Identification and characterization of novel marine oxasqualenoid yucatecone against Naegleria fowleri.

Naegleria fowleri is an opportunistic protozoan, belonging to the free-living amoeba group, that can be found in warm water bodies. It is causative agent the primary amoebic meningoencephalitis, a fulminant disease with a rapid progression that affects the central nervous system. However, no 100% effective treatments are available and those that are currently used involve the appearance of severe side effects, therefore, there is an urgent need to find novel antiamoebic compounds with low toxicity. In this study, the in vitro activity of six oxasqualenoids obtained from the red algae Laurencia viridis was evaluated against two different strains of N. fowleri (ATCC® 30808 and ATCC® 30215) as well as their cytotoxicity against murine macrophages. Yucatecone was the molecule with the highest selectivity index (>2.98 and 5.23 respectively) and it was selected to continue with the cell death type determination assays. Results showed that yucatone induced programmed cell death like responses in treated amoebae causing DNA condensation and cellular membrane damage among others. In this family of oxasqualenoids, it seems that the most significative structural feature to induce activity against N. fowleri is the presence of a ketone at C-18. This punctual oxidation transforms an inactive compound into a lead compound as the yucatecone and 18-ketodehydrotyrsiferol with IC50 values of 16.25 and 12.70 µM, respectively. The assessment of in silico ADME/Tox analysis revealed that the active compounds showed good Human Oral Absorption and demonstrate that are found to be within the limit of approved drug parameter range. Hence, the study highlights promising potential of yucatone to be tested for therapeutic use against primary amoebic meningoencephalitis.

Ultraviolet - Chlorine combined treatment efficiency to eliminate Naegleria fowleri in artificial surf lagoons.

Naegleria. fowleri, a protozoa belonging to the free-living amoeba group, is the causative agent of a central nervous system affecting disease that is fatal in more than the 95% of the reported cases. This parasite can be found in warm water bodies such as lakes, rivers or inadequately disinfected swimming pools. On the other hand, chlorination and UV light treatment are two of the most extensively used disinfection methods in recreational water facilities. In this study the effect of chlorination and UV light on N. fowleri trophozoites was studied in a close water circuit with the aim to assess the efficacy of this disinfection methods in large pools. The obtained results showed that the chlorination was able to decrease the number of viable cells despite the elimination was not totally achieved. Nonetheless, the combination of the UV light with the chlorination allowed the complete removal of the N. fowleri trophozoites from the water in experimental testing conditions.

A Fluorometric Assay for the In Vitro Evaluation of Activity against Naegleria fowleri Cysts.

Primary amoebic meningoencephalitis (PAM) is a lethal and rapid infection that affects the central nervous system and is caused by the free-living amoeba Naegleria fowleri. The life cycle of this protozoa consists of three different stages: The trophozoite, flagellate and cyst stages. Currently, no fully effective molecules have been found to treat PAM. In the search of new antiamoebic molecules, most of the efforts have focused on the trophozoidal activity of the compounds. However, there are no reports on the effect of the compounds on the N. fowleri cyst viability. In the present study, the cysticidal activity of four different molecules was evaluated using an alamarBlue based fluorometric assay. All the tested compounds were active against the cyst stage of N. fowleri. In fact, all the molecules except the amphotericin B, showed highest activity toward the cyst stage than the trophozoite stage. This work could be an effective protocol to select molecules with cysticidal and trophozoidal activity that can be considered a future PAM treatment. IMPORTANCE In the search of new anti-Naegleria fowleri compounds, most of the works focus on the activity of different molecules against the trophozoite stage; however, none of them include the effect of those compounds on the cyst viability. This manuscript presents a solid and reliable assay to evaluate the activity of compounds against the cyst stage of N. fowleri.

Cyclolauranes as plausible chemical scaffold against Naegleria fowleri.

Primary amoebic meningoencephalitis (PAM) is a central nervous system (CNS) disease caused by Naegleria fowleri that mainly affects children and young adults with fatal consequences in most of the cases. Treatment protocols are based on the combination of different antimicrobial agents, nonetheless there is the need to develop new anti-Naegleria compounds with low toxicity and full effects compared to the currently used drug combination. The marine environment is a well-established source of bioactive natural products. In this work, we have focused on the structure of Laurencia cyclolaurane-type sesquiterpenes as potential chemical model against Naegleria species. The effects of debromolaurinterol (1) to induce PCD/apoptosis-like events in Naegleria fowleri have been evaluated, revealing that this compound induced reduction of ATP production showing a decrease of 99.98% in treated parasite cells. A SAR analysis have been supported with molecular modeling and analysis of the in silico ADME/Tox properties of the Laurencia sesquiterpenes debromolaurinterol (1), laurinterol (2) and allolaurinterol (3), which reinforce cyclolaurane metabolites as plausible molecular models to develop PAM treatments.

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.

Naphthyridine Derivatives Induce Programmed Cell Death in Naegleria fowleri.

Primary amoebic encephalitis (PAM) caused by the opportunistic pathogen Naegleria fowleri is characterized as a rapid and lethal infection of the brain which ends in the death of the patient in more than 90% of the reported cases. This amoeba thrives in warm water bodies and causes infection after individuals perform risky activities such as splashing or diving, mostly in non-treated water bodies such as lakes and ponds. Moreover, the infection progresses very fast and no fully effective molecules have currently been found to treat PAM. In this study, naphthyridines fused with chromenes or chromenones previously synthetized by the group were tested in vitro against the trophozoite stage of two strains of N. fowleri. In addition, the most active molecule was evaluated in order to check the induction of programmed cell death (PCD) in the treated amoebae. Compound 3 showed good anti-Naegleria activity (61.45 ± 5.27 and 76.61 ± 10.84 µM, respectively) against the two different strains (ATCC® 30808 and ATCC® 30215) and a good selectivity compared to the cytotoxicity values (>300 µM). In addition, it was able to induce PCD, causing DNA condensation, damage at the cellular membrane, reduction in mitochondrial membrane potential and ATP levels, and ROS generation. Hence, naphthyridines fused with chromenes or chromenones could be potential therapeutic agents against PAM in the near future.

The therapeutic potential of novel isobenzofuranones against Naegleria fowleri.

The Free-Living Amoeba species, Naegleria fowleri is the causative agent of a lethal encephalitis known as Primary Amoebic Encephalitis (PAM). Moreover, most of the reported cases are often related to swimming and/or diving in aquatic environments. In addition, the current therapeutic options against PAM are not fully effective and hence, there is an urgent need to develop novel therapeutic agents against this disease. Previously isobenzofuranones compounds have been reported to present antiprotozoal and antifungal activity among others. However, to the best of our knowledge, these molecules have not been previously tested against N. fowleri. Therefore, the aim of this study was to evaluate the activity of 14 novel isobenzofuranones against this pathogenic amoeba. The most active and less toxic molecules, were assayed in order to check induction of Programmed Cell Death (PCD) in the treated amoebae. The obtained results showed that these molecules were able to eliminate N. fowleri trophozoites and also induced PCD. Therefore, the tested isobenzofuranones could be potential therapeutic candidates for the treatment of PAM.

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.

Exploring the Anti-Infective Value of Inuloxin A Isolated from Inula viscosa against the Brain-Eating Amoeba (Naegleria fowleri) by Activation of Programmed Cell Death.

Primary amoebic meningoencephalitis (PAM), caused by the pathogenic free-living amoeba Naegleria fowleri, is a rare but fatal disease. Nowadays, no fully effective therapy is available to erradicate or prevent this disease. Natural products could constitute a promising source of useful bioactive compounds in drug discovery. The present study is a characterization of main active compounds from the ethanolic extract of Inula viscosa (Asteraceae) leaves against N. fowleri trophozoites. Four compounds (1-4) were successfully identified by spectroscopic techniques, but only inuloxin A displayed a potential antiamoebic activity with an IC50 of 21.27 µM. The specificity of this compound toward the studied strain leads us to analyze the insight into its mechanism of action by performing in vitro assays of programmed cell death markers and to discuss the structure-activity relationship (SAR). The obtained results demonstrated that inuloxin A interferes with various processes leading to membrane damage, mitochondria alteration, chromatin condensation, and ROS accumulation, which highlight features specific to apoptosis. The current findings could be a promising step for developing new effective drugs against PAM.

Free living amoebae isolation in irrigation waters and soils of an insular arid agroecosystem.

The use of freshwater in agricultural systems represents a high percentage of total water consumption worldwide. Therefore, alternative sources of water for irrigation will need to be developed, particularly in arid and semi-arid areas, in order to meet the growing demand for food in the future. The use of recycled wastewater (RWW), brackish water (BW) or desalinated brackish water (DBW) are among the different non-conventional water resources proposed. However, it is necessary to evaluate the health risks for humans and animals associated with the microbiological load of these waters. Protozoa such as free-living amoebae (FLA) are considered an emerging group of opportunistic pathogens capable to cause several diseases in humans (e.g. cutaneous and ocular infections, lung, bone or adrenal gland conditions or fatal encephalitis). In the present study we evaluate FLA presence in three different irrigation water qualities (RWW, BW and DBW) and its survival in irrigated agricultural soils of an extremely arid insular ecosystem (Fuerteventura, Canary Islands, Spain). Samples were cultured on 2% Non-Nutrient Agar (NNA) plates covered with a thin layer of heat killed E. coli and checked daily for the presence of FLA. According to the prevalence of FLA, Vermamoeba vermiformis (53,8%), Acanthamoeba spp. (30,8%), Vahlkampfia avara (7,7%) and Naegleria australiensis (7,7%) were detected in the analysed water samples, while Acanthamoeba (83,3%), Cercozoa spp. (8,3%) and Vahlkampfia orchilla (8,3%) were isolated in irrigated soils. Only Acanthamoeba strains were isolated in no irrigated soils used as control, evidencing the capability of these protozoa to resist environmental harsh conditions. Additionally, all analysed water sources and the irrigated soils presented growth of several pathogenic bacteria. Therefore, the coexistence in water and soils of pathogenic bacteria and FLA, can mean an increased risk of infection in agroecosystems.

Laurinterol from Laurencia johnstonii eliminates Naegleria fowleri triggering PCD by inhibition of ATPases.

Primary amoebic encephalitis (PAM) is a lethal disease caused by the opportunistic pathogen, Naegleria fowleri. This amoebic species is able to live freely in warm aquatic habitats and to infect children and young adults when they perform risk activities in these water bodies such as swimming or splashing. Besides the need to increase awareness of PAM which will allow an early diagnosis, the development of fully effective therapeutic agents is needed. Current treatment options are amphotericin B and miltefosine which are not fully effective and also present toxicity issues. In this study, the in vitro activity of various sesquiterpenes isolated from the red alga Laurencia johnstonii were tested against the trophozoite stage of a strain of Naegleria fowleri. Moreover, the induced effects (apoptotic cell death) of the most active compound, laurinterol (1), was evaluated by measuring DNA condensation, damages at the mitochondrial level, cell membrane disruption and production of reactive oxygen species (ROS). The obtained results demonstrated that laurinterol was able to eliminate the amoebae at concentrations of 13.42 ± 2.57 µM and also to induced programmed cell death (PCD) in the treated amoebae. Moreover, since ATP levels were highly affected and laurinterol has been previously reported as an inhibitor of the Na+/K+-ATPase sodium-potassium ion pump, comparison with known inhibitors of ATPases were carried out. Our results points out that laurinterol was able to inhibit ENA ATPase pump at concentrations 100 times lower than furosemide.

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.

Evaluation of Indolocarbazoles from Streptomyces sanyensis as a Novel Source of Therapeutic Agents against the Brain-Eating Amoeba Naegleria fowleri.

Naegleria fowleri is an opportunistic pathogenic free-living amoeba which is able to rapidly colonize the central nervous system (CNS) and causes a lethal infection known as primary amoebic meningoencephalitis (PAM). Furthermore, more than 98% of the known cases of PAM are fatal and affect mainly children under 12 and young adults. Until now, no fully effective therapeutic agents against N. fowleri are available and hence the urgent need to find novel agents to treat PAM. At present, PAM therapy is based on the combination of amphotericin B, miltefosine, among others, with unwanted toxic effects. Recently, our team isolated various indolocarbazoles (ICZs) from the culture of a mangrove strain of Streptomyces sanyensis which showed activity against kinetoplastids and the Acanthamoeba genus. Hence, in this study, the activity of the previously isolated ICZs, staurosporine (STS), 7-oxostaurosporine (7OSTS), 4'-demethylamino-4'-oxostaurosporine, and streptocarbazole B, was evaluated against two type strains of N. fowleri. Furthermore, the performed activity assays revealed that STS was the most active ICZ presenting an inhibitory concentration 50 (IC50) of 0.08 ± 0.02 µM (SI 109.3). Moreover, STS induced programmed cell death (PCD) in the treated amoebae by triggering DNA condensation, mitochondrial disfunction, cell membrane disruption, and reactive oxygen species (ROS) generation. Therefore, STS could be a promising therapeutic agent against PAM.

Silver Nanoparticles as a Novel Potential Preventive Agent against Acanthamoeba Keratitis.

Free living, cosmopolitan amoebae from Acanthamoeba genus present a serious risk to human health. As facultative human parasites, these amoebae may cause Acanthamoeba keratitis (AK). Acanthamoeba keratitis is a severe, vision-threatening corneal infection with non-specific symptoms. The number of reported AK cases worldwide has been increasing every year. Moreover, 90% of Acanthamoeba keratitis cases are related to contact lens use. Wearing and storage contact lenses not in accordance with the physicians and manufacturers recommendations are the primary key risk factors of this disease. Amoebae can easily adhere to the contact lens surface and transmit to the corneal epithelium. Preventing amoebae adhesion to the contact lens surface could significantly decrease the number of AK infections. Until now, the effective therapy against AK is still under development. Currently proposed therapies are mainly limited to the chlorhexidine digluconate combined with propamidine isethionate or hexamidine applications, which are insufficient and very toxic to the eye. Due to lack of effective treatment, looking for new potential preventive agents is crucial to decrease the number of Acanthamoeba keratitis infections, especially among contact lens users. Nanoparticles have been already included in several novel therapies against bacteria, viruses, fungi, and protist. However, their anti-amoebic potential has not been fully tested yet. The aim of this study was to assess silver nanoparticles (AgNPs) and platinum nanoparticles (PtNPs) anti-amoebic activity and influence on the amoebae adhesion to the surface of four different groups of contact lenses-classified according to the Food and Drugs Administration (FDA) guidelines. The obtained results show that both tested nanoparticles were effective against Acanthamoeba trophozoites and decreased the amoebae adhesion to the contact lens surface. AgNPs showed better anti-amoebic activity to cytotoxicity dependence and reduced amoebae adhesion in a wider spectrum of the tested contact lenses. Our studies also confirmed that ionization next to hydration of the contact lens material is a crucial parameter influencing the Acanthamoeba adhesion to the contact lens surface. In conclusion, silver nanoparticles might be considered as a novel preventive agent against Acanthamoeba keratitis infection.

In vitro activity of 1H-phenalen-1-one derivatives against Leishmania spp. and evidence of programmed cell death.

BACKGROUND: The in vitro activity against Leishmania spp. of a novel group of compounds, phenalenone derivatives, is described in this study. Previous studies have shown that some phenalenones present leishmanicidal activity, and induce a decrease in the mitochondrial membrane potential in L. amazonensis parasites, so in order to elucidate the evidence of programmed cell death occurring inside the promastigote stage, different assays were performed in two different species of Leishmania. METHODS: We focused on the determination of the programmed cell death evidence by detecting the characteristic features of the apoptosis-like process, such as phosphatidylserine exposure, mitochondrial membrane potential, and chromatin condensation among others. RESULTS: The results showed that four molecules activated the apoptosis-like process in the parasite. All the signals observed were indicative of the death process that the parasites were undergoing. CONCLUSIONS: The present results highlight the potential use of phenalenone derivatives against Leishmania species and further studies should be undertaken to establish them as novel leishmanicidal therapeutic agents.

Withanolides from Withania aristata as Antikinetoplastid Agents through Induction of Programmed Cell Death.

Leishmaniasis and American trypanosomiasis are parasitic diseases that cause significant clinical, social and economic impact on the population of tropical and subtropical countries. Their current treatment is limited and presents multiple drawbacks, including high toxicity, high cost, lengthy treatment plans, as well as the emergence of resistant species. Therefore, there is a need to find new lead compounds with high potency against parasites and low toxicity in patients. In the present work, the bioguided fractionation of an endemic plant from the Canary Islands, Withania aristata, led to the identification of withanolide-type metabolites (1-3) with leishmanicidal and trypanocidal activities. Compounds 1 and 3 showed a significant dose-dependent inhibition effect on the proliferation of L. amazonensis promastigotes and T. cruzi epimastigotes, higher than the reference drugs, miltefosine and benznidazole, respectively. Moreover, compounds 1-3 were more potent (IC50 0.055-0.663 µM) than the reference drug against the intracellular amastigote stage of L. amazonensis, with a high selectivity index on murine macrophage cells (SI 58.66-216.73). Studies on the mechanism of death showed that the compounds induced programmed cell death or that which was apoptosis-like. The present findings underline the potential of withanolides as novel therapeutic antikinetoplastid agents.

In Vitro Activity of Statins against Naegleria fowleri.

Naegleria fowleri causes a deadly disease called primary amoebic meningoencephalitis (PAM). Even though PAM is still considered a rare disease, the number of reported cases worldwide has been increasing each year. Among the factors to be considered for this, awareness about this disease, and also global warming, as these amoebae thrive in warm water bodies, seem to be the key factors. Until present, no fully effective drugs have been developed to treat PAM, and the current options are amphotericin B and miltefosine, which present side effects such as liver and kidney toxicity. Statins are able to inhibit the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is a key enzyme for the synthesis of ergosterol of the cell membrane of these amoebae. Therefore, the in vitro activity of a group of statins was tested in this study against two types of strains of Naegleria fowleri. The obtained results showed that fluvastatin was the most effective statin tested in this study and was able to eliminate these amoebae at concentrations of 0.179 ± 0.078 to 1.682 ± 0.775 µM depending on the tested strain of N. fowleri. Therefore, fluvastatin could be a potential novel therapeutic agent against this emerging pathogen.

Ursolic Acid Derivatives as Potential Agents Against Acanthamoeba Spp.

The current chemotherapy of Acanthamoeba keratitis relies on few drugs with low potential and limited efficacy, for all this there is an urgent need to identify new classes of anti-Acanthamoeba agents. In this regard, natural products play an important role in overcoming the current need and medicinal chemistry of natural products represents an attractive approach for the discovery and development of new agents. Ursolic acid, a natural pentacyclic triterpenoid compound, possesses a broad spectrum of activities including anti-Acanthamoeba. Herein, we report on the development by chemical transformation of an ursolic acid-based series of seven compounds (2-8), one of them reported for the first time. The structure-activity relationship (SAR) analysis of their anti-Acanthamoeba activity revealed that acylation/ether formation or oxidation enhances their biological profile, suggesting that the hydrophobic moiety contributes to activity, presumably by increasing the affinity and/or cell membrane permeability. These ursolic acid derivatives highlight the potential of this source as a good base for the development of novel therapeutic agents against Acanthamoeba infections.

In Vitro Evaluation of Combined Commercialized Ophthalmic Solutions Against Acanthamoeba Strains.

Acanthamoeba is a free-living amoebae genus which is present worldwide in natural and artificial environments. These amoebae are clinically important as causative agents of diseases in humans and other animals such as a fatal encephalitis or a sight threatening Acanthamoeba keratitis (AK). Lately; studies have focused on the search of novel therapeutic options for AK but also to prevent infections. Furthermore; the evaluation of commercialized products seems to be an option for this case since not clinical assays would be required. Thus; we aimed to test the amoebicidal activity of different mixtures of two commercial ophthalmic solutions: Systane® Ultra; which has already shown anti-Acanthamoeba properties; and Naviblef® Daily Care. In addition, we tested their cytotoxic effect against murine macrophages. At the individual level; Naviblef® Daily Care showed to be the most active product against Acanthamoeba spp. Nevertheless; the combinations of Systane® Ultra and Naviblef® Daily Care; showed an improvement in the activity against trophozoites and cysts of Acanthamoeba castellanii Neff. Moreover; the concentration necessary to generate cytotoxic effect against murine macrophages (J774.1) was much higher than the required for the amoebicidal and cysticidal effect achieved in the most effective mixtures.

Screening of the pathogen box for the identification of anti-Acanthamoeba agents.

Acanthamoeba are free living amoeba that have been isolated from different environments like soil, water, air dust. Moreover, they are also able to act as opportunist pathogens, mainly causing a fatal encephalitis and also keratitis in both human and animals. This study was aimed to evaluate the activity of the Medicines for Malaria Venture (MMV) compounds against the trophozoite stage of Acanthamoeba castellanii Neff. Sixteen compounds showed ≥90% inhibition of parasite growth in the initial screen (10 µM). Those set were further evaluated to determine the inhibitor concentration that inhibit the 50% of the initial population and cytotoxicity against murine macrophages. Among the compounds included in the pathogen box, pentamidine and posaconazole were the most effective against this parasite with an of IC50 of 0.567 ±â€¯0.04 and 0.630 ±â€¯0.11, respectively.