During last three decades, a great effort was invested to the development of polymer conjugates of low molecular drugs with the aim to improve the specific targeting of drugs to diseased tissues, cells and organs. The main reason for this effort was the fact that high molecular weight copolymers have a favourite distribution profile in tissues and organisms. A linker between a polymer backbone and drug has very important role: it is possible to synthesize a biodegradable linker, which can be enzymatically hydrolyzed. Conversely, there is a possibility to synthesize an inert linker, resistant to the hydrolysis. Proper choice of the suitable precursor- polymer is also essential, hence it has to accomplish all of the stringent demands for biocompatibility. Macromolecular polymer-drug conjugates tend to accumulate in solid tumors because of the so called enhanced permeability and retention (EPR) effect. There is a whole range of possible applications of high molecular polymer-drug conjugates. In the introduction part of this thesis, I summarize potential use of drugs based on poly(N-(2-hydroxypropyl)methacrylamide) (HPMA) copolymers. Moreover, I introduce some therapeutically important proteins used in experimental drug discovery. In our laboratory, we have developed a concept of HPMA copolymers...

Résumé français manquant / Scientific background: GAGs mimetics properties on regenerative process.Glycosaminoglycans (GAGs) are sulfated polysaccharides actually considered as major structural components of the extracellular matrix as well as regulators of cells functions during homeostatic and pathological processes. These GAGs activities are based on their ability to interact with heparin binding growth-factors (HBGF), chemokines and enzymes, to protect them from proteolytic degradation and to potentialyze their interaction with cell surface specific receptors and/or other components of the ECM. GAGs are characterized by their extensive structural diversity, based on the number and location of sulfate or acetylate groups, that would determine specific biological interactions.As comparative tool to study the relationship between the complexity of GAGs chemical structures and their biological functions, we used synthetic GAGs mimetics, derivate from a polymer of dextran and functionalized with carboxylate, sulfate and/or acetate groups. They are structurally and functionally related to natural heparan sulfates. These compounds improved both the rate and quality of regenerative process in numerous animal models of injury after topical treatment.Our hypothesize is that specific HS cooperative interactions with HBGF and ECM compounds could influence both therapeutic progenitors and stem cells properties by compartmentalizing them to specific microenvironment niches, and protecting them against deleterious signals. Such abilities to modulate stem cell biology could be a new way to explain and to take advantage of regenerative properties of these compounds. The principal aim of this work was to demonstrate the effects of GAGs mimetics on Mesenchymal Stem Cells (MSC) properties for application in bone repair. GAGs mimetics as new potentializing agents of mesenchymal stem cells propertiesDuring osteogenesis, a controlled expression of functional HS is required to interact and regulate the activity of growth promoting and osteogenic differentiation factors. However effects of GAGs on MSC properties remain to be analyzed. We focus on two GAGs mimetics leader molecules [OTR4131] and [OTR4120], with distinct chemical characteristics, since sulfated mimetic [OTR4120] was previously shown to stimulate bone repair in vivo. We demonstrate that its acetylated and sulfated counterpart [OTR4131] enhances proliferation, whereas [OTR4120] clearly stimulates migration and osteogenic differentiation properties of rat MSC in vitro, that could explain its bone regenerative effect in vivo. This indicates that GAGs mimetics would be of great interest for potential application in therapy, since according to their structural signature they could modulate specific activities of progenitors and stem cells, and represent an alternative to exogenous growth factor treatments. New matricial strategy for bone repair associating GAGs mimetics to biomaterials and human MSCCell based therapy associated to biomaterials for repair of bone defects are promising but not enough efficient. We proposed to develop matricial strategy, associating efficient micro-environment molecules such as GAGs mimetics, to optimize cell therapeutic approaches. First we validated that GAGs mimetics are effective on human MSC proliferation, migration and differentiation properties in vitro. We demonstrated that colonization efficiency of hydroxyapatite/β-tricalcium phosphate biomaterial scaffolds by human MSC was improved when scaffolds are functionalized with GAGs mimetics in vitro. Finally osteoformation in vivo was evaluated after ectopic transplantation of functionalized and/or cellularized biomaterials in nude mice: few effects were observed on bone formation, whereas osteoclastogenesis and vascularization were clearly modulated by GAGs mimetics immobilized. GAGs mimetics as new mobilizing agents of stem cells...

Many peptide receptors are of pharmaceutical interest and there is thus a need for new ligands for such receptors. Unfortunately, peptides are not suitable as orally administrated drugs since they are associated with poor absorption, rapid metabolism and low sub-receptor selectivity. One approach that should allow identification of more drug-like ligands is to use the structural information of the endogenous ligand to develop peptidomimetic compounds. The main objective of the work described in this thesis was to convert angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) to small drug-like compounds with retained bioactivity at the AT2 receptor. The study was performed step-wise via incorporation of well-defined secondary structure mimetics and repeated truncation of the peptide. Five scaffolds, comprising a benzene ring as a central element, suitable as a γ-turn or dipeptide mimetics were designed and synthesized. In order to decorate the scaffolds, a method of microwave-assisted alkoxycarbonylation was developed. After incorporation of the scaffolds into Ang II-related peptides or peptide fragments, the affinities for both the AT1 and the AT2 receptor were determined. In the first series of ligands, two tyrosine-related scaffolds were introduced as γ-turn mimetics in Ang II. All five pseudopeptides exhibited good affinities for the AT2 receptor. One compound was chosen for functional studies and was shown to act as an AT2 receptor agonist. After truncation of Ang II it was shown that C-terminal pentapeptide analogs were AT2 receptor selective agonists. A series of pseudopeptides comprising tyrosine-related scaffolds, derived from the pentapeptides, displayed high AT2 receptor affinities. Two compounds had agonistic effect at the AT2 receptor. This study revealed that the N-terminal part was of less importance while a C-terminal Ile residue was a key element for enhanced AT2 receptor affinity. In the final set of compounds, the peptide was truncated to tripeptide C-terminal fragments. After replacing His-Pro by a histidine-related scaffold small drug-like peptidomimetic compounds with nanomolar affinity for the AT2 receptor were identified.

Les glioblastomes (GBs) sont les tumeurs primitives du SNC les plus agressives de l’adulte. Les causes d’échec thérapeutiques sont multiples, comme une résistance des cellules tumorales à l’apoptose, l’existence de cellules souches cancéreuses ou un microenvironnement pro-tumoral. La découverte de molécules thérapeutiques qui pourraient avoir une action pléiotrope est particulièrement attractive. Dans ce contexte nous nous sommes intéressés aux mimétiques de Smac (MS), antagonistes des inhibiteurs de l’apoptose (IAP), qui inhibent le plus souvent cIAP1, cIAP2, XIAP et ML-IAP. Nous avons recherché si les IAP pouvaient être des cibles thérapeutiques dans les GBs humains en étudiant leur expression et leurs valeurs pronostiques éventuelles : les IAP sont exprimés dans les GBs et ML-IAP est associé à un plus mauvais pronostic. Nous avons choisi d’utiliser pour la suite de nos expériences, un MS qui avait une action sur les IAP et en particulier ML-IAP : le GDC-0152. Le GDC-0152 induit l’apoptose in vitro, augmente la survie des souris porteuses de GB et ralentit la croissance tumorale in vivo. Ensuite nous avons recherché si l’effet du GDC-0152 pouvait être différent en fonction du taux d'oxygène. En effet, les GBs sont des tumeurs hypoxiques. Nous avons cultivé en normoxie et en hypoxie, quatre lignées de cellules souches de GBs. En normoxie, le GDC-0152 induit la différenciation des cellules souches (voie NF-κB) et en hypoxie il induit l’apoptose et diminue la prolifération cellulaire (voie ATR).Ces travaux soulignent l’importance du modèle préclinique utilisé dans la caractérisation de l'effet de nouvelles molécules et le potentiel thérapeutique des MS dans les GBs. / Glioblastomas (GBs) are the most aggressive primary brain tumors in adults. The causes of therapeutic failure are unknown and are multiples, such as tumor cell resistance to apoptosis, the presence of cancer stem cells or a pro-tumor microenvironment. Thus, the discovery of therapeutic molecules with pleiotropic action is particularly interesting. In this context, we are interested in smac mimetics (SM), antagonists of inhibitor of apoptosis proteins (IAPs) and most often antagonize cIAP1, cIAP2, XIAP and ML-IAP.We investigated whether IAPs could be attractive therapeutic targets in human GBs by studying their expression and their possible prognostic values. All IAPs were expressed in various degrees in GBs and ML-IAP was associated with a worse prognosis. Therefore, we chose GDC-0152 for the rest of our experiments because it antagonizes the different IAPs and in particular ML-IAP. We showed that GDC-0152 induces apoptosis in vitro, increases the survival of GB-bearing mice and slows tumor growth in vivo.We investigated whether the effect of GDC-0152 could be different depending on the oxygen level. Indeed, GBs are part of the most hypoxic tumors. For this purpose, four GB stem cell lines were grown in normoxia and hypoxia. We found that GDC-0152 has an anti-tumor effect regardless of oxygen level, but the signaling pathways involved were different. In normoxia, GDC-0152 induces differentiation of GB stem cells (NF-κB pathway) and in hypoxia it induces apoptosis and decreases cell proliferation (ATR pathway).This work highlights the importance of the preclinical model used in the characterization of a new molecule effects and the therapeutic potential of SM in GBs.

The Human Immunodeficiency Virus (HIV) is a member of the retroviridae family from lentivirus genus which primarily infects CD4+ T cells and also to lesser degree monocytes, macrophages, and dendritic cells causing progressive failure of the immune system, ultimately leading to development of acquired immunodeficiency syndrome (AIDS). Currently ~ 37 million people are infected with HIV-1 with approximately 2 million new infections occurring every year (UNAIDS, 2016). Developing safe, effective, and affordable vaccines to prevent HIV infection is the best hope for controlling the HIV/AIDS pandemic. Envelope glycoprotein (Env) on the HIV-1 virion surface is synthesized as a single precursor protein gp160 which is cleaved by furin to form the gp120 and gp41 subunits. gp41 is inserted into the membrane, while gp120 remains non-covalently associated with the ectodomain of gp41 to form a trimer of heterodimers. gp120 binds to the CD4 receptor on CD4+ T cells, which triggers a series of conformational changes leading to the exposure of co-receptor binding sites on gp120. Subsequent binding to the co-receptor (CXCR4 or CCR5) on T-cells initiates fusion of cellular and viral membranes via gp41 subunit. The envelope glycoprotein gp120, on the virion surface is the most accessible component of HIV-1 to the host immune system, and the target of most of the neutralization response. However, the virus has evolved many efficient ways to escape this immune surveillance. Extensive glycosylation of gp120 is one way by which it masks critical neutralization epitopes and the presence of immunodominant long variable loops focuses the immune response away from conserved regions. Certain conserved epitopes are cryptic and get exposed only after gp120 binds to its receptor. Also gp120 and gp41 are highly flexible molecules, attached in a non-covalent fashion to form a trimer of heterodimers, leading to inherent metastability of the Env. This results in exposure of a large number of non-native conformations to the immune system and thus minimizes elicitation of neutralizing antibodies. Despite these defense mechanisms, about 20-30% of HIV-1 patients do generate a broad neutralization response. Although these bNAbs and their epitopes have been identified, eliciting similar bNAbs through immunization is challenging. Monomeric gp120 when used as an immunogen elicits non neutralizing antibodies. This indicates that the epitopes of bNAbs are not present in the right conformation on this molecule. A rational design approach which focuses the immune response towards specific epitopes targeted by bNAbs is required, with the aim to maximize the exposure of conserved neutralization epitopes and to simultaneously ensure minimal exposure of variable non neutralizing epitopes. This can likely be achieved either by (a) stabilization of native Env trimers, or/and by (b) protein fragment design. Chapter 1 gives a brief description of HIV-1 virus. Structural features of the Env protein are described along with epitopes targeted by various bNAbs. Various strategies employed towards structure based vaccine design are discussed. One of the strategies towards rational vaccine design is using protein fragment based approaches. Grafting epitopes onto heterologous scaffolds is a promising approach which can provide more structural stability to the epitope, helps focus immune response on the epitope of interest and can be employed in a prime boost strategy for immunization studies. In a scaffold based approach we used crystal structure information of gp120 in complex with bNAb b12 to define the epitope of this antibody. In Chapter 2 we use this epitope information to graft the epitope on an unrelated scaffold protein to design unique epitope scaffolds. We report a computational strategy to graft the discontinuous epitope of b12 antibody onto different scaffold proteins. Our strategy focuses on identifying the best match of the target scaffold to the query protein so as to cause the least structural disturbance in the scaffold protein. The best hits were screened for binding to b12 using Yeast Surface Display (YSD). Random mutant libraries were also generated to screen for better b12 binders using YSD. We further characterized a few of these epitope scaffolds after purifying them from bacterial systems. One of the epitope scaffolds 1mkh_E2 bound to b12 with a KD value of 7.5µM. 2bodx_03, an unoptimized epitope scaffold reported previously (Azoitei et al, 2011) binds b12 with a KD value of 300μM. Thus our epitope scaffold 1mkh_E2 shows reasonable binding to b12 without any optimization. We are currently purifying other b12 epitope scaffolds and will be characterizing them for binding to b12. We have previously used a protein minimization strategy to design fragments of gp120, called b122a and b121a comprising a compact beta barrel on the lower part of the outer domain in order to focus the immune response towards the b12 epitope. (Bhattacharyya et al, 2013). These were bacterially expressed, found to be partially folded, however, could bind the broadly neutralizing antibody b12 with micromolar affinity. In rabbit immunization studies sera obtained following four primes with the b122a fragment protein and two boosts with full-length gp120 showed broad neutralization of a panel of multiple viruses across different clades (Bhattacharyya et al, 2013). In the present work, These designs were further stabilised by introducing various disulphides. One of the disulphide mutants b122a1-b showed better binding to b12 compared to b122a and increased protection to protease digestion. However these are partially structured as assessed by CD. In Chapter 3 we attempted to evolve stabilized versions of b122a1-b by using a genetic selection based on antibiotic resistance described previously (Foit et al, 2009). We were successfully able to show an in-vivo stability difference between b122a and b122a1-b. From the library generated in the background of b122a1-b using random mutagenesis, a few apparently stabilized mutants were isolated. Most of these mutations were hydrophobic to polar substitutions at exposed positions while a few of the mutations were substitutions with similar side chain chemistry as in wildtype. In future studies we will measure mutant stabilities and binding affinity to b12. A set of similar fragment immunogens were also designed based on subtype C CAP210 gp120 sequences. In Chapter 4 we describe various immunization studies comprising of different sets of b12 epitope based fragment immunogens. In one study we displayed some of these immunogens on Qβ VLPs. In another study, we tested subtype C based fragment immunogens. The humoral immune response was probed in terms of generation of antibodies against the immunogens using ELISA. Neutralization activity of the sera was measured in a standard TZM-bl assay. Sera raised against these particles in rabbit immunization studies could neutralize Tier1 viruses across different subtypes. The group primed with particles displaying b122a1-b and the group primed with b122a conjugated to particle in the presence of adjuvant contained significantly higher amounts of antibodies directed towards the CD4bs than sera from the group primed with empty particles and boosted with gp120. This study demonstrates the overall utility of the particle based display approach. In immunization studies with subtype C derived fragment immunogens as primes, no significant neutralization was seen even for Tier 1 viruses. In this study, the group primed and boosted with full length gp120 performed better than other groups suggesting that antibodies elicited against regions present in these subtype C priming immunogens are non-neutralizing. One of the rational vaccine design strategies is by stabilization of native Env trimers. In previous studies, a disulfide bond was engineered between gp120 and gp41 of Env to stabilize the interactions (SOS gp140). An I559P mutation was also introduced to stabilize the native gp41 conformation in the context of disulfide engineered Env (SOSIP gp140). The purified, soluble SOSIP gp140 immunogens were trimeric and cleaved properly and are believed to be one of the closest mimics of native Env trimers. However, these immunogens have so far failed to elicit broad neutralizing responses. In Chapter 5, we use structural information derived from high resolution atomic structure of native like cleaved gp140 BG505-SOSIP, to provide an alternate strategy to form uncleaved trimeric gp140s by cyclic permutation to design molecules that mimic cleaved trimers. The structure reveals that the gp41 C-terminus is in very close proximity (~8Å) to the N-terminus of gp120 from an adjacent subunit. We have designed a cyclic permutant of gp140 from JRFL strain where the gp41 C terminus is now connected to the gp120 N-terminus with a short linker. This novel connectivity results in preservation of the native gp41 N-terminus along with a much shorter linker length than in conventional gp140. This might promote trimer folding and stabilization because of the resulting decreased magnitude of conformational entropy change during folding. The structure also reveals that the gp120 C-terminus is close to the trimer axis, and due to cyclic permutation, this becomes the new C-terminus of gp140. To further stabilize the trimeric form, we have attached a foldon trimerization domain at the C terminus. The protein has been expressed and purified from mammalian cells. The protein exists primarily as a trimer in solution as assessed by SEC-MALS. It shows better binding to broadly neutralizing antibody b12 when compared to b6, a non-neutralizing antibody. Further biophysical characterization of the protein is in progress. We have previously described design of a bacterially expressed outer domain derivative of gp120 (ODEC) that had V1/V2 and V3 loops deleted and bound CD4 (Bhattacharyya et al, 2010). To improve the initial ODEC design, three different rational design strategies were used. In the first approach, residue frequency based methods were used to design a construct named ODECConsensus. In another approach, a cyclic permutant of ODEC (CycV4OD) was designed with new N and C termini in the flexible V4 loop. In the third approach the bridging sheet (BS) region was deleted from ODEC to form ODECΔBS. In Chapter 6 we have used hydrogen deuterium exchange-mass spectrometric analysis (HDX-MS) to study conformational flexibility of these fragment immunogens. These studies revealed that all the three immunogens show reduced conformational flexibility compared to ODEC. 5-7 protons remain protected up to 2 hours whereas for ODEC, exchange completes at 20 minutes. This reduced flexibility correlates with 6-20 fold tighter VRC01 binding relative to ODEC. In rabbit immunizations, all three constructs elicit significant gp120 titers as early as week 6 in the absence of any gp120 boost whereas ODEC shows significant gp120 titers only after two gp120 boosts. Week 24 sera elicited after immunization with ODECΔBS, ODECConsensus and CycV4OD boosted with gp120 show neutralization of multiple Tier 1 viruses from subtype B and C, whereas corresponding ODEC immunized animals failed to show a neutralizing response. This study demonstrates that reduced conformational flexibility correlates with better antigenicity and an improved immunogenicity profile for these fragment immunogens. Also we have used HDX-MS studies to one of the stem based HA fragment immunogen pH1HA10-foldon described previously (Mallajosyula et al, 2014) to do peptide finger printing and find regions of protein showing increased protection to hydrogen deuterium exchange and thus derive some structural insights about this trimeric fragment immunogen. Peptide mapping experiments show that the HA stem fragment peptides are exchanging rapidly with more than 90% exchange completing by 30 s for most of the peptides. The well folded foldon trimerization domain peptide shows a very slow exchange profile. A few of the HA peptides exchange slowly with 1-2 protons exchanging after 30 s. Fast exchange seen for this fragment immunogen may be due to truncation of the stem region leading to greater solvent accessibility of the trimer interface.

Le travail décrit dans ce manuscrit est consacré à la synthèse et à la caractérisation structurale d'une nouvelle famille de γ-amino acides hétérocycliques contraints, les ATCs (acides 4-Amino-(méthyl)-1,3-Thiazole-5-Carboxyliques). Ces monomères sont construits autour d'un noyau thiazole inséré entre les carbones Cα-Cβ permettant de limiter la valeur de l'angle dièdre ζ à 0°. La présence de deux points de substitution, sur le carbone γ asymétrique et en position 2 du noyau aromatique, autorise une large diversification structurale des ATCs. Des séries d'oligomères, i.e. dimères, tétramères et hexamères solubles dans les solvants organiques halogénés, dans les alcools et dans l'eau ont été synthétisées par couplages peptidiques. La conformation adoptée par ces séquences a été déterminée en solution par RMN 1D et 2D associées à des techniques de modélisation sous contraintes RMN. Nous avons montré que les oligomères d'ATCs s'arrangeaient en hélices 39 droites, présentant un pas de 11,8 Å. La conformation hélicoïdale est stabilisée par un réseau de liaisons hydrogène de type CO(i)…NH(i+2) s'établissant tout au long de la séquence, faisant entrer les oligomères d'ATCs dans le monde des foldamères. Des mesures de dichroïsme circulaire ont permis d'apprécier la stabilité conformationnelle des édifices et l'analyse à l'état solide par diffraction des rayons X a confirmé l'arrangement observé par RMN. Dans une seconde partie nous avons étudié le rôle structurant d'un motif ATC placé au sein d'une petite séquence peptidique. Nous montrons au travers d'expériences RMN et de calculs théoriques que la configuration du monomère d'ATC conditionne la conformation de peptides hybrides αγαα en solution. En termes d'application nous décrivons l'utilisation du motif ATC comme mime de coude pour concevoir un analogue fonctionnel de la gramicidine S, un cyclodécapeptide symétrique antibactérien. La dernière partie de ce travail concerne nos efforts pour développer, à partir des connaissances acquises quant à la structure tridimensionnelle des oligomères d'ATCs, des inhibiteurs de l'interaction protéine-protéine STAT6-NCoA-1. / The work described herein is devoted to the synthesis and the structural characterization of a new family of heterocyclic constrained γ-amino acids, named ATCs (4-Amino-(methyl)-1,3-Thiazole-5-Carboxylic acids). These building-blocks are built around a thiazole ring inserted between the Cα-Cβ carbons allowing the limitation of the ζ dihedral angle value to 0°. The presence of two diversification points both on the γ asymmetric carbon and on the position 2 of the aromatic ring, allows a large structural diversification of the ATCs. Series of oligomers consisting in dimers, tetramers and hexamers soluble in halogenated solvents, alcools and water have been synthesized according to peptide chemistry. The conformations of the sequences have been studied by various NMR experiments associated to modelling studies led under NMR constraints. The ATC oligomers adopt a right 39 helical shape, owning a pitch of 11.8 Å which has been confirmed by crystallography. The helix is stabilized by a conserved hydrogen bond pattern between CO(i)…NH(i+2) occurring all along the sequence axis. Circular dichroism measures have been done to check the conformational stability of the architectures. In the second part of the manuscript, we demonstrate by NMR and theoretical computing that when included in a short peptide sequence, ATCs could act as turns. The derived application consists in optimizing the biological behaviour of the ATC moiety as a turn mimetic thanks to the design and the antibacterial evaluation of a gramicidin S analogue. Based on our knowledge about the three-dimensional structure of ATC oligomers, the last part of this work deals with our efforts to develop inhibitors of protein-protein interaction STAT6-NCoA-1.

Thyroid hormones (THs; T4 and T3), secreted from thyroid gland, play an important role in human growth and development. T3 (3,5,3′-triiodothyronine) is the active hormone and the conversion of T4 (3,3′,5,5′-tetraiodothyronine) to T3 in cells is mediated by iodothyronine deiodinases enzymes (DIOs). DIOs are selenocysteine containing enzymes and are classified into three types (DIO1, DIO2 and DIO3). DIO1 catalyzes the outer-ring deiodination (ORD; T3 formation) and inner-ring deiodination (IRD; rT3 formation) reactions, involving in the activation (T4 to T3 conversion) and inactivation (T4 to rT3 conversion), respectively. DIO2 and DIO3 catalyse the ORD and IRD reactions, respectively. This homeostasis is regulated tightly and any deviation would lead to diseases like hyperthyroidism or hypothyroidism. Recently it is of interest to many research groups to develop iodothyronine deiodinase mimics and we have developed naphthalene-based peri-substituted thioselenol pair at 1,8-positions (1.25), which remove iodine selectively from inner-ring of T4. When selenium atom is substituted in place of sulfur (selenol-selenol pair; 1.26), the deiodination activity was ca. 90 times faster than with 1.25. This thesis deals with various aspects of the effect of substituents on the naphthalene-1,8-diselenol and solvent effect on the thyroid hormone deiodination by naphthalene-based iodothyronine deiodinase mimics. Figure 1. (A) Deiodination reactions by DIOs. (B) Chemical structure of 1.25 and 1.26. The thesis consists of five chapters. The first chapter provides a general overview about sialoproteins, thyroid hormone biosynthesis, thyroid hormone metabolism, halogen bonding, iodothyronine deiodinase mimics and proposed mechanisms for the deidoination of thyroid hormones. This chapter also introduces peri-naphthalene-1,8-diselenol (1.26), which is the key compound in this thesis and discusses about proposed mechanism for the deiodination of thyroxine involving co-operative halogen bonding and chalcogen bonding mechanism. Figure 2. (A) TH action. (B) Proposed mechanism for the deiodination of T4 by 1.26 involving cooperative halogen bonding and chalcogen bonding. Chapter 2 discusses about the synthesis, characterization and deiodination activity of a series of naphthalene-based peri-substituted-1,8-diselenols (Figure 3). These diselenols regioselectivity remove iodine from inner ring of thyroxine and other thyroid hormones, (T3 and 3, 5-T2). Substitution with different groups on the naphthalene ring did not change the regioselectivity of deiodination, indicating that the deiodination activity does not depend on the nature of substituents. Secondary or tertiary amine side chain group attached at the 2nd position of the naphthalene ring showed better activity. It is due to the secondary interaction, which facilitates the iodine removal. It was further confirmed with the substitutions at the 4th position of the ring to discriminate the possibility of electronic effect. The higher deiodination rate owing to the t-butyl group at second position of the ring also suggests that the steric effect may also play a role in the deiodination reaction (Figure 4). It is proposed that peri substituted naphthalene-1,8-diselenols remove iodine from thyroid hormones through halogen bonding-chalcogen bonding mechanism (Figure 2). The investigation of Se···Se bond distance from the crystal structures and through DFT calculation and NMR experiment showed that the stronger chalcogen bond could be the reason for the increase in the reactivity observed with substituted peri-naphthalene-1,8-diselenols. Figure 3. peri-substituted naphthalene-1,8-diselenols used for the study. Figure 4. Relative deiodinase activity of substituted-peri-naphthalene-1,8-diselenols with T4. In Chapter 3, we have discussed about the effect of chalcogen atom substitution in a series of deiodinase mimics on the deiodination of thyroid hormones. Moving from thiol-selenol pair (1.25) to selenol-selenol pair (1.26) in naphthalene based peri-substituted mimics, an increase in the activity was observed. In this chapter, we have shown that substituting with tellurium, as tellurium-thiol pair (3.3) and ditellurol (3.4) increases the reactivity of deiodination to several times and also regioselectivity of deiodination is changed from IRD in the case of 1.26 to both IRD and ORD for 3.3 and 3.4. The presence of two tellurol moieties (3.4) or a thiol-tellurol pair (3.3) can mediate sequential deiodination of T4, to produce all the possible thyroid hormone derivatives under physiologically relevant conditions (Figure 5). This study provided the first experimental evidence that the regioselectivity of the thyroid hormone deiodination is controlled by the nucleophilicity and the strength of halogen bond between the iodine and chalcogen atoms. Figure 5. (A) HPLC chromatograms of deiodination reaction of T4 with 3.3 and 3.4. (B) Chemical structure of 3.3 and 3.4. (C) Sequential deiodination reaction of T4 by 3.3 and 3.4. Chapter 4 describes the effect of alkyl conjugation at 4′-OH position of THs on the deiodination by iodothyronine mimics. In addition to the deiodination, iodothyronines undergo conjugation with sulfate and glucuronic acid group at 4′-hydroxyl position. Conjugation alters the physico-chemical properties of iodothyronines. For example, it is known that sulfate conjugation increases the rate of deiodination to a large extend. We have conjugated alkyl group at 4′-hydroxyl position of iodothyronines and investigated the deiodination reactions with reported peri-substituted naphthalene-1,8-diselenols. We observed that similar to sulfated thyroid hormones O-methylthyroxine also undergoes both phenolic and tyrosyl ring deiodination reactions and overall the rate of deiodination is increased at least by 5 times as compared with T4 under identical conditions. The phenolic iodine removal is favored by conjugation as compared to the tyrosyl ring iodine, which is similar to the observation made for T4S. Interestingly, when the acetamide group is conjugated at 4′-OH position, the regioselectivity of deiodination is changed exclusively to 5′-iodine. DFT calculations show that the positive potential on the iodine increase upon conjugation, which leads to stronger halogen bonding interaction with selenol, might be the reason for the change in the regioselectivity of deiodination. Figure 6. (A) HPLC chromatogram of deiodination reaction of T4(Me) with 1.26. (B) Initial rate comparison of T4 and T4(Me).(C) HPLC chromatogram of deiodination reaction of T4(AA) with 1.26 showing the formation of T3(AA) (ORD product). (D) Electron potential map of T4, T4(Me) and T4(AA) showing the increase in electro positive potential on 5′-iodine upon conjugation. Chapter 5 deals with the solvent effect on the deiodination reactions of THs by iodothyronine deiodinase mimics. As discussed in the earlier chapters, the deiodination reaction of thyroxine by naphthalene based-1,8-diselenols under physiological conditions produce, rT3 (IRD) as the only observable products. Surprisingly, when the deiodination reaction was performed in DMF or DMSO in the presence of 1.26, the regioselectivity of reaction was changed and the formation of both T3 (ORD) and rT3 was observed. In DMF or in DMSO, the deiodination reactivity of 1.26 was found to be 1000 fold higher than the reaction performed in phosphate buffer at pH 7.4. Figure 7. (A) HPLC chromatogram for the deiodination reaction of T4 in DMF by 1.26 showing both IRD and ORD. (B) A comparison of initial rate for the deiodination reactions of T4, T3 and 3,5-T2 in DMF and in DMSO by 1.26. (C) HPLC chromatograms for the deiodination reaction of T4 in DMF by 1.26 in the presence of TEMPO, showing the inhibition of deiodination (i) 0 mM TEMPO (ii) 10 mM of TEMPO (iii) 30 mM TEMPO. (D) HPLC chromatograms for the deiodination reaction of T4 in DMSO by 1.26 in the presence of TEMPO showing the inhibition of deiodination (i) 0 mM TEMPO (ii) 10 mM of TEMPO (iii) 30 mM TEMPO. 3,5-DIT was not denominated under physiological conditions, however, in DMF and in DMSO, 3,5-DIT was deiodinated by 2.4 to produce 3-MIT. We also observed that the control reactions in DMF or DMSO also showed a little deiodination activity. The very high reactivity observed in the presence of DMF or DMSO implied that the mechanism of denomination in these solvents may be different. It has been reported that DMSO or DMF radicals can be formed with small amounts of a base. Reaction mixture consisting of NaBH4 (for generating selenol from diselenide) and NaOH (T4 solution) may facilitate the radical formation. We also performed the reaction in the presence of TEMPO (free radical scavenger) and observed the inhibition of deiodination reaction. However, it is not clear whether the radical pathway could be one of the possible mechanisms of deiodination in these solvents by compounds 1.26 and 2.4. Further studies are required to propose a radical mechanism in different solvents such as DMF and DMSO.

Les cancers de l’ovaire demeurant particulièrement meurtriers du fait de leur capacité à développer une résistance aux chimiothérapies conventionnelles, il est donc indispensable de mettre en place de nouvelles stratégies thérapeutiques susceptibles de surmonter la chimiorésistance pour améliorer leur prise en charge. Les travaux antérieurs de l’Unité ont démontré que les protéines anti-apoptotiques Bcl-xL et Mcl-1 coopèrent pour protéger les cellules cancéreuses ovariennes contre l’apoptose et que leur inhibition concomitante conduit à la mort des cellules chimiorésistantes. A ce jour, seuls les inhibiteurs de Bcl-xL/Bcl-2 ont démontré une efficacité en clinique, en particulier l’ABT-263 (Navitoclax). En revanche, l’inhibition de Mcl-1 reste problématique dans un contexte clinique. La recherche d’outils pharmacologiques conduisant à l’inhibition ou à l’inactivation de Mcl-1, utilisables en clinique, reste donc un enjeu majeur, d’autant que cette protéine est désormais désignée comme une cible thérapeutique prioritaire dans de nombreuses localisations tumorales. C’est dans ce contexte que mon projet de thèse s’est inscrit, l’objectif étant d’identifier et d’évaluer de nouveaux inhibiteurs pharmacologiques de Mcl-1 synthétisés par des équipes de chimistes. Dans cette optique, mon projet de thèse s’est scindé en deux parties : Dans une première étude, je me suis attachée à cribler in vitro des molécules appartenant à différentes familles chimiques (Oligopyridines dérivées du Pyridoclax, « Lead » de la première génération des oligopyridines précédemment identifié par notre équipe, ou analogues de MIM1) dans le but d’identifier de nouveaux inhibiteurs de Mcl-1 plus actifs que les molécules dont ils dérivent. Ce travail a permis d’identifier la MR31367, une molécule issue du Pyridoclax qui présente une activité pro-apoptotique plus forte que ce dernier sur plusieurs lignées tumorales ovariennes chimioresistantes. Nous avons également pu mettre en évidence sur ces mêmes modèles que plusieurs molécules issues de MIM1 exhibaient une activité pro-apoptotique amplifiée. Cette étude nous a également permi de mettre en évidence une relation structure activité permettant de classer ces molécules en inhibiteurs spécifiques de Mcl-1 et « dual-inhibiteur » de Mcl-1 et Bcl-xL. Dans une seconde étude, mon travail a consisté en l’évaluation préclinique du sel de Pyridoclax. Nous avons étudié l’effet de différentes doses de Pyridoclax administré par différentes voies d’administration, en agent seul ou en combinaison avec l’ABT-263 sur différents modèles tumoraux établis à partir des lignées chimiorésistantes de cancers ovariens. Nous avons ainsi pu mettre en évidence un effet anti-tumoral du Pyridoclax (20 mg/kg/j) administré par voie IV en agent seul sur des 2 modèles des 3 modèles de xénogreffes, et cela sans toxicité avérée. Ces résultats prometteurs ouvrent des perspectives intéressantes quant à l’utilisation d’inhibiteurs pharmacologiques de Mcl-1 pour le traitement des cancers de l’ovaire / Ovarian cancer is the most leading cause of death from gynecologic malignancies because of its late diagnosis and its ability to develop chemoresistance to conventional therapies. It is now essential to develop new therapeutic strategies to overcome this chemoresistance and improve patient care. Our laboratory has demonstrated the overexpression of the Bcl-2 anti-apoptotic proteins Bcl-xL and Mcl-1 and their cooperation to protect ovarian cancer cells from apoptosis. Currently, the clinically relevant pharmacologic inhibition of Bcl-xL is available using ABT-263 (Navitoclax). However, selective direct Mcl-1 inhibition remained a challenge. This protein is one of the most important anti-apoptotic member which is expressed in multiple cancer types and is at the origin of the acquired resistance to chemotherapy and Bcl-2 family inhibitors (Navitoclax, Venetoclax). Thus, Mcl-1 inhibition represents a major challenge for the clinical success of the Bcl-2 family inhibitors. In this context, I was interested in identifying and evaluating new Mcl-1 inhibitors designed and synthetized by different chemistry research teams. My project was focused on two aspects. In the first part, we have evaluated the cytotoxic effect of different molecules derived from 2 Mcl-1 inhibitors, the Pyridoclax from the oligopyridine family and MIM1. The screening of 8 Noxa-mimetic molecules derived from Pyridoclax allowed us to identify MR31367, one of the most potent oligopyridine molecules that shows a stronger anti-apoptotic activity than Pyridoclax (15μM) and sensitizes different chemoresistant ovarian cancer cell lines (IGROV1-R10, SKOV-3 and A2780) to anti-Bcl-xL strategies (ABT-737, siRNA). Furthermore, we have evaluated the pro-apoptotic activity of 14 MIM1 derivative molecules using the same cellular model. This study has demonstrated that most of these derivatives have greater pro-apoptotic activity than MIM1. We have also established the structure-activity relationship leading to classify these molecules as “Mcl-1 inhibitors” or as “dual inhibitors” of Mcl-1 and Bcl-xL.The second part of my project was focused on the preclinical evaluation of Pyridoclax hydrochlorid. We have analyzed the antitumor activity of Pyridoclax hydrochlorid in several subcutaneous xenografts derived from human ovarian cancer cell lines (IGROV1-R10, SKOV-3 and A2780). Different routes of Pyridoclax hydrochlorid administration were tested (oral, IV and IT) and its antitumor effect was analyzed at different doses as single agent or in combination with ABT-263 (100mg/kg). This study highlighted an effective antitumor activity of 20mg/kg of Pyridoclax hydrochlorid administered intravenously as single agent in two of three xenograft models without any side effects. This results open up interesting perspectives for the clinical use of Mcl-1 inhibitors to improve the clinical management of ovarian cancer

Ce travail de thèse a pour objectif la simplification de la préparation d’un nouveau type d’inhibiteur d’entrée du VIH conçu, synthétisé et validé dans le cadre d’une collaboration entre le laboratoire, l’Institut de Biologie Structurale de Grenoble et l’Institut Pasteur de Paris. Ce prototype est constitué d’un mime fonctionnel de CD4 lié de façon covalente à un fragment dodécasaccharidique d’Héparane Sulfate dont la synthèse est complexe. Nous avons donc proposé de préparer des oligomaltosides sulfatés afin de déterminer si ils pouvaient se comporter comme des mimes d’Héparane Sulfate. Dans un premier temps, nous avons mis au point la synthèse d’un précurseur trisaccharidique oligomérisable à partir de maltotriose, un trisaccharide biosourcé commercial. Au cours de ce travail, nous avons résolu trois points particulièrement délicats : l’allylation de l’extrémité réductrice du maltotriose, l’installation d’un groupement paraméthoxybenzylidène en position O-4ᴵᴵᴵ et O-6ᴵᴵᴵ et la protection sélective des positions O-6ᴵ et O-6ᴵᴵ par un groupement silylé. Les optimisations menées nous ont permis de limiter la formation de produits secondaires, d’augmenter le rendement de chaque étape. La synthèse définitive du trisaccharide oligomérisable comprend 8 étapes, a un bon rendement global de 44 % et peut être menée sans problème à des échelles allant jusqu’à 10 g. Dans un deuxième temps, nous avons étudié les réactions de glycosylation α(1→4) visant à oligomériser la brique maltotrioside : nous avons constaté qu'une activation des donneurs sous forme de N-phényltrifluoroacétimidate (PTFA) est un meilleur choix que sous forme de trichloroacétimidate (TCA). En effet, ces derniers subissent une réaction parasite du réarrangement de l’imidate en trichloroacétamide. Nous avons aussi observé que le groupement benzoyle (Bz) en position O-6ᴵᴵᴵ du donneur est préférable à l’acétyle (Ac) qui est moins stable dans les conditions acides et basiques. Ensuite, nous avons synthétisé différents donneurs PTFA possédant différents groupements protecteurs en position O-6ᴵ (TBDPS, Bn, pNO2Bz, Piv, Ac et Bz). Nos études de glycosylation avec ces différents donneurs nous ont permis de conclure qu'un groupement TBDPS en position O-6ᴵ permet d'obtenir de hauts rendements et une totale stéréosélectivité 1,2-cis dans les réactions de glycosylation et, de plus, est parfaitement indiqué comme groupement protecteur dans les étapes de diversifications ultérieures. Pour finir, nous avons optimisé différents paramètres comme : le promoteur, la température, l’ordre d’addition des réactifs, le type de tamis moléculaire, l’effet de solvant, le rapport donneur/accepteur, la concentration de l’accepteur et l’échelle de la réaction. Les conditions optimisées nous ont permis d’accéder efficacement, avec de bons rendements et une stéréosélectivité α totale, aux hexa, nona et dodecasaccharide protégés ciblés. Dans un troisième temps, nous avons réussi à réaliser les étapes de déprotection et fonctionnalisation des oligomaltosides synthétisés : débenzoylation et désilylation chimiosélective (hexa et nonasaccharide), sulfatations (hexa et nonasaccharides), puis déprotection des groupements protecteurs résiduels (hexasaccharides). La finalisation de ce projet impliquera de sulfater les nonasaccharides et de déprotéger les groupements protecteurs résiduels, puis d'introduire un espaceur fonctionnalisé sur tous les membres de la chimiothèque ainsi obtenue (hexa et nonasaccharides) et enfin de réaliser l'hydrogénolyse finale des groupements benzyles pour conduire aux oligomaltosides sulfatés libres dont les activités biologiques seront déterminées en collaboration avec nos partenaires. / This work aims at simplifying the preparation of a new type of HIV entry inhibitor, conceived, synthesized and validated within a collaboration between our group, the "Institut de Biologie Structurale" (Grenoble) and the Institut Pasteur (Paris). This prototype is composed of a CD4 functional mimetic linked to a dodecasaccharide fragment of Heparan Sulfate, whose synthesis is complex. In order to determine if Heparan Sulfate may be replaced by simpler sulfated oligosaccharides, we decided to prepare a set of sulfated oligomaltosides. To this goal, we first optimized the synthesis of an oligomerizable maltotrioside building block in eight steps and 44% global yield from maltotriose, a commercial and biosourced trisaccharide. In this work, we had to address three major points: the allylation of the reducing end of maltotiose, the introduction of a paramethoxybenzylidene group between positions O-4ᴵᴵᴵ and O-6ᴵᴵᴵ and the selective protection of the remaining primary positions O-6ᴵ and O-6ᴵᴵ by a silylated protecting group. Each step has been optimized to minimize the amount of secondary products and thus to enhance its yield. The resulting synthesis was thus shown to be highly reproducible up to ten grams scale. Then, glycoside acceptors and donors were prepared from the oligomerizable maltotrioside building block and we studied their behaviors in glycosylation reactions. We found that trichloroacetimidate activation led to poor glycosylation yields, due to the competitive formation of trichloroacetamidyl glycoside rearrangement product. Gratifyingly, N-phenyltrifluroacetimidate activation solved the rearrangement problem. We demonstrate that benzoyl group (Bz) at position O-6ᴵᴵᴵ in the donor should be preferred to acetyl (Ac) which is less stable both in acidic or basic conditions. Then, we synthesized various PTFA donors bearing different protecting groups at O-6ᴵ position (TBDPS, Bn, pNO2Bz, Piv, Ac et Bz) in order to study their influence on the yields and stereochemical outcome of the glycosylation reactions. We concluded that TBDPS represent the best compromise for efficient glycosylation and later protecting group manipulation strategies. Then we optimized various reaction parameters: promotor nature, temperature, reagents addition order, molecular sieve type, solvent nature, donor/acceptor ratio, concentrations and scale of the reaction. The optimized conditions allowed efficient access, in high yields and full α stereoselectivity, to the targeted protected hexa, nona and dodecasaccharides. Then we validated the deprotection and functionnalization steps: chimioselective debenzoylation and desilylation (hexa and nonasaccharide), sulfations (hexa and nonasaccharides), the final deprotection of the residual protecting groups (hexasaccharides).

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