Conception et synthese de nouvelles sondes ciblees pour l'imagerie moleculaire

Advance in personalized medicine requires probes development for the detection of pathology markers at the earliest possible stage. This thesis describes the synthesis and the evaluation of targeted probes to produce images in MRI or scintigraphy. These probes are constructed by functionalizing a molecular scaffold prepared by a three-component reaction. The resulting propargylamine has the necessary structure to introduce targeting ligands and two chelating agents for imaging. It also has a group in the terminal position to connect it on structures like small polyfunctional molecules, dendrimers or proteins. A high throughput screening technique based on small molecule microarrays was used for the detection of ligands. Molecules spotted on the chip came from either complex C-glycosides prepared according to the concept of diversity-oriented synthesis or from the laboratory chemical library. These microarrays have enable ligand identification for several proteins such as Vpr a protein from the capsid of AIDS virus. A secondary screening by NMR confirmed the interaction between protein and small molecules. Probes carrying a flavonoid for targeting tumor endothelial cells were treated with a gadolinium salt solution and MRI evaluated their properties. Initial results indicated that these compounds does offer of two to four folds contrast than the contrast agents usually used. Their biological evaluation will be conducted soon.Le développement d'une médecine personnalisée nécessite la mise au point de sondes permettant la détection des marqueurs d'une pathologie au stade le plus précoce possible. Ce mémoire décrit la synthèse et l'évaluation de sondes ciblées permettant de réaliser des images en IRM ou en scintigraphie. Ces sondes sont construites par fonctionnalisation d'un châssis moléculaire préparé par une réaction à trois composants. La propargylamine résultante possède plusieurs points d'ancrage pour introduire les ligands destinés au ciblage et deux groupes chélatants pour l'imagerie. Ce châssis possède aussi en position terminale un groupement réactif pour le fixer sur des petites molécules polyfonctionnelles, des dendrimères ou des protéines. Une technique de criblage à flux rapide, basée sur l'utilisation des puces à petites molécules, a été utilisée pour la détection des ligands. Les molécules déposées sur la puce proviennent soit des C-glycosides complexes préparés selon le concept de la synthèse orientée vers la diversité, soit de la chimiothèque du laboratoire. Ces puces ont permis l'identification de ligands de plusieurs protéines comme la protéine VPR de la capside du virus du sida. Un criblage secondaire par RMN a confirmé l'interaction protéine-petite molécule. Les sondes portant un flavonoïde pour cibler les cellules tumorales de l'endothélium ont été traitées par une solution de sel de gadolinium puis leurs propriétés ont été évaluées par IRM. Les premiers résultats indiquent que ces composés offrent un contraste de 2 à 4 fois supérieurs aux agents de contraste usuellement utilisés. Leur évaluation biologique sera réalisée prochainement

Conception et synthèse de nouvelles sondes ciblées pour l'imagerie moléculaire

Le développement d une médecine personnalisée nécessite la mise au point de sondes permettant la détection des marqueurs d une pathologie au stade le plus précoce possible. Ce mémoire décrit la synthèse et l évaluation de sondes ciblées permettant de réaliser des images en IRM. Ces sondes sont construites par fonctionnalisation d un châssis moléculaire préparé par une réaction à 3 composants. La propargylamine résultante possède plusieurs points d ancrage pour introduire les ligands destinés au ciblage et 2 groupes chélatants pour l imagerie. Une technique de criblage, basée sur l'utilisation des puces à petites molécules, a été utilisée pour la détection des ligands. Les molécules déposées sur la puce proviennent soit des C-glycosides complexes préparés par synthèse orientée vers la diversité, soit de la chimiothèque du laboratoire. Ces puces ont permis l'identification de ligands de plusieurs protéines comme la protéine VPR de la capside du virus du sida. Un criblage secondaire par RMN a confirmé l'interaction protéine-petite molécule. Les sondes portant un flavonoïde pour cibler les cellules tumorales de l endothélium ont été traitées par du gadolinium (III) puis leurs propriétés ont été évaluées par IRM. Les premiers résultats indiquent que ces composés offrent un contraste de 2 à 4 fois supérieurs aux agents de contraste usuellement utilisésPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Competition of Ligands and the 18‐mer Binding Domain of the RHAU Helicase for G‐Quadruplexes: Orthosteric or Allosteric Binding Mechanism?

Stabilizing the DNA and RNA structures known as G‐quadruplexes (G4s) using specific ligands is a strategy that has been proposed to fight cancer. However, although G‐quadruplex:ligand (G4:L) interactions have often been investigated, whether or not ligands are able to disrupt G‐quadruplex:protein (G4:P) interactions remains poorly studied. In this study, using native mass spectrometry, we have investigated ternary G4:L:P complexes formed by G4s, some of the highest affinity ligands, and the binding domain of the RHAU helicase. Our results suggest that RHAU binds not only preferentially to parallel G4s, but also to free external G‐quartets. We also found that, depending on the G4, ligands could prevent the binding of the peptide, either by direct competition for the binding sites (orthosteric inhibition) or by inducing conformational changes (allosteric inhibition). Notably, the ligand Cu–ttpy (ttpy=4′‐tolyl‐2,2′:6′,2′′‐terpyridine) induced a conformational change that increased the binding of the peptide. This study illustrates that it is important to not only characterize drug–target interactions, but also how the binding to other partners is affected. © 2020 Wiley‐VCH GmbH.ISSN:0947-6539ISSN:1521-376

In Vivo Evaluation of Magnetic Targeting in Mice Colon Tumors with Ultra-Magnetic Liposomes Monitored by MRI

International audiencePurposeThe development of theranostic nanocarriers as an innovative therapy against cancer has been improved by targeting properties in order to optimize the drug delivery to safely achieve its desired therapeutic effect. The aim of this paper is to evaluate the magnetic targeting (MT) efficiency of ultra-magnetic liposomes (UML) into CT26 murine colon tumor by magnetic resonance imaging (MRI).ProceduresDynamic susceptibility contrast MRI was applied to assess the bloodstream circulation time. A novel semi-quantitative method called %I0.25, based on the intensity distribution in T2*-weighted MRI images was developed to compare the accumulation of T2 contrast agent in tumors with or without MT. To evaluate the efficiency of magnetic targeting, the percentage of pixels under the intensity value I0.25 (I0.25 = 0.25(Imax − Imin)) was calculated on the intensity distribution histogram.ResultsThis innovative method of processing MRI images showed the MT efficiency by a %I0.25 that was significantly higher in tumors using MT compared to passive accumulation, from 15.3 to 28.6 %. This methodology was validated by ex vivo methods with an iron concentration that is 3-fold higher in tumors using MT.ConclusionsWe have developed a method that allows a semi-quantitative evaluation of targeting efficiency in tumors, which could be applied to different T2 contrast agents

Functional Characterization of the N-Acetylmuramyl-l-Alanine Amidase, Ami1, from Mycobacterium abscessus

International audiencePeptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells. Numerous autolysins with various substrate specificities participate in PG remodeling. Expression of these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human macrophages and zebrafish. The capacity of purified Ami1 to hydrolyze muramyl-dipeptide was demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for these inhibitors

Multimodal RM and Optic Imaging using ultramagnetic liposomes to monitor a cancer therapy

Congrès sous l’égide de la Société Française de Génie Biologique et Médical (SFGBM)National audienceMagnetic liposomes present an opportunity for the development of theranostic systems. Ultra-magnetic liposomes (UML) are highly loaded with ultra-small particles of iron oxide (USPIO) and can accumulate in a biological area thanks to a magnet. The incorporation of a fluorophore such as cyanine 5.5 in the formulation of the UML forms a bimodal imaging agent (MRI and optic) to monitor the magnetic accumulation in the tumor. The addition of an antitumoral drug into the UML combined with the application of High Intensity Focused Ultrasounds (HIFU) in the tumor can be used for a specific release of the drug in the region of interest

The zebrafish embryo as an in vivo model for screening nanoparticle-formulated lipophilic anti-tuberculosis compounds

International audienceWith the increasing emergence of drug-resistant Mycobacterium tuberculosis strains, new and effective antibiotics against tuberculosis (TB) are urgently needed. However, the high frequency of poorly water-soluble compounds among hits in high-throughput drug screening campaigns is a major obstacle in drug discovery. Moreover, in vivo testing using conventional animal TB models, such as mice, is time consuming and costly, and represents a major bottleneck in lead compound discovery and development. Here, we report the use of the zebrafish embryo TB model for evaluating the in vivo toxicity and efficacy of five poorly water-soluble nitronaphthofuran derivatives, which were recently identified as possessing anti-TB activity in vitro. To aid solubilization, compounds were formulated in biocompatible polymeric micelles (PMs). Three of the five PM-formulated nitronaphthofuran derivatives showed low toxicity in vivo, significantly reduced bacterial burden and improved survival in infected zebrafish embryos. We propose the zebrafish embryo TB-model as a quick and sensitive tool for evaluating the in vivo toxicity and efficacy of new anti-TB compounds during early stages of drug development. Thus, this model is well suited for pinpointing promising compounds for further development

Cytidine deaminase deficiency in tumor cells is associated with sensitivity to a naphthol derivative and a decrease in oncometabolite levels

International audienceIdentifying new molecular targets for novel anticancer treatments is a major challenge in clinical cancer research. We have shown that cytidine deaminase (CDA) expression is downregulated in about 60% of cancer cells and tissues. In this study, we aimed to develop a new anticancer treatment specifically inhibiting the growth of CDA-deficient tumor cells. High-throughput screening of a chemical library led to the identification of a naphthol derivative, X55, targeting CDA-deficient tumor cells preferentially, without affecting the growth of non-tumoral cells regardless of CDA expression status. Metabolomic profiling revealed that CDA-deficient HeLa cells differed markedly from control HeLa cells. X55 treatment had a moderate effect on control cells, but greatly disturbed the metabolome of CDA-deficient HeLa cells, worsening the deregulation of many metabolites. In particular, the levels of the three oncometabolites, fumarate, succinate and 2-hydroxyglutarate, were significantly lower in CDA-depleted cells, and this decrease in levels was exacerbated by X55 treatment, revealing an unexpected link between CDA deficiency, mitochondrial function and X55 response. Finally, we identified strong downregulation of MAPT (encoding Tau, a microtubule associated protein) expression as a reliable predictive marker for tumor cell X55 sensitivity

Selectivity of Terpyridine Platinum Anticancer Drugs for G-quadruplex DNA

Guanine-rich DNA can form four-stranded structures called G-quadruplexes (G4s) that can regulate many biological processes. Metal complexes have shown high affinity and selectivity toward the quadruplex structure. Here, we report the comparison of a panel of platinum (II) complexes for quadruplex DNA selective recognition by exploring the aromatic core around terpyridine derivatives. Their affinity and selectivity towards G4 structures of various topologies have been evaluated by FRET-melting (Fluorescence Resonance Energy Transfert-melting) and Fluorescent Intercalator Displacement (FID) assays, the latter performed by using three different fluorescent probes (Thiazole Orange (TO), TO-PRO-3, and PhenDV). Their ability to bind covalently to the c-myc G4 structure in vitro and their cytotoxicity potential in two ovarian cancerous cell lines were established. Our results show that the aromatic surface of the metallic ligands governs, in vitro, their affinity, their selectivity for the G4 over the duplex structures, and platination efficiency. However, the structural modifications do not allow significant discrimination among the different G4 topologies. Moreover, all compounds were tested on ovarian cancer cell lines and normal cell lines and were all able to overcome cisplatin resistance highlighting their interest as new anticancer drugs