Identification of plicamycin, TG02, panobinostat, lestaurtinib, and GDC-0084 as promising compounds for the treatment of central nervous system infections caused by the free-living amebae Naegleria, Acanthamoeba and Balamuthia.

The free-living amebae Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis. Acanthamoeba can also cause chronic keratitis and both Balamuthia and Acanthamoeba can cause skin and systemic infections. There are minimal drug development pipelines for these pathogens despite a lack of available treatment regimens and high fatality rates. To identify anti-amebic drugs, we screened 159 compounds from a high-value repurposed library against trophozoites of the three amebae. Our efforts identified 38 compounds with activity against at least one ameba. Multiple drugs that bind the ATP-binding pocket of mTOR and PI3K are active, highlighting these compounds as important inhibitors of these parasites. Importantly, 24 active compounds have progressed at least to phase II clinical studies and overall 15 compounds were active against all three amebae. Based on central nervous system (CNS) penetration or exceptional potency against one amebic species, we identified sixteen priority compounds for the treatment of meningoencephalitis caused by these pathogens. The top five compounds are (i) plicamycin, active against all three free-living amebae and previously U.S. Food and Drug Administration (FDA) approved, (ii) TG02, active against all three amebae, (iii and iv) FDA-approved panobinostat and FDA orphan drug lestaurtinib, both highly potent against Naegleria, and (v) GDC-0084, a CNS penetrant mTOR inhibitor, active against at least two of the three amebae. These results set the stage for further investigation of these clinically advanced compounds for treatment of infections caused by the free-living amebae, including treatment of the highly fatal meningoencephalitis.

Amphotericin B induces apoptosis-like programmed cell death in Naegleria fowleri and Naegleria gruberi.

Naegleria fowleri and Naegleria gruberi belong to the free-living amoebae group. It is widely known that the non-pathogenic species N. gruberi is usually employed as a model to describe molecular pathways in this genus, mainly because its genome has been recently described. However, N. fowleri is an aetiological agent of primary amoebic meningoencephalitis, an acute and fatal disease. Currently, the most widely used drug for its treatment is amphotericin B (AmB). It was previously reported that AmB has an amoebicidal effect in both N. fowleri and N. gruberi trophozoites by inducing morphological changes that resemble programmed cell death (PCD). PCD is a mechanism that activates morphological, biochemical and genetic changes. However, PCD has not yet been characterized in the genus Naegleria. The aim of the present work was to evaluate the typical markers to describe PCD in both amoebae. These results showed that treated trophozoites displayed several parameters of apoptosis-like PCD in both species. We observed ultrastructural changes, an increase in reactive oxygen species, phosphatidylserine externalization and a decrease in intracellular potassium, while DNA degradation was evaluated using the TUNEL assay and agarose gels, and all of these parameters are related to PCD. Finally, we analysed the expression of apoptosis-related genes, such as sir2 and atg8, in N. gruberi. Taken together, our results showed that AmB induces the morphological, biochemical and genetic changes of apoptosis-like PCD in the genus Naegleria.

Corifungin, a new drug lead against Naegleria, identified from a high-throughput screen.

Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

Naegleria gruberi metabolism.

The completion of the genome project for Naegleria gruberi provides a unique insight into the metabolic capacities of an organism, for which there is an almost complete lack of experimental data. The metabolism of Naegleria seems to be extremely versatile, as can be expected for a free-living amoeboflagellate, but although considered to be fully aerobic, its genome also predicts important anaerobic traits. Other predictions are that carbohydrates are oxidised to carbon dioxide and water when oxygen is not limiting and that in the absence of oxygen the end-products will be succinate, acetate and minor quantities of ethanol and D-lactate. The hybrid mitochondrion/hydrogenosome has both cytochromes and an [Fe] hydrogenase, but seems to lack pyruvate-ferredoxin oxidoreductase. Genomic information also provides the possibility to identify drugs with a possible mode of action in the fatal primary amoebic meningoencephalitis caused by the closely related opportunistic pathogen Naegleria fowleri.

Protozoan-induced regulation of cyclic lipopeptide biosynthesis is an effective predation defense mechanism for Pseudomonas fluorescens.

Environmental bacteria are exposed to a myriad of biotic interactions that influence their function and survival. The grazing activity of protozoan predators significantly impacts the dynamics, diversification, and evolution of bacterial communities in soil ecosystems. To evade protozoan predation, bacteria employ various defense strategies. Soil-dwelling Pseudomonas fluorescens strains SS101 and SBW25 produce the cyclic lipopeptide surfactants (CLPs) massetolide and viscosin, respectively, in a quorum-sensing-independent manner. In this study, CLP production was shown to protect these bacteria from protozoan predation as, compared to CLP-deficient mutants, strains SS101 and SBW25 exhibited resistance to grazing by Naegleria americana in vitro and superior persistence in soil in the presence of this bacterial predator. In the wheat rhizosphere, CLP-producing strains had a direct deleterious impact on the survival of N. americana. In vitro assays further showed that N. americana was three times more sensitive to viscosin than to massetolide and that exposure of strain SS101 or SBW25 to this protozoan resulted in upregulation of CLP biosynthesis genes. Enhanced expression of the massABC and viscABC genes did not require physical contact between the two organisms as gene expression levels were up to threefold higher in bacterial cells harvested 1 cm from feeding protozoans than in cells collected 4 cm from feeding protozoans. These findings document a new natural function of CLPs and highlight that bacterium-protozoan interactions can result in activation of an antipredator response in prey populations.

An examination of the antibiotic effects of cylindrospermopsin on common gram-positive and gram-negative bacteria and the protozoan Naegleria lovaniensis.

The importance of the toxin cylindrospermopsin to the function and fitness of the cyanobacteria that produce it remains a matter of conjecture. Given that the structure of cylindrospermopsin has commonalities with other antibacterial protein synthesis inhibitors, such as streptomycin, authors tested the possibility that the toxin might act as an antibacterial compound that can kill competing microbes. Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa were tested by the minimal inhibitory concentration method and significant antibacterial activity was only observed at a cylindrospermopsin concentration of 300 microg mL(-1) after exposure for 5 days. No effect on log phase growth of E. coli was observed for this same toxin concentration. Protein synthesis was inhibited by cylindrospermopsin in E. coli 70S extracts, reduced by 25% compared with controls when treated with 41.5 microg mL(-1) of the toxin; however, a much greater reduction of 97% was observed for chloramphenicol in the same experiment. Naegleria lovaniensis, a phagotrophic protozoan, was more susceptible to cylindrospermopsin, with a decrease in the number of N. lovaniensis plaques after 24-h treatment with 5-50 microg mL(-1) of toxin and an LC(50) of approximately 60 microg mL(-1). Given these results, cylindrospermopsin is clearly not antibacterial at concentrations found in environmental waters, nor will it adversely affect N. lovaniensis at these concentrations. For organisms that are able to ingest cylindrospermopsin-producing cells, the response of N. lovaniensis to the toxin suggests that only a few ingested cells would be enough to kill predatory organisms with similar susceptibility.

Effect of antifungal drugs on pathogenic Naegleria spp isolated from natural water sources.

UNLABELLED: Five of 16 strains of pathogenic Naegleria spp isolated from 350 natural water sources in Taling Chan District, Bangkok had similar molecular weights and zymogram patterns to those of Naegleria fowleri CDCVO 3081 and Thai strain. The in vitro effects of antifungal drugs (amphotericin B, ketoconazole, fluconazole and itraconazole) were tested at the following concentrations: amphotericin B 0.01-0.55 microg/ml, ketoconazole 0.01-0.3 microg/ml, fluconazole 0.75-3.5 mg/ml and itraconazole 4-12 mg/ml respectively. Aliquots (15,000 cells/ml) of the amoebae were placed in the cells of the microtiter plate and incubated at 37 degrees C. Amoebae from each treatment sample were exposed to one of the four antifungal drugs. Statistical analysis was done by dependent t-test. The sensitivity of the antifungal drugs (MIC50) was as follows: amphotericin B 0.03-0.035 microg/ml ketoconazole 0.05-0.15 microg/ml fluconazole 1.75 mg/ml and itraconazole 8-9 mg/ml respectively (p < 0.005). CONCLUSION: Amphotericin B and ketoconazole are more active against Naegleria fowleri in vitro. The results of the present study should be used as an in vitro screening test for drugs that have potential amebicidal activity.

In vitro effect of antifungal drugs on pathogenic Naegleria spp.

An ameba of the genus Naegleria causing fatal meningoencephalitis in human subjects was investigated for its sensitivity to antifungal drugs: amphotericin B, ketoconazole, fluconazole and itraconazole. The efficacy of these antifungal drugs for pathogenic Naegleria spp was investigated in three strains isolated from patients who had died of primary amebic meningoencephalitis infection at Siriraj Hospital (1986), Ramathibodi Hospital (1987) and Chachoengsao Hospital (1987). All of the isolates were maintained in axenic culture in the Department of Parasitology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand. The sensitivities of the antifungal drugs (MIC50) were: amphotericin B (0.05-0.5 microg/ml), ketoconazole (0.125 microg/ml), fluconazole (0.5-2.0 mg/ml), and itraconazole (10 mg/ml) (p < 0.05). It is important to explain that ketoconazole is slightly more effective than amphotericin B because its action is directed of the permeability of the amebic membrane. The amebae were more resistant ot fluconazole and itraconazole due to the action of the cytochrome P450 multienzyme (in the case of fluconazole) and the direct effect on heme-iron, blocking cytochrome P450-dependent chitin synthesis (in the case of itraconzole). We conclude that amphotericin B and ketoconazole remain the main drugs with proven activity against pathogenic Naegleria spp.

The effects of diffusates from the spores of Aspergillus fumigatus and A. terreus on human neutrophils, Naegleria gruberi and Acanthamoeba castellanii.

Diffusates from dormant spores of Aspergillus fumigatus are inhibitory to certain functions of immune phagocytic cells and soil protozoa. An assay of human neutrophil phagocytosis and an in vitro method of measuring inhibition of the growth of free living amoebae are described. The anti-phagocytic and anti-amoebal effects of diffusates from clinical and environmental isolates of A. fumigatus and of A. terreus were measured using these methods. Spore diffusates from all isolates of A. fumigatus and A. terreus tested significantly inhibited neutrophil phagocytosis. Spore diffusates from A. fumigatus, but not A. terreus, significantly inhibited the growth of Naegleria gruberi. Spore diffusate from A. fumigatus did not inhibit the growth of Acanthamoeba castellanii. The relevance of these findings to the pathogenicity of A. fumigatus and A. terreus is discussed.

Purification of amoebolytic substances from Bacillus licheniformis M-4.

Three antibiotic peptides with amoebolytic activity have been purified from culture supernatants of Bacillus licheniformis M-4 (amoebicins m4-A, m4-B, and m4-C). They were hydrophilic peptides consisting of six different amino acids (Asp, Glu, Ser, Thr, Pro, Tyr). Their molecular weights ranged from 3,000 to 3,200. Purified amoebicins were active against human pathogenic and non-pathogenic strains of Naegleria. They also showed a broad antifungal spectrum, but a narrow antibacterial activity.

Repair of damage caused by UV- and X-irradiation in the amoeboflagellate Naegleria gruberi.

Cysts of Naegleria gruberi have a normal UV- and an extremely high X-ray resistance compared to other protozoans. Caffeine and 3-aminobenzamide applied to excysting amoeba after irradiation in the encysted state (UV and X-rays) by feeding with drug-containing bacteria increased lethality, while fractionated irradiation (UV) and liquid-holding (UV and X-rays) increased survival. Illumination with visible light after UV-irradiation restored almost 100% viability. The results are discussed in regard to the activity of repair mechanisms.

Amoeboid locomotion of Naegleria gruberi: the effects of cytochalasin B on cell-substratum interactions and motile behavior.

The major manifestations of amoeboid locomotion in Naegleria-cytoplasmic streaming, pseudopod production, cell polarity and focal contact production-require that the actin-based cytoskeleton be extremely dynamic. Whether these features are causally linked is unclear. In an attempt to answer this question we have used the fungal product cytochalasin B (cyt B) to dissect the motility process. This drug can perturb the organisation of actin filaments both in vivo and in vitro. Essentially cyt B acts as a molecule which can cap the barbed ends of actin filaments. Not surprisingly, therefore cyt B has an effect on rates of actin polymerization and the dynamic state of actin in the cytoplasm. We have found that cyt B has a profound effect on focal contact production and breakdown. Within minutes of addition of cyt B focal contact production ceases, existing focal contacts are stabilised but cytoplasmic streaming and pseudopod production are not blocked. In conclusion it is now clear that the state of actin required for focal contact production is different from that required for pseudopod extension and cytoplasmic streaming.

Naegleria lovaniensis tarasca new subspecies, and the purepecha strain, a morphological variant of N. l. lovaniensis, isolated from natural thermal waters in Mexico.

Amoebae were isolated from a natural thermal water source in Michoacán, Mexico, in September 1986. Two 500-ml samples were taken from pools with water at 45 degrees C and 46 degrees C and concentrated at 2,000 g for 15 min. The sediment was seeded on nonnutritive agar plates and incubated at 42 degrees C. The isolates were axenized in bactocasitone-serum medium. The identification of the isolates was based on their morphology, total protein and isoenzyme patterns by agarose isoelectric focusing, serology, fine structure, agglutination with Concanavalin A, sensitivity to trimethoprim, capacity to kill mice, and their cytopathic effect in Vero cells. The results showed several morphophysiological, biochemical and serological differences between the isolates and the type strain Aq/9/1/45D of Naegleria lovaniensis. These remarkable differences provide sufficient evidence to consider one of the isolates a new subspecies, and the other one a morphological variant of N. l. lovaniensis, which can be differentiated from other Naegleriae by their morphology, biochemistry, serology and physiology. The authors propose the name tarasca for the subspecies and purepecha for the morphological variant.

Cytopathic action of Naegleria fowleri amoebae on rat neuroblastoma target cells.

The axenically cultured, weakly pathogenic Naegleria fowleri LEE and the highly pathogenic, mouse passaged N. fowleri LEEmp are cytopathic for B103 rat nerve cells in culture. Cytopathogenicity was measured by release of radiolabeled rubidium or radiolabeled chromium from B103 target cells. Cytopathogenicity was time-dependent for up to 18 h and dependent upon amoebae effector to nerve cell target ratios of less than 1:1. Release of 51Cr from B103 cells by either LEE or LEEmp amoebae was enhanced by addition of calcium or magnesium to medium free of these divalent cations but the ion-channel inhibitor, verapamil, or the ionophore A23187 and phorbol myristate acetate did not alter release of 51Cr from B103 cells cocultured with the amoebae. Cycloheximide or actinomycin D impaired release of 51Cr from B103 target cells injured by either LEE or LEEmp amoebae. Both strains of amoebae were fractionated by glass bead disruption and high speed centrifugation into membrane and soluble fractions. Each fraction was incubated with either 86Rb or 51Cr labeled nerve cells. The membrane fraction from LEEmp was more active than the soluble fraction in facilitating rubidium and chromium release. In contrast, the soluble fraction from LEE was more active than the membrane fraction in facilitating rubidium release from radiolabeled target cells. The sequential release of 86Rb and 51Cr from target cells rather than the simultaneous release of the two isotopes indicates that target cell death is due to the release of ions followed later by the release of large macromolecules. The results indicate that N. fowleri amoebae injure nerve cells by two alternate mechanisms, trogocytosis or contact-dependent lysis.

Resistance of highly pathogenic Naegleria fowleri amoebae to complement-mediated lysis.

Weakly pathogenic and nonpathogenic Naegleria spp. are readily lysed by human and guinea pig complement. Highly pathogenic Naegleria fowleri are resistant to complement-mediated lysis. Electrophoretic analysis of normal human serum (NHS) incubated with pathogenic or nonpathogenic Naegleria spp. demonstrates that amoebae activate the complement cascade, resulting in the production of C3 and C5 complement cleavage products. To determine whether surface constituents play a role in resistance to complement lysis, trophozoites of Naegleria spp. were subjected to enzymatic treatments prior to incubation in NHS. Treatment of trophozoites with papain or trypsin for 1 h, but not with neuraminidase, increased susceptibility of highly pathogenic Naegleria fowleri to complement lysis. Treatment of trophozoites with actinomycin D or cycloheximide during incubation with NHS or pretreatment with various protease inhibitors for 4 h did not increase the susceptibility of N. fowleri amoebae to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appears to account for the increased resistance of N. fowleri amoebae to complement-mediated lysis.

An in vitro comparative study on the effect of amphotericin B, econazole, and 5-fluorocytosine on Naegleria fowleri, Naegleria australiensis, and Naegleria australiensis s.sp. italica.

We tested in vitro amphotericin B (AMP-B), econazole (ECO), and 5-fluorocytosine (5-FC) on pathogenic Naegleria fowleri (KUL strain), Naegleria australiensis s.sp. italica (AB-T-F3, original strain) to assess their sensitivity to chemotherapeutic compounds. Previous reports have shown the polyene antibiotic AMP-B to be the most active agent. It was, however, much more active on N. fowleri than on N. australiensis and N. australiensis s.sp italica. 5-FC and ECO gave rise to non appreciable effect at non-toxic corresponding dosages in vivo. The results of these in vitro tests are discussed.

Effects of cyclophosphamide and a metabolite, acrolein, on Naegleria fowleri in vitro and in vivo.

Mice challenged intranasally with Naegleria fowleri died of primary amoebic meningoencephalitis. Mice given 30 mg of cyclophosphamide per kg of body weight daily for 10 days starting 2 days before challenge were protected. Neither cyclophosphamide nor serum from cyclophosphamide-treated mice inhibited N. fowleri in vitro. A metabolic product of cyclophosphamide, acrolein, inhibited growth and enflagellation of N. fowleri. Acrolein at 40 microM was amoebicidal. Acrolein injured starved cells and amoebae at 5 degrees C and growing N. fowleri.

New antiamebic acridines. II. Synthesis and DNA binding of a series of 9-acridanones and 9-iminoacridines.

A series of novel 9-acridanones and 9-iminoacridines has been prepared and investigated by a number of spectroscopic techniques in order to determine the nature and extent of the binding of these compounds to DNA. Results are discussed with reference to antiamebic activity in vitro.