Fügi, Matthias. Membrane therapy for chagas' disease. 2014, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_11900
In the framework of this PhD thesis various aspects of sterol anabolism in eukaryotes and its potential exploitation as drug target in parasites were analysed. First, using genome profiling, I did a comparative genomics study of sterol biosynthesis (SB) focusing on eukaryotic parasites. In vitro testing of known SBIs and quantifying the expression levels of SB genes during the different life stages of T. cruzi and Trypanosoma brucei completed this part of the thesis. Then, I used genetically modified yeast strains as a tool to assess selectivity of SBIs to ergosterol-containing cells. Finally, integrating the results from my work led to a specific proposition how to advance drug development in Chagas’ disease.
For the genome profiling an in silico pipeline was developed to globally evaluate sterol metabolism and perform comparative genomics. Hidden Markov model-based profiles for 42 SB enzymes allowed to represent the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally grouped eukaryote proteomes and revealed convergent evolution, in particular metabolic reduction in obligate endoparasites. The only obligate endoparasites found to possess SB genes were the trypanosomatids, Trypanosoma spp. and Leishmania spp. However, the origin of SB genes in trypanosomatids remains obscure, as there was no evidence for horizontal transfer.
SBIs are generally thought to act by inhibition of ergosterol anabolism. To investigate this more closely, I developed an assay using genetically modified yeast strains that either synthesize ergosterol or cholesterol. Different efficiencies of a given molecule in inhibiting ergosterol- or cholesterol-producing yeast can thus be attributed to sterol content. Nystatin concentrations required to inhibit growth in the cholesterol-producing yeast strain were 10-fold higher than in the ergosterol-producing strain, demonstrating the validity of the approach. Like amphotericin B, nystatin binds to ergosterol and forms pores in the membrane that lead to death of the target cell. This clear-cut result was only observed for molecules that bind to the finished end product of SB. Inhibitors of enzymes involved in SB did not exclusively inhibit growth of ergosterol-producing yeast strain, showing that the selectivity of SBIs for fungi is not based on differences between cholesterol and ergosterol anabolism. Two possible explanations why SBIs are selective inhibitors of fungal and trypanosomatid growth are brought forward: i) mammalian cells can salvage cholesterol from the environment and thus circumvent inhibition of sterol de novo synthesis whereas trypanosomatids and fungi require the presence of ergosterol and other 24-alkylated sterols, which cannot be replaced by the host’s sterols or ii) fungal and protozoan orthologs of SB enzymes are more susceptible to SBIs than the respective mammalian orthologs.
Even though azoles have been used as antifungals for decades, their use against trypanosomatids is still not implemented. Even worse, the most advanced candidate – posaconazole – could not confirm its initial potential in a recent phase II clinical trial for chronic Chagas’ disease. Based on my findings and integrating the work of others, posaconazole should not be abandoned but partnered with another drug for combination therapy. In the concluding chapter I elaborate on why a sphingolipid biosynthesis inhibitor is probably the best match.
|Advisors:||Mäser, Pascal and Conzelmann, Andreas|
|Faculties and Departments:||09 Associated Institutions > Swiss Tropical and Public Health Institute (Swiss TPH) > Department of Medical Parasitology and Infection Biology > Parasite Chemotherapy (Mäser)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||1 Online-Ressource (78 Seiten)|
|Last Modified:||06 Dec 2016 11:58|
|Deposited On:||06 Dec 2016 11:58|
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