugs have in vitro activity against FLA, mortality from these infections remains>90% despite treatment with combinations of drugs.
Assuntos
Amebíase/tratamento farmacológico , Antiprotozoários/uso terapêutico , Infecções Protozoárias do Sistema Nervoso Central/tratamento farmacológico , Drogas em Investigação/uso terapêutico , Encefalite/tratamento farmacológico , Fosforilcolina/análogos & derivados , Acanthamoeba/efeitos dos fármacos , Animais , Balamuthia mandrillaris/efeitos dos fármacos , Centers for Disease Control and Prevention, U.S. , Humanos , Naegleria fowleri/efeitos dos fármacos , Fosforilcolina/uso terapêutico , Resultado do Tratamento , Estados UnidosRESUMO
The free-living amoeba Balamuthia mandrillaris causes usually fatal encephalitis in humans and animals. Only limited studies have investigated the efficacy of antimicrobial agents against the organism. Assay methods were developed to assess antimicrobial efficacy against both the trophozoite and cyst stage of B. mandrillaris (ATCC 50209). Amphotericin B, ciclopirox olamine, miltefosine, natamycin, paromomycin, pentamidine isethionate, protriptyline, spiramycin, sulconazole and telithromycin had limited activity with amoebacidal levels of > 135-500 µM. However, diminazene aceturate (Berenil(®) ) was amoebacidal at 7.8 µM and 31.3-61.5 µM for trophozoites and cysts, respectively. Assays for antimicrobial testing may improve the prognosis for infection and aid in the development of primary selective culture isolation media.
Assuntos
Anti-Infecciosos/farmacologia , Balamuthia mandrillaris/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Testes de Sensibilidade Parasitária , Esporos de Protozoários/efeitos dos fármacosRESUMO
The opportunist free-living protists such as Acanthamoeba spp. and Balamuthia mandrillaris have become a serious threat to human life. As most available drugs target functional aspects of pathogens, the ability of free-living protists to transform into metabolically inactive cyst forms presents a challenge in treatment. It is hoped, that the development of broad spectrum antiprotist agents acting against multiple cyst-forming protists to provide target-directed inhibition will offer a viable drug strategy in the treatment of these rare infections. Here, we present a comprehensive report on upcoming drug targets, with emphasis on cyst wall biosynthesis along with the related biochemistry of encystment pathways, as we strive to bring ourselves a step closer to being able to combat these deadly diseases.