Increasing the fungicidal action of Amphotericin B by inhibiting the Nitric Oxide-Dependent tolerance pathway

Amphotericin B (AmB) induces oxidative and nitrosative stresses, characterized by production of reactive oxygen and nitrogen species, in fungi. Yet, how these toxic species contribute to AmB-induced fungal cell death is unclear. We investigated the role of superoxide and nitric oxide radicals in AmB's fungicidal activity in Saccharomyces cerevisiae, using a digital microfluidic platform, which enabled monitoring individual cells at a spatiotemporal resolution, and plating assays. The nitric oxide synthase inhibitor L-NAME was used to interfere with nitric oxide radical production. L-NAME increased and accelerated AmB-induced accumulation of superoxide radicals, membrane permeabilization, and loss of proliferative capacity in S. cerevisiae. In contrast, the nitric oxide donor S-nitrosoglutathione inhibited AmB's action. Hence, superoxide radicals were important for AmB's fungicidal action, whereas nitric oxide radicals mediated tolerance towards AmB. Finally, also the human pathogens Candida albicans and Candida glabrata were more susceptible to AmB in the presence of L-NAME, pointing to the potential of AmB-L-NAME combination therapy to treat fungal infections.Kim Vriens acknowledges the receipt of a predoctoral grant from the Flanders Innovation & Entrepeneurship Agency (IWT-SB 111016); Karin Thevissen acknowledges the receipt of a mandate of Industrial Research Fund (KU Leuven). In addition, the research leading to these results has received funding from the Research Foundation - Flanders (FWO G086114N and G080016N) and the KU Leuven (OT 13/ 058 and IDO 10/012, IOF KP/12/009 Atheromix, IOF KP/ 12/002 Nanodiag). This work was partially developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Belém Sampaio-Marques is supported by the fellowship SFRH/BPD/90533/2012 funded by Fundação para a Ciência e Tecnologia (FCT, Portugal).info:eu-repo/semantics/publishedVersio

The plant decapeptide OSIP108 can alleviate mitochondrial dysfunction induced by cisplatin in human cells

We investigated the effect of the Arabidopsis thaliana-derived decapeptide OSIP108 on human cell tolerance to the chemotherapeutic agent cisplatin (Cp), which induces apoptosis and mitochondrial dysfunction. We found that OSIP108 increases the tolerance of HepG2 cells to Cp and prevents Cp-induced changes in basic cellular metabolism. More specifically, we demonstrate that OSIP108 reduces Cp-induced inhibition of respiration, decreases glycolysis and prevents Cp-uptake in HepG2 cells. Apart from its protective action against Cp in human cells, OSIP108 also increases the yeast Saccharomyces cerevisiae tolerance to Cp. A limited yeast-based study of OSIP108 analogs showed that cyclization does not severely affect its activity, which was further confirmed in HepG2 cells. Furthermore, the similarity in the activity of the D-stereoisomer (mirror image) form of OSIP108 with the L stereoisomer suggests that its mode of action does not involve binding to a stereospecific receptor. In addition, as OSIP108 decreases Cp uptake in HepG2 cells and the anti-Cp activity of OSIP108 analogs without free cysteine is reduced, OSIP108 seems to protect against Cp-induced toxicity only partly via complexation. Taken together, our data indicate that OSIP108 and its cyclic derivatives can protect against Cp-induced toxicity and, thus, show potential as treatment options for mitochondrial dysfunction- and apoptosis-related conditions

Embry-Riddle Fly Paper 1943-01-15

https://commons.erau.edu/fly-paper/1159/thumbnail.jp

Identification of survival-promoting OSIP108 peptide variants and their internalization in human cells

The plant-derived decapeptide OSIP108 increases tolerance of yeast and human cells to apoptosis-inducing agents, such as copper and cisplatin. We performed a whole amino acid scan of OSIP108 and conducted structure-activity relationship studies on the induction of cisplatin tolerance (CT) in yeast. The use of cisplatin as apoptosis-inducing trigger in this study should be considered as a tool to better understand the survival-promoting nature of OSIP108 and not for purposes related to anti-cancer treatment. We found that charged residues (Arg, His, Lys, Glu or Asp) or a Pro on positions 4–7 improved OSIP108 activity by 10% or more. The variant OSIP108[G7P] induced the most pronounced tolerance to toxic concentrations of copper and cisplatin in yeast and/or HepG2 cells. Both OSIP108 and OSIP108[G7P] were shown to internalize equally into HeLa cells, but at a higher rate than the inactive OSIP108[E10A], suggesting that the peptides can internalize into cells and that OSIP108 activity is dependent on subsequent intracellular interactions. In conclusion, our studies demonstrated that tolerance/survival-promoting properties of OSIP108 can be significantly improved by single amino acid substitutions, and that these properties are dependent on (an) intracellular target(s), yet to be determined

Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa.

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies

Identification and characterization of novel compounds that can alleviate mitochondrial dysfunction using yeast as a model

Mitochondrial dysfunction is related to various human pathologies including inborn errors of mitochondrial metabolism such as Leber s hereditary optic neuropathy, age-related neurodegenerative disorders such as Alzheimer s disease and Parkinson s disease but also rare diseases such as Wilson disease and Niemann Pick disease type C1. In addition, mitochondrial dysfunction is not exclusively related to inborn/acquired mutations, but can also be caused by for instance drugs such as the chemotherapeutic agents cisplatin (Cp) or excess copper (Cu) as is the case for Wilson disease. Wilson disease is characterized by Cu accumulation in the liver, leading to acute liver failure or cirrhosis, but also causing neurodegeneration. Next to Cu-induced mitochondrial dysfunction, excess Cu is known to induce oxidative stress and apoptosis. In addition, mitochondrial dysfunction-related conditions are also often characterized by aberrancies in sphingolipid metabolism, a class of lipid species that are important membrane constituents, but also have crucial signaling roles in orchestrating a wide range of biological processes. Current treatment options for mitochondrial dysfunction-related disorders, however, are inadequate, emphasizing the need for novel treatment options. The major goal of this PhD was to identify and characterize novel compounds that can alleviate mitochondrial dysfunction upon treatment with excess Cu or Cp, thereby using the budding yeast Saccharomyces cerevisiae as a model eukaryote. To this end, we focused on the bioactive Arabidopsis thaliana-derived decapeptide OSIP108, which is characterized by anti-oxidant activity, and on a repositioning library (Pharmakon 1600 Repositioning library) that comprises 1600 off-patent drugs and bioactive agents. Such repositioned drugs are characterized by a well-known toxicity profile and mode of action of their primary aimed activity. This work shows that the OSIP108 peptide can prevent Cu-induced oxidative stress and apoptosis in yeast and human cells, and this effect is likely mediated by an effect on sphingolipid metabolism. Subsequently, we successfully translated these data to relevant in vitro and in vivo models applicable to Wilson disease. To this end we show that OSIP108 prevents Cu-induced cell death of human neuroglioblastoma cells and decreases Cu-induced changes in liver morphology (indicative for liver damage) of zebrafish larvae. Furthermore, we extrapolated our analysis of the bioactive properties of OSIP108 to Cp-induced toxicity, as we describe the protective effect of OSIP108 against Cp-induced toxicity in yeast and human cells, as well as a direct effect on basic cellular metabolism in human cells. Moreover, we describe the screening of the Pharmakon 1600 Repositioning library for agents that can prevent Cu-induced toxicity in yeast, resulting in the identification of the drug class of Angiotensin II Type 1 Receptor blockers. Moreover, in line with our research on OSIP108, we show that several Angiotensin II Type1 Receptor blockers can prevent Cu-induced toxicity in S. cerevisiae, as well as Cp-induced toxicity. In summary, the results of this PhD research indicate that OSIP108 and specific Angiotensin II Type 1 Receptor blockers show promise as therapeutic options in treatment of Wilson disease, or of mitochondrial dysfunction-related disorders in general. Furthermore, not only does our research shed light onto sphingolipids as a direct target in treatment of Wilson disease, knowledge of the pro-survival mode of action of OSIP108 and the Angiotensin II Type 1 Receptor blockers in S. cerevisiae will likely reveal putative novel therapeutic targets for treatment of human mitochondrial dysfunction-related disorders.status: publishe

Sphingolipids and mitochondrial function, lessons learned from yeast

Background: Sphingolipids (SLs) are key components of cellular membranes, but also play an important role as signaling molecules in orchestrating both cell growth and apoptosis. In Saccharomyces cerevisiae, three complex SLs are present and hydrolysis of either of these species is catalyzed by the inositol phosphosphingolipid phospholipase C (Isc1p). Strikingly, mutants deficient in Isc1p display several hallmarks of mitochondrial dysfunction such as the inability to grow on a non-fermentative carbon course, increased oxidative stress sensitivity and aberrant mitochondrial morphology. Scope of Review: In this review, we focus on the pivotal role of Isc1p in regulating mitochondrial function via SL metabolism, and on Sch9p as central signal transducer. Sch9p is one of the main effectors of the target of rapamycin complex 1 (TORC1), which is regarded as a crucial signaling axis for regulation of Isc1p-mediated events. Finally, we describe the retrograde response, a signaling event originating from dysfunctional mitochondria to the nucleus that results in the induction of nuclear target genes. Intriguingly, the retrograde response is associated with the induction of SL biosynthetic genes. Major Conclusions: All the above suggests a pivotal signaling role for SLs in maintaining correct mitochondrial function in budding yeast. General Significance: Studies with budding yeast provide insight on SL signaling events that affect mitochondrial function.status: publishe

Sphingolipids and mitochondrial function, lessons learned from yeast

Mitochondrial dysfunction is a hallmark of several neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, but also of cancer, diabetes and rare diseases such as Wilson’s disease (WD) and Niemann Pick type C1 (NPC). Mitochondrial dysfunction underlying human pathologies has often been associated with an aberrant cellular sphingolipid metabolism. Sphingolipids (SLs) are important membrane constituents that also act as signaling molecules. The yeast Saccharomyces cerevisiae has been pivotal in unraveling mammalian SL metabolism, mainly due to the high degree of conservation of SL metabolic pathways. In this review we will first provide a brief overview of the major differences in SL metabolism between yeast and mammalian cells and the use of SL biosynthetic inhibitors to elucidate the contribution of specific parts of the SL metabolic pathway in response to for instance stress. Next, we will discuss recent findings in yeast SL research concerning a crucial signaling role for SLs in orchestrating mitochondrial function, and translate these findings to relevant disease settings such as WD and NPC. In summary, recent research shows that S. cerevisiae is an invaluable model to investigate SLs as signaling molecules in modulating mitochondrial function, but can also be used as a tool to further enhance our current knowledge on SLs and mitochondria in mammalian cells

Een 76-jarige vrouw met een anterieure mediastinale massa

info:eu-repo/semantics/publishe

Anti-apoptotic compounds

status: publishe