The anti-amoebic activity of Pinus densiflora leaf extract against the brain-eating amoeba Naegleria fowleri.

Naegleria fowleri invades the brain and causes a fatal primary amoebic meningoencephalitis (PAM). Despite its high mortality rate of approximately 97%, an effective therapeutic drug for PAM has not been developed. Approaches with miltefosine, amphotericin B, and other antimicrobials have been clinically attempted to treat PAM, but their therapeutic efficacy remains unclear. The development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated the anti-amoebic activity of Pinus densiflora leaf extract (PLE) against N. fowleri. PLE induced significant morphological changes in N. fowleri trophozoites, resulting in the death of the amoeba. The IC50 of PLE on N. fowleri was 62.3±0.95 µg/ml. Alternatively, PLE did not significantly affect the viability of the rat glial cell line C6. Transcriptome analysis revealed differentially expressed genes (DEGs) between PLE-treated and non-treated amoebae. A total of 5,846 DEGs were identified, of which 2,189 were upregulated, and 3,657 were downregulated in the PLE-treated amoebae. The DEGs were categorized into biological process (1,742 genes), cellular component (1,237 genes), and molecular function (846 genes) based on the gene ontology analysis, indicating that PLE may have dramatically altered the biological and cellular functions of the amoeba and contributed to their death. These results suggest that PLE has anti-N. fowleri activity and may be considered as a potential candidate for the development of therapeutic drugs for PAM. It may also be used as a supplement compound to enhance the therapeutic efficacy of drugs currently used to treat PAM.

Next generation imidazothiazole and imidazooxazole derivatives as potential drugs against brain-eating amoebae.

Managing primary amoebic meningoencephalitis, induced by Naegleria fowleri poses a complex medical challenge. There is currently no specific anti-amoebic drug that has proven effectiveness against N. fowleri infection. Ongoing research endeavours are dedicated to uncovering innovative treatment strategies, including the utilization of drugs and immune modulators targeting Naegleria infection. In this study, we explored the potential of imidazo[2,1-b]thiazole and imidazooxazole derivatives that incorporate sulfonate and sulfamate groups as agents with anti-amoebic properties against N. fowleri. We assessed several synthesized compounds (1f, 1m, 1q, 1s, and 1t) for their efficacy in eliminating amoebae, their impact on cytotoxicity, and their influence on the damage caused to human cerebral microvascular endothelial (HBEC-5i) cells when exposed to the N. fowleri (ATCC 30174) strain. The outcomes revealed that, among the five compounds under examination, 1m, 1q, and 1t demonstrated notable anti-parasitic effects against N. fowleri (P ≤ 0.05). Compound 1t exhibited the highest anti-parasitic activity, reducing N. fowleri population by 80%. Additionally, three compounds, 1m, 1q, and 1t, significantly mitigated the damage inflicted on host cells by N. fowleri. However, the results of cytotoxicity analysis indicated that while 1m and 1q had minimal cytotoxic effects on endothelial cells, compound 1t caused moderate cytotoxicity (34%). Consequently, we conclude that imidazo[2,1-b]thiazole and imidazooxazole derivatives containing sulfonate and sulfamate groups exhibit a marked capacity to eliminate amoebae viability while causing limited toxicity to human cells. In aggregate, these findings hold promise that could potentially evolve into novel therapeutic options for treating N. fowleri infection.

Flucofuron as a Promising Therapeutic Agent against Brain-Eating Amoeba.

Primary amoebic meningoencephalitis (PAM) is a rare and fulminant neurodegenerative disease caused by the free-living amoeba Naegleria fowleri. Currently, there is a lack of standardized protocols for therapeutic action. In response to the critical need for effective therapeutic agents, we explored the Global Health Priority Box, a collection of 240 compounds provided by the Medicines for Malaria Venture (MMV). From this pool, flucofuron emerged as a promising candidate, exhibiting high efficacy against trophozoites of both N. fowleri strains (ATCC 30808 IC50 : 2.58 ± 0.64 µM and ATCC 30215 IC50: 2.47 ± 0.38 µM), being even active against the resistant cyst stage (IC50: 0.88 ± 0.07 µM). Moreover, flucofuron induced diverse metabolic events that suggest the triggering of apoptotic cell death. This study highlights the potential of repurposing medications for treating challenging diseases, such as PAM.

Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

Sesquiterpene lactones as potential therapeutic agents against Naegleria fowleri.

Naegleria fowleri is the causative agent the primary amoebic meningoencephalitis (PAM), a fatal disease in more than the 90% of the reported cases that affects the central nervous system. The amoeba infects the nasal cavity of mostly children and young adults who report previous aquatic exposure in warm water sources. The rapid progression of the disease and the lack of effective and safety therapeutic options make the search of new anti-amoebic compounds an urgent issue. In this study, twelve sesquiterpene lactones isolated from the zoanthid Palythoa aff. clavata were tested against the trophozoite stage of Naegleria fowleri. Anhydroartemorin (2) and 1(10)Z,4E,14-acetoxy-costunolide (3) showed the best anti-amoeboid activity values with IC50 23.02 ± 1.26 and 28.34 ± 6.27, respectively. In addition, the mechanisms of programmed cell death induction of these two molecules were evaluated with positive results for both compounds. Finally, a structure-activity relationship was analyzed to reveal the dependence of reactivity and lipophilicity on the biological activity. The log P values of the compounds were calculated to postulate them as good candidates to cross the blood-brain barrier, a limiting factor in the development of new anti-Naegleria treatments. Therefore, the mentioned sesquiterpene lactones could be considered as potential PAM therapeutic options in the future.

Differential Growth Rates and In Vitro Drug Susceptibility to Currently Used Drugs for Multiple Isolates of Naegleria fowleri.

The free-living amoeba Naegleria fowleri, which typically dwells within warm, freshwater environments, can opportunistically cause primary amoebic meningoencephalitis (PAM), a disease with a mortality rate of >97%. The lack of positive treatment outcomes for PAM has prompted the discovery and development of more effective therapeutics, yet most studies utilize only one or two clinical isolates. The inability to assess possible heterogenic responses to drugs among isolates from various geographical regions hinders progress in the discovery of more effective drugs. Here, we conducted drug efficacy and growth rate determinations for 11 different clinical isolates by applying a previously developed CellTiter-Glo 2.0 screening technique and flow cytometry. We found significant differences in the susceptibilities of these isolates to 7 of 8 drugs tested, all of which make up the cocktail that is recommended to physicians by the U.S. Centers for Disease Control and Prevention. We also discovered significant variances in growth rates among isolates, which draws attention to the differences among the amoeba isolates collected from different patients. Our results demonstrate the need for additional clinical isolates of various genotypes in drug assays and highlight the necessity for more targeted therapeutics with universal efficacy across N. fowleri isolates. Our data establish a needed baseline for drug susceptibility among clinical isolates and provide a segue for future combination therapy studies as well as research related to phenotypic or genetic differences that could shed light on mechanisms of action or predispositions to specific drugs. IMPORTANCE Naegleria fowleri, also known as the brain-eating amoeba, is ubiquitous in warm freshwater and is an opportunistic pathogen that causes primary amoebic meningoencephalitis. Although few cases are described each year, the disease has a case fatality rate of >97%. In most laboratory studies of this organism, only one or two well-adapted lab strains are used; therefore, there is a lack of data to discern if there are major differences in potency of currently used drugs for multiple strains and genotypes of the amoeba. In this study, we found significant differences in the susceptibilities of 11 N. fowleri isolates to 7 of the 8 drugs currently used to treat the disease. The data from this study provide a baseline of drug susceptibility among clinical isolates and suggest that new drugs should be tested on a larger number of isolates in the future.

Primary Amoebic Meningoencephalitis by Naegleria fowleri: Pathogenesis and Treatments.

Naegleria fowleri is a free-living amoeba (FLA) that is commonly known as the "brain-eating amoeba." This parasite can invade the central nervous system (CNS), causing an acute and fulminating infection known as primary amoebic meningoencephalitis (PAM). Even though PAM is characterized by low morbidity, it has shown a mortality rate of 98%, usually causing death in less than two weeks after the initial exposure. This review summarizes the most recent information about N. fowleri, its pathogenic molecular mechanisms, and the neuropathological processes implicated. Additionally, this review includes the main therapeutic strategies described in case reports and preclinical studies, including the possible use of immunomodulatory agents to decrease neurological damage.

In silico analysis of Naegleria fowleri cathepsin B paralogs: important drug targets.

Naegleria fowleri is a deadly human pathogen that causes primary amoebic meningoencephalitis (PAM). In this study, in silico investigations of two important N. fowleri cathepsin B paralogs, i.e., copies of genes resulting from a gene duplication event, were carried out using comparative modeling and molecular dynamics (MD) simulations. Comparative models of both paralogs showed significant architectural similarity with their template, i.e., rat cathepsin B. However, in N. fowleri cathepsin B (UniProt ID: X5D761) and putative cathepsin B (UniProt ID: M1HE19) enzymes, eleven and fifteen residues in the occluding loop regions were deleted, respectively, suggesting that these enzymes have a short occluding loop. Thus, it is concluded that N. fowleri cathepsin B and putative cathepsin B enzymes lack exopeptidase activity but possess enhanced endopeptidase activity and an affinity for macromolecular inhibitors. MD simulations further confirmed that prosegments (macromolecular inhibitors) bond more tightly with both enzymes than with wild-type cathepsin B. Additionally, a mutation was identified at an important N-glycosylation site; this mutation is believed to affect cathepsin B targeting inside the cell and make cathepsin B available in the extracellular environment. Due to this important N-glycosylation site mutation, these enzymes are secreted in the extracellular environment via an alternative, still unknown, posttranslational processing strategy. The present study is the first to predict the three-dimensional folds of N. fowleri cathepsin B paralogous enzymes, including a detailed description of the active site architecture and information about propeptide binding mode. This information can contribute to the discovery of novel and selective treatments that are effective against N. fowleri.

The type 2 statins, cerivastatin, rosuvastatin and pitavastatin eliminate Naegleria fowleri at low concentrations and by induction of programmed cell death (PCD).

Primary Amoebic Encephalitis due to Naegleria fowleri species is a fatal infection of the Central Nervous System mostly affecting children and young adults. Infections often occur after performance of risk activities in aquatic habitats such as swimming and splashing. PAMs therapy remain a key issue to be solved which needs an urgent development. Recently, statins have been highlighted as possible novel compounds to treat PAM. Furthermore, type 2 statins due to improved pharmacological properties and lower toxicity could be use in the future. In the present work, three type 2 statins were checked for their activity against two type strains of N. fowleri. In addition, the effects at the cellular level triggered in treated amoebae were checked in order to evaluate if programmed cell death was induced. The obtained results showed that the tested statins, rosuvastatin, pitavastatin and cerivastatin were able to eliminate N. fowleri trophozoites and also induced PCD. Therefore, type 2 statins could be used in the near future for the treatment of PAM.

Drug discovery for primary amebic meningoencephalitis: from screen to identification of leads.

Introduction: Naegleria fowleri is responsible for primary amebic meningoencephalitis (PAM) which has a fatality rate of >97%. Because of the rarity of the disease, pharmaceutical companies do not pursue new drug discovery for PAM. Yet, it is possible that the infection is underreported and finding a better drug would have an impact on people suffering from this deadly infection.Areas covered: This paper reports the efforts undertaken by different academic groups over the last 20 years to test different compounds against N. fowleri. The drug discovery research encompassed synthesis of new compounds, development and use of high-throughput screening methods and attempts to repurpose clinically developed or FDA-approved compounds for the treatment of PAM.Expert opinion: In absence of economic investment to develop new drugs for PAM, repurposing the FDA-approved drugs has been the best strategy so far to identify new leads against N. fowleri. Increasing use of high-throughput phenotypic screening has the potential to accelerate the identification of new leads, either in monotherapy or in combination treatment. Since phase II clinical trial is not possible for PAM, it is critical to demonstrate in vivo efficacy of a clinically safe compound to translate the discovery from lab to the clinic.

Synthetic nanoparticle-conjugated bisindoles and hydrazinyl arylthiazole as novel antiamoebic agents against brain-eating amoebae.

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.

Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae.

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.

Fluvastatin and atorvastatin induce programmed cell death in the brain eating amoeba Naegleria fowleri.

Naegleria fowleri is the causative agent of a type of encephalitis called Primary Amoebic Encephalitis (PAM). Almost 98 % of PAM cases reported worldwide are fatal and affect mostly immunocompetent children and young adults. The current therapeutic option against PAM cases includes a combination of miltefosine, amphotericin B and other drugs which are unfortunately associated with severe side effects. In a recent study in our group, statins were tested in vitro against Naegleria fowleri trophozoites showing activity against these pathogens at low concentrations causing low toxicity. Consequently, there is an urgent need to develop novel PAM therapeutic options. Therefore, this study was undertaken to evaluate the pathway of cell death induced by two of the previously tested molecules, fluvastatin and atorvastatin. Moreover, these statins were compared to miltefosine and amphotericin B. Furthermore, the induction of Programmed Cell Death (PCD) instead of necrosis in treated amoebae would be the ideal situation since necrosis could lead to non-desired inflammation processes in the infected individual. The obtained results revealed that both statins induced PCD in the treated amoebae after the observation of condensed chromatin, cell membrane damages, mitochondrial membrane potential and ATP levels collapse and ROS generation. In conclusion, both fluvastatin and atorvastatin could be potential new candidates for PAM therapy since they are active at low concentrations, induce low toxicity and cause PCD in the treated amoebae, hence avoiding the activation of inflammation pathways.

Naegleria fowleri: Sources of infection, pathophysiology, diagnosis, and management; a review.

Naegleria fowleri, a thermophilic flagellate amoeba known as a "brain-eating" amoeba, is the aetiological agent of a perilous and devastating waterborne disease known as primary amoebic meningoencephalitis (PAM), both in humans as well as in animals. PAM is a rare but fatal disease affecting young adults all around the world, particularly in the developed world but recently reported from developing countries, with 95%-99% mortality rate. Swimmers and divers are at high risk of PAM as the warm water is the most propitious environment adapted by N. fowleri to cause this infection. Infective amoeba in the trophozoite phase enter the victim's body through the nose, crossing the cribriform plate to reach the human brain and cause severe destruction of the central nervous system (CNS). The brain damage leads to brain haemorrhage and death occurs within 3-7 days in undiagnosed cases and maltreated cases. Though the exact pathogenesis of N. fowleri is still not known, it has exhibited two primary mechanisms, contact-independent (brain damage through different proteins) and contact-dependent (brain damage through surface structures food cups), that predominantly contribute to the pathogen invading the host CNS. For the management of this life-threatening infection different treatment regimens have been applied but still the survival rate is only 5% which is ascribed to its misdiagnosis, as the PAM symptoms closely resembled bacterial meningitis. The main objectives of this review article are to compile data to explore the sources and routes of N. fowleri infection, its association in causing PAM along with its pathophysiology; latest techniques used for accurate diagnosis, management options along with challenges for Pakistan to control this drastic disorder.

Identification of Four Amoebicidal Nontoxic Compounds by a Molecular Docking Screen of Naegleria fowleri Sterol Δ8-Δ7-Isomerase and Phenotypic Assays.

Naegleria fowleri is a free-living amoeba causing primary amoebic meningoencephalitis, a rapid-onset brain infection in humans with over 97% mortality rate. Despite some progress in the treatment of the disease, there is no single, proven, evidence-based treatment with a high probability of cure. Here we report the chemical library screening and experimental identification of four new compounds with amoebicidal effects against N. fowleri. The chemical library was screened by molecular docking against a homology model of sterol Δ8-Δ7 isomerase (NfERG2). Thirty top-ranking hits were then tested in a cell-based assay for antiproliferative/amoebicidal activities. Eight chemicals exhibited nearly 100% inhibition of N. fowleri at 50 µM, with the EC50 values ranging from 6 to 25 µM. A cell toxicity assay using human HEK-293 cells was also performed. Four of the compounds preferentially kill amoeba cells with no apparent human cell toxicities. These compounds fall into two distinct chemical scaffolds with druglike properties.

Repositioning of Guanabenz in Conjugation with Gold and Silver Nanoparticles against Pathogenic Amoebae Acanthamoeba castellanii and Naegleria fowleri.

Brain-eating amoebae cause devastating infections in the central nervous system of humans, resulting in a mortality rate of 95%. There are limited effective therapeutic options available clinically for treating granulomatous amoebic encephalitis and primary amoebic meningoencephalitis caused by Acanthamoeba castellanii (A. castellanii) and Naegleria fowleri (N. fowleri), respectively. Here, we report for the first time that guanabenz conjugated to gold and silver nanoparticles has significant antiamoebic activity against both A. castellanii and N. fowleri. Gold and silver conjugated guanabenz nanoparticles were synthesized by the one-phase reduction method and were characterized by ultraviolet-visible spectrophotometry and atomic force microscopy. Both metals were facilely stabilized by the coating of guanabenz, which was examined by surface plasmon resonance determination. The average size of gold nanoconjugated guanabenz was found to be 60 nm, whereas silver nanoparticles were produced in a larger size distribution with the average diameter of around 100 nm. Guanabenz and its noble metal nanoconjugates exhibited potent antiamoebic effects in the range of 2.5 to 100 µM against both amoebae. Nanoparticle conjugation enhanced the antiamoebic effects of guanabenz, as more potent activity was observed at a lower effective concentration (2.5 and 5 µM) compared to the drug alone. Moreover, encystation and excystation assays revealed that guanabenz inhibits the interconversion between the trophozoite and cyst forms of A. castellanii. Cysticdal effects against N. fowleri were also observed. Notably, pretreatment of A. castellanii with guanabenz and its nanoconjugates exhibited a significant reduction in the host cell cytopathogenicity from 65% to 38% and 2% in case of gold and silver nanoconjugates, respectively. Moreover, the cytotoxic evaluation of guanabenz and its nanoconjugates revealed negligible cytotoxicity against human cells. Guanabenz is already approved for hypertension and crosses the blood-brain barrier; the results of our current study suggest that guanabenz and its conjugated gold and silver nanoparticles can be repurposed as a potential drug for treating brain-eating amoebic infections.

trans-Cinnamic Acid Conjugated Gold Nanoparticles as Potent Therapeutics against Brain-Eating Amoeba Naegleria fowleri.

Primary amoebic meningoencephalitis (PAM), a deadly brain infection, is caused by brain-eating amoeba Naegleria fowleri. The current first line of treatment against PAM is a mixture of amphotericin B, rifampin, and miltefosine. Since, no single effective drug has been developed so far, the mortality rate is above 95%. Moreover, severe adverse side effects are associated with these drugs. Nanotechnology has provided several advances in biomedical applications especially in drug delivery and diagnosis. Herein, for the first time we report antiamoebic properties of cinnamic acid (CA) and gold nanoparticles conjugated with CA (CA-AuNPs) against N. fowleri. CA-AuNPs were successfully synthesized by sodium borohydride reduction of tetrachloroauric acid. Size and morphology were determined by atomic force microscopy (AFM) while the surface plasmon resonance band was analyzed by ultraviolet-visible (UV-vis) spectrophotometry for the characterization of the nanoparticles. Amoebicidal and cytopathogenicity (host cell cytotoxicity) assays revealed that both CA and CA-AuNPs displayed significant anti- N. fowleri properties ( P < 0.05), whereas nanoparticles conjugation further enhanced the anti- N. fowleri effects of CA. This study established a potential drug lead, while CA-AuNPs appear to be promising candidate for drug discovery against PAM.

Brain-eating Amoebae Infection: Challenges and Opportunities in Chemotherapy.

Pathogenic free-living amoeba are known to cause a devastating infection of the central nervous system and are often referred to as "brain-eating amoebae". The mortality rate of more than 90% and free-living nature of these amoebae is a cause for concern. It is distressing that the mortality rate has remained the same over the past few decades, highlighting the lack of interest by the pharmaceutical industry. With the threat of global warming and increased outdoor activities of public, there is a need for renewed interest in identifying potential anti-amoebic compounds for successful prognosis. Here, we discuss the available chemotherapeutic options and opportunities for potential strategies in the treatment and diagnosis of these life-threatening infections.

Antiparasitic Activity of Auranofin against Pathogenic Naegleria fowleri.

We report that the gold containing antirheumatoid drug auranofin is amoebicidal against human pathogenic Naegleria fowleri. Treatment of N. fowleri cultures at biologically relevant concentrations of 0.75-3.0 µg/ml auranofin reduced amoeba counts, metabolic activity, and increased cell permeability. These results suggest that the addition of auranofin may benefit the treatment of N. fowleri-infected patients afflicted by the rapidly fatal disease primary amoebic meningoencephalitis.

Clinically Approved Drugs against CNS Diseases as Potential Therapeutic Agents To Target Brain-Eating Amoebae.

Central nervous system (CNS) infections caused by free-living amoebae such as Acanthamoeba species and Naegleria fowleri are rare but fatal. A major challenge in the treatment against the infections caused by these amoebae is the discovery of novel compounds that can effectively cross the blood-brain barrier to penetrate the CNS. It is logical to test clinically approved drugs against CNS diseases for their potential antiamoebic effects since they are known for effective blood-brain barrier penetration and affect eukaryotic cell targets. The antiamoebic effects of clinically available drugs for seizures targeting gamma-amino butyric acid (GABA) receptor and ion channels were tested against Acanthamoeba castellanii belonging to the T4 genotype and N. fowleri. Three such drugs, namely, diazepam (Valium), phenobarbitone (Luminal), phenytoin (Dilantin), and their silver nanoparticles (AgNPs) were evaluated against both trophozoites and cysts stage. Drugs alone and drug conjugated silver nanoparticles were tested for amoebicidal, cysticidal, and host-cell cytotoxicity assays. Nanoparticles were synthesized by sodium borohydride reduction of silver nitrate with drugs as capping agents. Drug conjugated nanoconjugates were characterized by ultraviolet-visible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopies and atomic force microscopy (AFM). In vitro moebicidal assay showed potent amoebicidal effects for diazepam, phenobarbitone, and phenytoin-conjugated AgNPs as compared to drugs alone against A. castellanii and N. fowleri. Furthermore, both drugs and drug conjugated AgNPs showed compelling cysticidal effects. Drugs conjugations with silver nanoparticles enhanced their antiacanthamoebic activity. Interestingly, amoeba-mediated host-cell cytotoxicity was also significantly reduced by drugs alone as well as their nanoconjugates. Since, these drugs are being used to target CNS diseases, their evaluation against brain-eating amoebae seems feasible due to advantages such as permeability of the blood-brain barrier, established pharmacokinetics and dynamics, and United States Food and Drug Administration (FDA) approval. Given the limited availability of effective drugs against brain-eating amoebae, the clinically available drugs tested here present potential for further in vivo studies.