UMR9187 / U1196 – Chemistry, Modelling and Imaging for Biology (CMIB)

Theses

2017

Molecular tools for the study of G-quadruplex in the human genome
Joël Lefebvre

Abstract

Deoxyribonucleic acid has different structures in human beings. The most known is the double helix but a lot of secondary structures exist and particularly G-quadruplex. It consists of guanine-rich nucleic acid sequences. The association of four guanines through hydrogen bonds forms a plan called G-quartet. This set of hydrogen bonds is called Hoogsteen base pairs. The stacking of at least two quartets around a monovalent cation like potassium or sodium establishes the G-quadruplex. These structures have been much studied over the past twenty years. They are involved in numerous biological mechanisms like replication, transcription, translation and also telomere maintenance. G-quadruplex presence can cause an important genetic as well as epigenetic instability. That is why many methods have been developed in order to localize these structures and to understand their role in vivo. To this end, a broad panel of molecular tools has been used. However, it is still difficult to bring an answer to all the questions about the involvement of G-quadruplex at the genomic level with this panel. In this thesis work, we developed new molecular tools able to target selectively G-quadruplex in a complex biological medium from two benchmark ligands, PhenDC3 and PDC, which have very good affinity and selectivity for G-quadruplex.On the one hand, functionalized ligands have been synthetized with a biotin and/or a photoactivatable group in order to trap and pull-down G-quadruplex in various cellular contexts. On the other hand, derivative compounds which are able to be functionalized in cellulo by bioorthogonal reactions have been obtained. Once the compound interacts with its cellular target, a function (fluorophore or biotin) can be added through an orthogonal reaction. The new panel of compounds has been evaluated by biophysical techniques, FRET-melting experiment and FID assay, in order to determine their affinity to G-quadruplex and their selectivity. We proposed a relation between the two biophysical experiments in order to have a good ranking of ligands for G-quadruplex structures.One of the most important objectives of this work was to localize G-quadruplex ligands in human cancer cells. First, a complete study in fixed cells has been performed using two reactions of click chemistry: reaction of copper-catalyzed-alkyne-azide-cycloaddition (CuAAC) and reaction of strain-promoted alkyne-azide cycloaddition (SPAAC). Secondly, the study has been pursued in living cells using SPAAC reaction because of the toxicity of copper in cells.These compounds have also been used to extract G-quadruplex from biological systems with cyclooctyne-coated magnetic beads. However, results obtained in this preliminary study are not decisive so it could be interesting to optimize the system before concluding. (Defended on December 15, 2017)


Therapeutic follow-up of photodynamic therapy treatment of retinoblastoma murine models

Stéphanie Lemaitre

Abstract

Retinoblastoma is the most common primary intraocular malignancy in children. Current retinoblastoma treatments have many adverse effects. New therapeutic approaches (like photodynamic therapy [PDT] or intravitreal injections [IVT] of chemotherapy) must therefore be evaluated on animal models, before a clinical application.In this thesis we first characterized an orthotopic xenograft murine model obtained with human retinoblastoma cells. We showed that intraocular tumor growth can be achieved in immunodeficient mouse strains (Swiss-nude and SCID [severe combined immunodeficiency]) and in an immunocompetent strain (B6Albino). Due to insufficient tumor engraftment rates (between 28.4 and 68.8% depending on the mouse strains) and to ocular complications after the injection of tumor cells (cataract, chronic retinal detachment) the treatments (PDT and IVT of chemotherapy) were performed on a transgenic retinoblastoma mouse model (LHBetaTag).In order to perform PDT, an MRI study (magnetic resonance imaging) of the photosensitizer (PS, DEG-mannose) coupled with manganese and a biodistribution study based on the dosage of the PS were performed. Both studies showed that the illumination of the tumor should be performed between 24 and 48h after the intraperitoneal injection of the PS (which corresponds to the “drug-to-light interval” of PDT). Using these parameters, PDT was effective on the retinal tumors of LHBetaTag mice. In the area treated with PDT we found 91.7% chorioretinal scars on OCT (optical coherence tomography) with a “drug-to-light interval” of 24h, and 100% chorioretinal scars with a “drug-to-light interval” of 48h. The retina outside the treated area had a normal aspect on histology, showing that PDT is not toxic on healthy tissues. Laser treatment alone had no anti-tumor effect.IVT of chemotherapy were also performed in LHBetaTag mice. We used melphalan, carboplatin and topotecan, alone or in association. We showed that 4 weekly IVT of carboplatin at the dose of 1.5 µg had the best anti-tumor effect (83.3% of eyes had no tumor mass on histology) and little retinal toxicity (21.4% of eyes had diminished retinal thickness on OCT). Carboplatin seems an interesting alternative to melphalan which is currently the most commonly used chemotherapy for IVT (but has a retinal toxicity).In conclusion, these preclinical studies on a retinoblastoma mouse model (LHBetaTag) show that PDT could be used to treat retinal tumors in human retinoblastoma. IVT of carboplatin could be used to treat vitreous seeds in this disease. Functional tests (electroretinogram, optokinetic reflex) should be performed in mice in order to evaluate more precisely the retinal toxicity of these treatments. (Defended on November 27, 2017)


Methodological and software development for X-ray scanning imaging at Nanoscopium beamline

Antoine Bergamaschi

Abstract

The subject of this thesis is the methodological and software development of tools for processing very large multimodal and tomographic datasets produced on Nanoscopium beamline in the SOLEIL synchrotron. Scanning hard X-ray imaging allows simultaneous acquisition of multimodal information, i.e. of images in which each pixel contains several types of data. Combining these scanning techniques with the FLYSCAN infrastructure, developed for fast data acquisition at Synchrotron SOLEIL, permits to perform multimodal tomographic imaging and tomographic reconstruction during routine user experiments. A main challenge of such imaging techniques is the online processing of the important amount of generated multimodal data. The main outcome of this thesis work is the MMX-I software which is dedicated to processing large 2D/3D multimodal dataset. This software includes an original algorithm for continuous reading of large data volumes, several reduction functions, two phase reconstruction algorithms (integration in Fourier space and iterative technics) and tomographic reconstruction technics (filtered back projection and iterative technics). Every implemented method as well as application allowing to validate the new developments and few evolution perspectives are presented in this thesis manuscript. (Defended on March 7, 2017)

2016

Autour du noyau imidazo[4,5-b]pyridine : inhibiteurs potentiels de la protéine kinase Tyro3 et fonctionnalisation directe de liaisons C – H
Tom Baladi

Résumé

Étant au quatrième rang des cancers les plus fréquents chez l’homme, le cancer de la vessie représente un enjeu médical important. Pourtant, à ce jour, seuls des traitements chirurgicaux handicapants et/ou chimiothérapiques non spécifiques peuvent être envisagés. Le projet de thèse s’inscrit dans le cadre de la recherche de thérapies ciblées du cancer de la vessie en ayant pour objectif le blocage, au niveau moléculaire et de manière sélective, des voies de signalisation mises en œuvre par la tyrosine kinase Tyro3 au sein des cellules cancéreuses. La mise en évidence de la surexpression de ce récepteur membranaire dans la majorité des tumeurs de vessie et son rôle dans la survie des cellules cancéreuses ont en effet permis de valider Tyro3 comme cible thérapeutique pour ce type de cancers. Le projet peut se diviser en trois parties : le développement de nouvelles méthodologies de synthèse autour du motif imidazo[4,5-b]pyridine, la synthèse d’une librairie de candidats inhibiteurs en utilisant les méthodes mises au point et enfin l’étude des relations structure-activité vis-à-vis de la protéine kinase Tyro3. (Defended on November 18, 2016)

Synthesis and photobiological evaluations of porphyrine dimers for targeted two-photon photodynamic therapy
Su Chen

Abstract

Photodynamic therapy (PDT) is a new potential treatment against retinoblastoma, which doesn’t induce mutations. The porphyrin derivatives used as Ps in PDT are widely studied since the birth of the first synthesis Ps (HpD). An important limitation of PDT comes from low penetration of light (λ<700 nm) used for excitation of the Ps. In order to provide enough energy to enable production of singlet oxygen in the phototherapeutic window between 700-1300, the absorption of two relatively low-energy photons simultaneously has been proposed. Ps excited by simultaneous absorption of two photons leads to the concept of two-photon PDT (TPE-PDT). This process has very low probability; its application in the PDT needs develop new Ps with intensive cross section. Another limitation is the low selectivity and specificity of current Ps for tumor cells. Active targeting to appropriate receptors expressed at the tumor cells give a possible solution. It has been reported that the lectin-like receptors recognizing certain sugars are overexpressed in malignant cells. We will present the synthesis and in vitro photocytotoxical results of asymmetric porphyrin dimers P-Y-P’, inspired by previous studies of our laboratory, introducing three para-phenoxy-diethylene glycol mannose chains at the meso positions of porphyrin core which optimized for two-photon absorption and targeted to membrane lectins. To circumvent the solubility problem of porphyrin dimers in aqueous medium and improve internalization of Ps into tumor cells, we analyzed the interfacial behavior of the porphyrin dimers in the air-buffer interface, studied the incorporation of porphyrin dimers in liposomes using the technique of fluorescence, and evaluated the interaction between porphyrin dimers and Concanavalin A (Canavalia ensiformis lectin derived from that specifically recognizes the alpha-D-mannose). (Defended on February 15, 2016)

2015

Trapping G-quadruplex DNA from molecular tools to cellular assays
Élodie Morel

Abstract

Nucleic acids secondary structures may form in guanine-rich regions by Hoogsteen base-pairing around a cation (K+ or Na+) and stacking of guanine quartets. Those nucleic acid secondary structures called G-quadruplex are believed to play regulatory roles in the main functions related to DNA processing. However, although numerous sequences, potentially forming G4-structures are present in genomes, evidence concerning their in vivo formation and biological role remains limited. Primary aim of our research is to provide new chemical biology tools for evaluating the biological impacts of quadruplexes and the potential of our compounds for quadruplex-targeted anticancer therapy. We have synthetized a set of compounds equipped with biotin and cross linking moieties in order to trap and pull-down G4-structures in various cellular contexts. The G4-ligands (PDC, PhenDC3 and Metal-ttpy) were evaluated thanks to FID and FRET-melting assays, and carefully chosen to efficiently target G-quadruplexes but also to display enough selectivity for cellular assays. Direct trapping of a G-quadruplex structures can also be done by metal complexes, thanks to coordination with DNA bases. Platinum tolylterpyridine derivatives have been studied on gel electrophoresis to map the platination sites and to evaluate the kinetics of the phenomenon. By adding photo crosslinking moieties to Pt-ttpy, efficient double-anchoring has been done on DNA G-quadruplex structure. Moreover, first cellular imaging evaluations were done by adding a fluorophore to this platinum tolylterpyridine complex. To eventually probe quadruplex DNA at the genome-wide scale, full control of the trapping protocol is indeed a key step. Full development of the pull-down step has been done, using streptavidin-coated magnetic beads. On-beads experiments indicate that efficacy of trapping can vary dramatically depending on quadruplex and G4-ligand topologies. Moreover, photo crosslinking moiety, introduced on some compounds, has not shown any improvement of the trapping. However, the development of this method and the design of the capture compounds have led to an optimal isolation of telomeric G-quadruplex forming sequences, from genomic DNA. (Defended on December 18, 2015)

Telomeres, potential targets of cis-platin derivatives : Binding and modification of their structure
Samar Ali

Abstract

Telomeres are specialized nucleoprotein complexes located at the end of chromosomes. They protect chromosomes from degradation, recombination and telomeric fusions and avoid them to be recognized as DNA breaks. They are composed of telomeric DNA consisting of repetitions of the sequence TTAGGG, which is extended by a 3 ‘single-stranded DNA, and of six telomeric proteins which TRF2 protein, that are essential to the maintenance of the integrity of telomeres. The guanine-rich strand is able to fold, in the presence of monovalent cations, in a four stranded structure, the G-quadruplexes. The stabilization of these structures is a promising anticancer strategy because it induces telomeric perturbations leading to cancer cell death. As telomeres are rich in guanines, they are potential targets for platinum complexes. Our aim is to increase the targeting of telomeres by associating within the same molecule, a ligand of G-quadruplex which stabilizes these structures with a platinum atom which then, will irreversibly block these structures. The tolyl-terpyridin-Pt(II) (Pt-ttpy) has been designed in this aim. It stabilizes and binds irreversibly to the G-quadruplex structures in vitro. We anlysed the telomeric perturbations induced by this G-quadruplex ligand (Pt-ttpy) in comparison with complexes which do not stabilize these structures (terpyridin-Pt(II) or Pt-tpy and cisplatin, an anti-tumor widely drug used in chemotherapy) and quantified the number of complexes bound to telomeres. We used two ovarian cancer cells lines, sensitive and resistant to cisplatin (A2780 and A2780-cis), and a non-cancer cell line (BJ-hTERT). Pt-ttpy and Pt-tpy inhibit cancer cell proliferation in doses at the µM range and show no cross-resistance with cisplatin. Our results, obtained by ChIP and immunofluorescence experiments, show that Pt-ttpy delocalize 50% of protein TRF2 from telomeres of cancer cells and increases the damage to telomeric DNA compared to the complexes that are not ligands of G quadruplexes, without inducing telomere shortening. Therefore, the association of a G-quadruplex ligand to a platinum atom within the same molecule allows to preferentially targeting telomeres of cancer cells compared to the platinum complex alone. However, telomeric perturbations induced by Pt-ttpy were not increased compared to the best known G-quadruplex ligands. Interestingly, and for the first time in the literature, we have shown that Pt-ttpy, Pt-tpy complexes directly target cancer cells since they bind irreversibly to them. They increase the preference of binding to telomeric DNA versus genomic DNA by a factor of 15 compared to cisplatin. This preference seems independent of the recognition of G-quadruplex structures but seems to depend on the nature of platinum complexes. On the other hand, because of the small amount of complexes bound to telomeres, their binding to telomeres cannot be at the origin, alone, of the delocalization of TRF2 from telomeres, suggesting that delocalization of telomeric TRF2 is not due to a physical impediment but rather to a biological response. Our work shows that the hybrid molecules G-quadruplex ligands-Pt (II) are an interesting strategy for targeting telomere of cancer cells. Therefore, it will be interesting to develop new complexes which would increase the preference for telomeric DNA and also the amount of platinum bound to telomeric DNA compared to Pt-ttpy. (Defended on November 24, 2015)

Development of fluorescent probes for the detection of quadruplex DNA
Xiao Xie

Abstract

Single-Stranded nucleic acids containing guanine repeats can form non-Canonical secondary structures called G-Quadruplexes. These structures are composed of several guanine quartets, maintained by hydrogen bonds and metal cations (K+ or Na+) coordinated between G-Quartets. In spite of being well-Studied in vitro, the evidence for the presence of quadruplex DNA structures in vivo remains mainly indirect. The objective of this work was research of fluorescent probes that can signal the presence of quadruplex DNA and detect its structure (topology).Two series of fluorescent probes were considered and prepared: styryls dyes (mostly distyryls) and PDC-Coumarin derivatives. The design of these two series is based on the molecular scaffold of bisquinolinium pyridodicarboxamide (PDC-360A), a selective ligand with good affinity for quadruplex DNA structures but which is not fluorescent. Inspired by this molecule and the styryl motif, which is known for its spectroscopic properties, we considered a library of distyryles dyes. A second series, the PDC-Coumarin derivatives, was developed to introduce the fluorescence property of coumarin in the PDC by a covalent link. The properties of dyes of these two libraries (65 compounds) were studied in the presence of a number of DNA structures (quadruplex and duplex) by a fluorescent screening using microplate and titration methods. Our results show that some of synthesized dyes display high fluorescence response (i.e. fluorescence increase factor from 200 to 600) for different quadruplex DNA and RNA structures, while having a very low fluorimetric response for duplex DNA. This allows a selective visualization of quadruplex DNA in solution or in electrophoresis gel. These results represent the first steps towards the use of these probes in a biological context, for example in fluorescence imaging. (Defended on January 28, 2015)

2012

Vinyl-triphenylamine dyes optimized for two-photon microscopy : Non coalent interactions with DNA or HSA and cellular imaging
Blaise Dumat

Abstract

Significant advances were made in the field of in vivo fluorescence imaging thanks to the recent development of biphotonic microscopy and super-resolution techniques, rendering intravital imaging and biological tissues analysis possible. Those techniques however require the use of new probes with optimized optical and biological properties.Several series of cationic dyes for DNA staining were developed based on the vinyl-triphenylamine (TP) scaffold. Those new switchable yellow or red fluorophores bind in the minor-groove of DNA and display high two-photon absorption cross-sections. Two anionic derivatives were also designed for staining HSA.In fixed or apoptotic cells, the cationic dyes stain nuclear DNA with a high brightness and contrast. They are non-cytotoxic, photostable and cell permeant. The molecule with the most optimized properties, TP-2Bzim, has one of the highest two-photon brightness to date (383 GM in DNA), allowing sensible detection in biphotonic microscopy at low concentration and excitation power. In live cells, the dyes are localized in the mitochondria, but it appears that upon constant mono- or bi-photonic excitation they trigger cell apoptosis within a few minutes and are released in the nucleus. Since the phenomenon can be imaged by fluorescence microscopy, the TP dyes could thus be used as photosensitizers for theranostics.A synthetic pathway was also developed to functionalize the TP-2Bzim. It was then coupled by “click-chemistry” to short oligonucleotides or PNA sequences for fluorescence in situ hybridization, and to folic acid and spermidine for cancer cells targeting. (Defended on December 7, 2012)

Synthesis and characterization of two-photon absorbing photosensitizers for application to photodynamic therapy : Targeting of retinoblastoma
Fabien Hammerer

Résumé

The Institut Curie is strongly committed to the development of new treatments ofchildhood eye cancer (or retinoblastoma). Existing therapies often lead to severeside effects which could be avoided by the use a milder treatment techniques. Webelieve Photodynamic Therapy (PDT) provides a promissing alternative as a nonmutagenic and efficient therapy. Over the last decade, our laboratory has developpeda new strategy for the targeting of retinoblastoma through the synthesis ofglycoconjugated photosensitizers (PS) displaying a strong affinity for membranereceptors known as lectins overexpressed by the tumor cells.In order to avoid the damages to healthy tissues close to the tumour, we haveadapted our targeting strategy to Two-Photon Absorption Photodynamic Therapy(TPA-PDT). Two-Photon Absorption (TPA) is a localized excitation mechanismwhich allows strict control of the PDT effect preventing the killing of the retiniancells next to the tumor.This work presents the progress in the development of retinoblastoma targetedTPA-PDT. Three fields have been investigated.Porphyrin oligomers have been reported to possess high two-photon cross sectionsand have shown high potential for their application to TPA-PDT.We synthesizedglycoconjugated analogs of these oligomers and analyzed their photophysicaland biological properties. These compounds show high TPA cross section as wellas high singlet oxygen production quantum yields making them good candidatesfor TPA-PDT. A supplementary study is underway to enhance their cellular internalisation.They also display temperaure dependent fluorescence emission that could allowtheir use as fluorescent ratiometric temperature probes.Porphyrin-triphenylamine hybrids (PTP) are also promissing as two-photonabsorbing photosensitizers (TPA-PS). We studied the influence of different moieties,such as triphenylamine substitutents, meso-phenyl substitutent (glycosylatedor triethylene glycol) and the intern zinc atom, on their photophysical and biologicalproperties. Excellent singlet oxygen generation in polar solvent as well asgood TPA cross sections were measured. Difficulties concerning their cellular internalisationwere encountered and are currently being solved.A second family of cationic hybrids was prepared. Their behaviour relies onthe fluorescence exaltation properties of methylated triphenylamines upon bindingto DNA. These compounds display low emitting and photosensitizing propoertiesin water which can be restored by interaction with DNA. An energy transfer fromthe triphenylamine to the porphyrin was observed in glycerol. The methylatedhybrids show good in vitro phototoxicity toward cancer cells and allow fluorescenceimaging applications. (Defended on December 4, 2012)