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Etablissement d’une approche de déconvolution de cibles pour des composés leishmanicides Ecole Pratique des Hautes Etudes Mémoire préparé sous la direction de Gérald Späth, Najma Rachidi et Sylvie Demignot

Abstract : Currently, medicines used to treat leishmaniasis have many serious side effects and their use is greatly compromised by the appearance of resistant parasites. It is therefore urgent and necessary to obtain new drugs to fight the disease effectively. In recent years, pharmaceutical companies have carried out screening campaigns that have identified thousands of compounds with a leishmanicidal effect. However, since their targets are unknown, these compounds are difficult to optimize, which in the long term could lead to side effects for the drugs derived from these compounds. Thus, deconvolution of targets has become a major issue, since it allows the identification of the primary and / or secondary targets of the compounds in order to understand their mechanism of action and to promote their optimization. We carried out a screening campaign which allowed us to identify a dozen potential inhibitors of kinases effective against the parasite. In order to determine their targets, I have developed a deconvolution strategy adapted to these inhibitors which consists initially in a selective enrichment of the Leishmania ATP binding proteins (ATPome) by ATP affinity chromatography , Then in a second stage in a competition by the compound of interest. The method of enrichment was very effective since it made it possible to obtain the first ATPome never realized in the trypanosomatids with the identification of 1177 proteins with potential ATP binding, including 205 annotated proteins for the binding function to ATP (Gene Ontology: 0005524), including 80 kinases and about 80 hypothetical proteins with an ATP binding domain or known ATPase function but not annotated. I then optimized the stage of competition and established the proof of concept using a known inhibitor-kinase pair. Finally, by combining this method with 2D-DiGE, I established a fast, visual and semi-quantitative method that allows me to determine whether inhibitors are broad-spectrum or specific; Only the latter will be optimized. This method thus makes it possible to visually determine the efficiency and the specificity of the competitive compounds of ATP. Using this approach, of the ten compounds initially obtained by screening, I was able to select two that were specific. For these two compounds, the targets will be identified by mass spectrometry.
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Contributor : Olivier Leclercq Connect in order to contact the contributor
Submitted on : Wednesday, February 15, 2017 - 11:02:08 AM
Last modification on : Tuesday, October 18, 2022 - 4:21:57 AM
Long-term archiving on: : Tuesday, May 16, 2017 - 1:22:04 PM

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Olivier Leclercq. Etablissement d’une approche de déconvolution de cibles pour des composés leishmanicides Ecole Pratique des Hautes Etudes Mémoire préparé sous la direction de Gérald Späth, Najma Rachidi et Sylvie Demignot. Biochimie [q-bio.BM]. 2016. ⟨hal-01468100⟩

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