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Identifier 000460430
Title ¨Ελεγχος της αντιλεϊσμανιακής δράσης νέων αιθερικών φωσφολιπιδίων : Μελέτη του μηχανισμού-στόχου των δραστικών ουσιών και του μηχανισμού ανάπτυξης της αντοχής του παρασίτου / Ειρήνη Φραγκιαδάκη.
Alternative Title Screening of the antileishmanial action of new ether lipid analogues
Author Φραγκιαδάκη, Ειρήνη
Thesis advisor Σκουλίκα, Ευσταθία
Reviewer Τσιώτης, Γεώργιος
Καλογεροπούλου, Θεοδώρα
Abstract Leishmaniasis, caused by the protozoan parasite Leishmania is considered one of the most important tropical parasitic diseases, affecting 12 million people worldwide1. The serious problems associated with the treatment of leishmaniasis, such as the development of resistance to available drugs, the need for low-cost treatments combined with the low funding for the study of the treatment of parasitic diseases, have prompted several research groups to synthesize new anti-leishmanial compounds based on modifications of already existing drugs2-7. Despite the fact that more than three decades have passed since the discovery of the leishmanicidal action of miltefosine, the first orally administered anti-leishmanial drug8, the knowledge of the structure-activity relationship of phospholipid analogues is relatively limited9. All the above make the field of designing and synthesizing new antileishmanial phospholipid analogues a continuous challenge, leading to the production of miltefosine analogues, whose biological activity was evaluated in this thesis. Specifically, 55 ether phospholipid analogues were studied in terms of their antiparasitic activity, in the promastigote and intracellular amastigte form of the parasite. Additionally, the cytotoxic activity of the analogues against the THP1 human macrophage cell line, as well as their hemolytic activity, was studied. The majority of the compounds were more active than miltefosine in both laboratory strains used. In addition, significantly improved cytotoxic/hemolytic activity was also observed, thus highlighting the structural characteristics that influence their properties10-13. Various studies have reported differences in the response of laboratory and clinical strains to antiparasitic drugs, which can be attributed to variations in the mechanisms that mediate drug uptake and/or the interaction with cellular targets14, 15. For this reason, the activity of selected analogues was evaluated against clinical strains isolated from dogs suffering from visceral leishmaniasis (kala azar), highlighting significant differences in the response of the clinical strains to the analogues used. However, no correlation was observed between the above differences and the existence of polymorphisms in the LdMT (Leishmania donovani Miltefosine Transporter) ATP-ase gene, which is responsible for reduced uptake of the drug and the development of resistance to miltefosine16. Therefore, it can be concluded that the action of the analogues and the final antiparasitic effect is determined by the combination of the chemical structure of the compound as well as the biotypic characteristics of each strain. Understanding the mode of action of a substance is crucial for the identification of potential drug targets and design of new therapeutic strategies. Therefore, we studied the mechanisms involved in the antiparasitic activity of the analogues. Cellular phenomena such as autophagy, apoptosis, loss of mitochondrial membrane polarization and induction of oxidative stress were studied, using selected active analogues. Additionally, the intracellular distribution of the drugs was studied using fluorescent analogues. During the assessment of the action of the analogues in the intracellular amastigote form, the existence of numerous vesicles was microscopically observed within the infected macrophages, but also within the parasitophorous vacuole, which was further confirmed by electron microscopy. In order to study whether the above vesicles are involved in the parasiticidal effect, through the induction of the autophagic process, staining of the vacuoles was performed using the specific marker for autophagy Monodancyl Cadaverine (MDC) and antibodies against the LAMP protein (Lysosome Associated Membrane Protein)17, 18. We were able to detect significant differences in the autophagic vesicle flux and autophagic vesicle maturation after exposure to selected active analogues, regarding the addition of chemical groups to the lipid chain of miltefosine. Trifluralin and triazole analogues induced a marked increase in autophagic flux in infected macrophages, in contrast to miltefosine and analogues TC19 and TCAN26 that contain other types of rings. Because the above assays do not directly correlate the final parasiticidal effect with the induction of phagosome and autophagosome maturation, the induction of apoptosis of infected macrophages after exposure to miltefosine and selected analogues was studied. Using the fluorescent protein Annexin V and propidium iodide on the one hand19, 20, and the mitochondrial homeostasis marker MitoTracker Red Dye on the other hand21, 22, we demonstrated the effect of miltefosine and phospholipid analogues on mitochondrial function, apoptosis and death in infected macrophages. We showed that miltefosine, at concentration x7.5 of the IC50 against the intracellular amastigote, kills ~78% of infected cells in contrast to TC95 analogue, where the respective percentage is <10%. The result of the strong cytotoxicity of miltefosine is reflected in the drastic reduction of mitochondrial function and the total loss of membrane potential. This effect is dose-dependent and can only be reduced at subtherapeutic concentrations in terms of the paraciticidal effect. In contrast, other analogues such as TC95, at concentrations many times higher than the IC50 do not affect host cell homeostasis. In addition, the mitochondrion functions as an important source for the production of reactive oxygen species (ROS) within cells, and specifically it has been suggested that miltefosine acts through programmed cell death associated with ROS production23. In our study, increased Nitric Oxide (NO) ion production was observed during the first hours of incubation with selected analogues, accompanied by a low survival rate of promastigotes. The above results raised the question of the intracellular distribution of alkylphophocholines in the promastigote and in the host cell. Three fluorescent analogues were used, containing triazole and adamantinyl rings in the lipid chain. Two of them had a strong fluorescence that allowed tracking under the fluorescence microscope. Cytoplasmic accumulation of fluorescence was observed in regions identical with the mitochondrial dye Mitotracker Red Dye, thus strengthening the hypothesis that the inhibition of mitochondrial functions caused by miltefosine is due to its accumulation in this specific promastigote organelle. However, the question of the distribution of analogues with reduced cytotoxicity is under consideration. Because the in vitro results were encouraging for some analogues, especially those containing trifluralin ring, we proceeded to study the in vivo effect in dogs with active kala azar. We applied the approved miltefosine treatment protocol to a dog receiving TC106, an analogue with similar characteristics to TC95 but less cytotoxic. The comparison was made with a dog that received the veterinary drug Milteforan®. The results were particularly encouraging in terms of the reduction of symptoms and the significant improvement of the clinical picture. The animal treated with TC106 had stable hematological and hepatic markers during treatment, significantly improved compared to the Milteforan®-treated animal. Most importantly, the dog that received the experimental drug did not experience side effects such as irritability, vomiting and diarrhea that are often seen with miltefosine treatment. Despite the interesting clinical results, the parasite was detectable in the lymph nodes of both dogs at least 40 days after the completion of treatment. Taking into account the above findings in the dog combined with the reduced effectiveness of the available treatments observed in recent years also in humans, the use of combination therapy to combat kala azar is becoming more and more common, as the synergistic action of the drugs leads to remission of the disease and reduction of relapses24-27. For this reason, we attempted to exploit the potential synergistic effect of a selected analogue and miltefosine, in combination with a class of antimicrobial peptides, called peptaibols. It was observed that the combination of peptaibols with the drugs had a superadditive effect against the intracellular parasite, induced changes in promastigote redox levels, led to mitochondrial membrane potential fragmentation and affected mitochondrial morphology. Therefore, the synergy observed between phopholipids and peptaibols leads to an improvement of the antiparasitic action and thereby to improvement of the therapeutic effect28. Failure to eradicate the pathogen from the clinical site of infection is often, due to the phenotypic development of drug-resistant/tolerant strains. We aimed to study possible mechanisms of adaptation of the parasite to alkylphosphocholines, with the ultimate goal of understanding the tolerant populations and finding new treatment targets for effective parasite eradication. We cultured L. donovani strain in subtherapeutic doses of miltefosine and the analogue TC19, and analyzed protein expression using proteomics. We reconstructed the main mechanisms of energy production, lipid metabolism and transport of alkylphosphocholines in the promastigote. We found similarities in the induction of expression of central metabolic machinery between the two analogues, such as biosynthesis of sterols and long-chain fatty acids and oxidative phosphorylation in mitochondria. However, we also found significant differences in energy production from glycolysis and fatty acid catabolism in mitochondria through β-oxidation. We also found that the two chemical substances do not use the same mechanism of entry into the cytoplasm. In conclusion, many miltefosine analogues have greatly improved characteristics in terms of effect against the parasite and the host cell. A deeper study of their ability to use mechanisms to remove the parasite from the host is important, to understand the effective mode of action. The study of the survival mechanisms of the parasite in vivo is critical for the identification of new pharmaceutical targets and the application of synergistic treatments for effective eradication of the parasite
Language Greek, English
Subject Autophagy
Canine therapy
Drug synergy
Leishmaniasis
Mechanism of action
Miltefosine analoques
Mitochondria
Oxidative stress
Ανάλογα μιλτεφοσίνης
Απόπτωση
Αυτοφαγία
Θεραπεία στο σκύλο
Λεϊσμανίαση
Μηχανισμός προσαρμογής
Οξειδωτικό στρεσ
Συνεργεία φαρμάκων
Issue date 2023-12-08
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/5/5/6/metadata-dlib-1700560967-377531-19541.tkl Bookmark and Share
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