CSP: Chemical Shift Perturbation CYPs: Cyclophilins DLS: Dynamic Light Scattering DMSO: Dimethylsulfoxyde DOSY: Diffusion-Ordered SpectroscopY DSF: Differential Scanning Fluorimetry DSS: Sodium trimethylsilylpropanesulfonate

Keywords: Heteronuclear Single Quantum Correlation IDP, Intrinsically Disordered Protein IPTG, Isopropyl thiogalactopyranoside NMR, Nuclear Magnetic Resonance NOESY, Nuclear Overhauser Effect SpectroscopY PFG-SE, Pulse Field Gradient Spin Echo

FPLC: Fast Protein Liquid Chromatograpphy HNCA: 3D triple-resonance NMR experiment with magnetization transfer pathway for an amino acid: amide proton to the amide nitrogen, to the alpha carbons of both the starting residue and the previous residue in the protein's amino acid sequence. HNCACB: 3D triple-resonance NMR experiment with magnetization transfer pathway for an amino acid residue: amide proton to the amide nitrogen, and then to the alpha carbons of both the starting residue and the previous residue and then to the beta carbons of both the starting residue and the previous residue in the protein's amino acid sequence. HNCO: 3D triple-resonance NMR experiment with magnetization transfer pathway for an amino acid residue: amide proton to the amide nitrogen, and then to the carbonyl of the previous residue in the protein's amino acid sequence. HN(CO)CA: 3D triple-resonance NMR experiment with magnetization transfer pathway for an amino acid residue: amide proton to the amide nitrogen, then to the carbonyl, then to the alpha carbons of the previous residue in the protein's amino acid sequence. HN(CO)CACB: 3D triple-resonance NMR experiment with magnetization transfer pathway for an amino acid residue: amide proton to the amide nitrogen, and then to the carbonyl of the previous residue in the protein's amino acid sequence, then to its alpha carbon and finally to its beta carbon. HPLC: High-performance liquid chromatography HSQC:

I wish to thank Dr. Rachel Lenoir-Capello, Edward Chalouhi, Paula Milan Rodriguez and Stephanie Davidson for their continued support and help to this project, notably Stephanie for helping me in the NMR experiments and protein expression and purification, and Edward for his support and for always finding the time to help me when I needed. I also thank the other members of the LBM whom I have not named here who have contributed by near or far to realization of my project.

And last but not least, I address my warmest thanks to my husband Robert who allowed me to achieve my project and to get where I am today. I would also thank him for his advices, encouragement and of course his presence at every moment during these years, but also to my parents, my brother Kayssar, and my sisters, Sahira, Racha and Sana who supported me until the end of this thesis and probably even beyond.

I dedicate this thesis to my husband and my daughters

General Discussion

Material and Methods

Bibliography

Usual amino acids

Abstract

Pin1 is a peptidyl-prolyl cis/trans isomerase specific for phosphorylated Ser/Thr-Pro (pS/T-P) motifs. Structurally, Pin1 is a relative small multidomain protein (18.2 kDa) composed of a PPIase catalytic domain (with preference to the substrates in cis conformation) and a WW domain (with preference to the substrates in trans conformation) that are connected by a flexible linker loop region. Previous studies showed that binding of phosphopeptide substrates to Pin1 WW domain could alter Pin1 inter-domain contacts, strengthening or weakening it depending on the substrate sequence. Thus, changes in inter-domain contacts may act as a trigger in the Pin1 mechanism. In cells, Pin1 plays a crucial role in numerous signaling pathways: it regulates the outcome of the proline-directed kinase signaling and consequently controls cell proliferation and survival. Importantly, Pin1 dysregulation can lead to a number of human diseases, including cancer and neurological disorders such as Alzheimer's disease. Pin1 has thus become a promising drug target and the development of potent Pin1 inhibitors has been an attractive topic. However, so far, the identified Pin1 peptide inhibitors lack specificity, cell permeability and/or stability.

In this project, we took advantage of the overall properties imparted by the fluorine atoms for the developpement of fluorinated peptides as a new class of Pin1 ligands. We have considered the fluorination of ligands at the C d position of pseudoproline residues to lower the rotational barrier of the cis-trans peptide bond isomerization and enhance the cis:trans population ratio.

The interaction of tens of fluorinated (or not) ligands with FL Pin1 and the isolated PPIase and WW domains were deeply studied by NMR titrations. We showed the ability of fluorinated peptides, mainly adopting the cis conformation, to bind selectively to the PPIase domain.

In a second step, we decided to take advantage of the domain binding preferences of our ligands to gain insight into the activity mechanism of Pin1. For this purpose, NMR relaxation experiments were performed in the presence of different ligands. In addition, biological assays, using fluorometric coupled tests and EXSY NMR measurements were carried out. We showed that one subset of WW ligands was able to reduce the overall dynamics of the Pin1 protein, while a second subset increased it. A similar classification can be made for the PPIase ligands. Interestingly, an allosteric mechanism was demonstrated where modification of Pin1 protein dynamics by ligation of a WW ligand correlated with an increase in affinity for the first subset of the PPIase domain ligands, while the affinity of the second subset was decreased. In this regard, our results established that the WW domain can provide a targeting role for PPIase activity, as Pin1 has more than 80 protein partners identified to date.

Another part of my work explored the oligomerization properties of the Pin1 protein in solution.

Based on several crystallographic structures of Pin1 which present a dimer in their asymmetric units and MD simulations to assess the stability of the dimer with or without ligands, we decided to test whether the dimeric form of Pin1 remained present in solution. For this purpose, three different techniques have been employed, DLS, DOSY and relaxation experiments by NMR. Interestingly, at concentration higher than 1 mM, results revealed the presence of oligomeric forms of Pin1 in solution. Additional data are still needed to confirm this result and evaluate its implication on the Pin1 catalytic mechanism.

Résumé

Pin1 est une peptidyl-prolyl cis/trans isomérase spécifique des motifs Ser/Thr-Pro (pS/T-P) phosphorylés. Pin1 est une protéine relativement petite (18,2 kDa) composée d'un domaine catalytique PPIase (avec une préférence pour les substrats en conformation cis) et d'un domaine WW (avec une préférence pour les substrats en conformation trans) qui sont reliés par une bras flexible. Des études antérieures ont montré que la liaison de substrats phosphopeptidiques au domaine WW pouvait modifier les contacts inter-domaines de Pin1, en les renforçant ou les affaiblissant selon la séquence du substrat. Ainsi, les changements dans les contacts interdomaines peuvent agir comme un modulateur du mécanisme catalytique de Pin1.Dans les cellules, Pin1 joue un rôle crucial dans de nombreuses voies de signalisation : elle régule l'issue de la signalisation des kinases dirigées par la proline et, par conséquent, contrôle la prolifération et la survie des cellules. Il est important de noter que la dérégulation de Pin1 peut conduire à un certain nombre de maladies humaines, y compris le cancer et les troubles neurologiques tels que la maladie d'Alzheimer. Pin1 est donc devenue une cible thérapeutique prometteuse et le développement d'inhibiteurs puissants de Pin1 est un sujet qui intéresse de nombreux chercheurs. Cependant, jusqu'à présent, les inhibiteurs peptidiques de Pin1 identifiés manquent de spécificité, de perméabilité cellulaire et/ou de stabilité.

Dans ce projet, nous avons choisi de profiter des propriétés particulières conférées par les atomes de fluor pour développer des peptides fluorés comme nouvelle classe d'inhibiteurs de Pin1. Nous avons tenté d'incorporer le groupe CF 3 sur la position C d d'acides aminés pseudoproline afin d'abaisser la barrière d'énergie de l'isomérisation de la liaison peptidique cis-trans et d'augmenter le rapport de population cis:trans. L'interaction de dizaines de ligands fluorés (ou non) avec FL Pin1 et ses domaines PPIase et WW isolés a été étudiée en profondeur par RMN. Nous avons montré la capacité des peptides fluorés, adoptant majoritairement une conformation cis, à se lier sélectivement au domaine PPIase. Dans un deuxième temps, nous avons décidé de profiter de la selectivité de nos ligands envers un domaine particulier pour mieux comprendre le mécanisme d'activité catalytique de Pin1. Dans ce but, des expériences de relaxation par RMN ont été réalisées en présence de différents ligands. De plus, des tests biologiques, utilisant des tests fluorimétriques et des mesures RMN EXSY ont été réalisés. Nous avons montré qu'un sous-ensemble de ligands WW était capable de réduire la dynamique globale de la protéine Pin1, tandis qu'un second sous-ensemble l'augmentait. Une classification similaire a pu être faite pour les ligands du domaine PPIase. De façon intéressante, un mécanisme allostérique a été démontré où la modification de la dynamique de la protéine Pin1 par la ligation d'un ligand WW est corrélée à une augmentation de l'affinité pour le premier sous-ensemble de ligands du domaine PPIase, alors que l'affinité du second sous-ensemble est diminuée. À cet égard, nos résultats établissent que le domaine WW peut jouer un rôle de ciblage pour l'activité PPIase, car Pin1 a plus de 80 partenaires protéiques identifiés à ce jour.

Une autre partie de mon travail a consisté à explorer les propriétés d'oligomérisation de la protéine Pin1 en solution. Sur la base de plusieurs structures cristallographiques de Pin1 qui présentent un dimère dans leur unité asymétrique et de simulations MD pour évaluer la stabilité du dimère avec ou sans ligands, nous avons décidé de tester si la forme dimérique de Pin1 reste présente ou non en solution. A cette fin, trois techniques différentes ont été employées, la DLS, des expériences DOSY et des mesures de relaxation par RMN. De manière intéressante, à une concentration supérieure à 1 mM, nos résultats ont révélé la présence de formes oligomériques de Pin1 en solution. Des données supplémentaires sont encore nécessaires pour confirmer ce résultat et évaluer son implication sur le mécanisme enzymatique de Pin1.

Résumé Français

Le repliement des protéines est un processus par lequel une chaîne polypeptidique se replie pour devenir une protéine biologiquement active basée sur sa structure 3D native. Ce processus est principalement guidé par des interactions hydrophobes, la formation de liaisons hydrogènes intramoléculaires, les forces de van der Waals, mais a un coût énergétique du fait de la perte d'entropie conformationnelle.

De plus, comme le processus de repliement des protéines peut rencontrer des obstacles cinétiques, il est souvent assisté par des protéines appelées chaperons. Les chaperons moléculaires correspondent à une classe de protéines qui aident au repliement correct d'autres protéines in vivo. Des chaperons existent dans tous les compartiments cellulaires. Ils interagissent avec la chaîne polypeptidique de la protéine partenaire afin de lui permettre d'adopter sa conformation tridimensionnelle native. On distingue plusieurs classes de protéines chaperonnes, capables d'accélérer des processus lents pendant le repliement, parmi lesquelles figurent les peptidyl prolyl cis/trans isomérases ou PPIases.

En effet, la nature double liaison partielle de la liaison peptidique ainsi que l'encombrement stérique des chaines latérales des acides aminés sont à l'origine de hautes barrières énergétiques pour l'isomérisation cis à trans et trans à cis des liaisons peptidiques [START_REF] Scherer | Barriers to rotation of secondary amide peptide bonds[END_REF][START_REF] Schutkowski | Role of phosphorylation in determining the backbone dynamics of the serine/threonine-proline motif and Pin1 substrate recognition[END_REF]. De manière générale, les liaisons peptidiques adoptent très majoritairement des conformations trans, plus stable en energie, mais la proline qui est le seul acide aminé comportant une amine secondaire (groupement amine lié à deux carbones Ca et Cd) possède une proportion de population cis non négligeable. Les analyses des structures protéiques montrent que jusqu'à 5-6% des liaisons peptidiques précédant les prolines sont en conformation cis, ce chiffre montant même à 20% dans les peptides [START_REF] Vitagliano | Preferred proline puckerings in cis and trans peptide groups: implications for collagen stability[END_REF].

L'isomérisation cis-trans de certaines de ces liaisons Xaa-Pro est nécessaire pour obtenir la structure native des protéines, mais ce processus reste lent ce qui crée un défi pour le repliement des protéines. Le rôle des PPIases, qui permettent d'accélérer d'un facteur cent à mille l'isomérisation cis-trans d'une liaison peptidique précédé une proline est donc central en biologie.

Au cours de l'évolution, les PPIases ont adopté différentes topologies et leurs structures se divisent en trois familles : les cyclophilines (CYPs), les FK-506 binding protéines (FKBPs) ainsi que les parvulines. La famille parvuline regroupe des protéines possédant un domaine catalytique de 90 à 110 résidus. Elle est en fait constituée d'uniquement trois protéines : la Le rôle du domaine WW dans l'activité de Pin1 est toujours un grand sujet d'étude. Il a été suggéré que le domaine WW servait à augmenter la concentration locale des substrats de Pin1 dans la cellule. Ainsi, bien que le domaine PPIase isolé soit capable de maintenir son activité in vitro, une perte totale de l'activité de Pin1 a été observée in vivo quand le domaine WW était absent, suggérant que le domaine WW était capable de réguler finement l'interaction et l'activité du domaine PPIase dans la cellule. Il a été proposé que cette régulation pouvait provenir d'effets allostériques médiés par les régions de contact entre les deux domaines.

De plus, il a été montré que selon la séquence du substrat se liant au domaine WW, l'interaction transitoire entre les deux domaines pouvait être favorisée ou perturbée (Jacobs et al., 2003).

Ainsi, des études RMN approfondies et des simulations par dynamique moléculaire ont pu montrer que l'allostérie entre les deux domaines se manifestait par un changement de la dynamique d'un conduit hydrophobe entre les deux domaines (allostérie dynamique) plutôt que par un réarrangement structural global de Pin1 [START_REF] Namanja | Substrate recognition reduces side-chain flexibility for conserved hydrophobic residues in human Pin1[END_REF][START_REF] Namanja | Stereospecific gating of functional motions in Pin1[END_REF][START_REF] Guo | Two pathways mediate interdomain allosteric regulation in pin1[END_REF].

Dans la cellule, Pin1 est impliqué dans différentes voies de signalisation. Pin1 joue notamment un rôle crucial dans la progression du cycle cellulaire, la transcription, la réparation de l'ADN et l'apoptose. Jusqu'à présent, plus que 82 partenaires ont été identifié comme interagissant avec Pin1 (Hoon Yu et al., 2020). Ainsi, Pin1 est impliquée dans une grande variété de maladies qui sont corrélées à des niveaux d'expression déséquilibrés de la protéine. Selon le type cellulaire, le niveau d'expression de Pin1 peut jouer un rôle essentiel dans la tumorigénèse, Alzheimer, la réponse immunitaire, l'infection microbienne ainsi que le syndrome métabolique.

Pin1 est donc devenu une cible thérapeutique prometteuse et le développement d'inhibiteurs puissants de Pin1 constitue un enjeu majeur. Cependant, jusqu'à présent, les inhibiteurs peptidiques de Pin1 identifiés manquent de spécificité, ne sont pas suffisamment affins, et/ou sont incapables de traverser la membrane dans les tests cellulaires. Notre projet vise à explorer l'impact des atomes de fluor sur des nouveaux ligands peptidomimétiques afin de cibler l'inhibition de Pin1. L'utilisation de peptides fluorés a pour objectif d'améliorer la sélectivité et le profil métabolique des inhibiteurs de Pin1, et de moduler leurs propriétés structurales et thermodynamiques. L'incorporation d'un groupe CF 3 sur le carbone C d de l'oxazolidine Ser(Y H,CF 3 Pro) diminue significative la barrière rotationnelle cis/trans (DG tc # perd environ 5

Kcal/mol par rapport à la liaison peptidique Xaa-Pro), ce qui la rapproche de la barrière d'énergie libre mesurée sur les peptides en présence de Pin1 (DG » 13 Kcal/mol) (Feytens et Pour l'expression simplement marqué 15 N-et doublement marqué 15 N, 13 C-de Pin1 FL et de ses deux domaines isolés, nous avons appliqué le protocole de Marley [START_REF] Lu | A method for efficient isotopic labeling of recombinant proteins[END_REF].

La purification de Pin1 a été faite en trois étapes, les deux premières étapes ont eu lieu par chromatographie NiNTA séparés par une étape de clivage de l'étiquette histidines. La troisième étape par chromatographie d'exclusion stérique a permis d'améliorer la pureté de la protéine et de vérifier son état d'oligomérisation. Le rendement de la protéine purifiée par litre de culture a été estimé à 14 mg/L. Malheureusement, les synthèses de ces peptides ne sont pas encore abouties au terme de mon travail de thèse.

Des études structurales [START_REF] Smet | Regulation of Pin1 peptidylprolyl cis/trans isomerase activity by its WW binding module on a multi-phosphorylated peptide of Tau protein[END_REF] La prochaine étape dans ce projet consistera à tester Pin1 en présence d'un ligand bidente capable de lier à l'une de ses extrémités le domaine WW d'un monomère et à l'autre extrémité, le domaine PPIase de l'autre monomère. Ces ligands sont actuellement en cours de synthèse.

Introduction to protein folding

Protein folding is the process by which a protein structure adopts its functional shape or conformation. This process is mainly guided by hydrophobic interactions, formations of intramolecular hydrogen bonds, van der Waals forces, and it is opposed to conformational entropy. The first most basic level of the protein structure is the sequence of amino acids themselves (Garett et al., 2010 Biochemistry fourth edition). It determines the types of stabilizing interactions in protein during their folding. Folding in vivo is in some cases cotranslational. In this case, the N-terminus of the protein begins to fold while the C-terminal of protein is still being synthesized [START_REF] Hardesty | Folding of a nascent peptide on the ribosome[END_REF]. However, other proteins undergo the major part of their folding in the cytoplasm after release from the ribosome, whereas other proteins fold in specific compartments, after translocation through membranes, such as mitochondria or the endoplasmic reticulum (ER) [START_REF] Hartl | Molecular chaperones in the cytosol: from nascent chain to folded protein[END_REF].

The configuration space of a protein folding can be visualized as an energy landscape.

According to Joseph Bryngelson and Peter Wolynes, proteins follow the principal of minimal frustration meaning that during evolution, proteins have optimized their folding energy landscapes and that nature has chosen amino acid sequences in order to obtained the most stable folded state of the protein (Bryngelson et al., 1995). In consequence, proteins are generally thought to have globally "funneled energy landscapes". They are directed toward the native state corresponding to the most thermodynamically stable structures under physiological conditions [START_REF] Leopold | Tein Folding funnels: a kinetic approach to the sequence-structure relationship[END_REF][START_REF] Dobson | Protein folding: a perspective from theory and experiment[END_REF]. Noting that this folding funnel landscape allows the protein to fold to the native state through a large number of intermediates rather than being restricted to a single mechanism [START_REF] Dill | From Levinthal to pathways to funnels[END_REF]. It is important to highlight a class of protein that is of increasing interest, which corresponds to "IDPs" for intrinsically disorder proteins, or proteins comprising intrinsically disorder domains (IDRs). This class of proteins is devoid of secondary and tertiary structures in cells, acting as a hub that can interact with several partners. The energy function of IDPs is very different from globular proteins. For IDPs, it consists of "shallow energy landscapes" with a whole continuum of conformations that have more or less the same energy.

Many details of the folding process depend on the particular environment in which folding takes place, for example the solvent (water or lipid bilayer), the concentration of salts, the pH, the temperature and the possible presence of cofactors. A particular importance in this context are the many molecular chaperones that are present in all types of cells and cellular compartments.

Chaperones may assist in protein folding as they emerge from ribosomes, whereas others are involved in guiding later stages of the folding process [START_REF] Hartl | Molecular chaperones in the cytosol: from nascent chain to folded protein[END_REF]. The main role of molecular chaperons is to stabilize an otherwise unstable structure of a protein in its folding pathway, but they do not contain the necessary information to guide the protein to its correctly native structure; rather, molecular chaperones work by preventing incorrect folding conformations. In this way, chaperones do not themselves increase the rate of individual steps in protein folding, but they enhance the overall process efficiency by reducing the probability of competing reaction, particularly aggregation and they provide a more competent pathway for the polypeptide chain to assume the correct conformations. In eukaryotic cells, many of synthesized proteins are destined for secretion to the extracellular environment. These proteins are translocated into the ER, before being secreted by the Golgi apparatus. The ER contains a wide range of molecular chaperones and folding catalysts. In addition, the proteins that fold in the ER must satisfy a 'quality control' check before being exported. Correctly folded proteins are delivered to the extra cellular environment while incorrectly folded proteins are ubiquitinated and then degraded in the cytoplasm by proteasomes [START_REF] Hammon | Quality control in the secretory pathway[END_REF].

From another side, there are several classes of folding protein that accelerate potentially slow steps in the folding process. The most important are protein disulfide isomerases, which enhance the rate of formation and reorganization of disulfide bonds, and the peptidyl prolyl isomerases, which increase the rate of cis/trans isomerization of peptides bonds involving proline residues [START_REF] Schiene | Enzymes that catalyze the restructuring of proteins[END_REF].

In humans, there are 18 FKBPs, 24 cyclophilins, and 3 parvulins. These proteins generally exhibit a modular architecture and can contain one or several PPIase domains.

The structure of a PPIase domain is typically composed of anti-parallel b-sheets that are packed against one or several a-helices. The loop between the a-helix and b-sheets forms a solvent exposed active site that binds to the target proline. The names "Cyclophilin" and "FKBP" (FK506-binding proteins) originate from their ability to bind 2 different types of immunosuppressors agents: cyclosporin A (CsA) for cyclophilins, and FK506 for FKBPs. The name of each FKBP also indicates their molecular weight (for instance 52 kDa for FKBP52).

Cyclophilins and FKBP, which are called immunophilins, play a role in the immune system during the formation of the cyclophin/CsA or FKBP/FK506 complex. These complexes helped understanding certain signal transduction pathways involving T cells. In particular, Cyclophilin and FKBP12 have been shown to act as immunosuppressive agents by blocking the activation of T cells. However, this activity does not depend on the isomerase activity, but from the interruption of signals within transductional mechanisms. Immunosuppressants such as CsA can bind to cyclophilins, inhibit their binding to other substrates, and induce an immune response. Despite their conservation over the evolution, cyclophilins and FKBPs are not essential for cell life.

Parvulins form the smallest family of PPIases. Their catalytic domain consists of approximately 90 to 110 residues. The name "parvulin" originates from the latin word "parvulus" which means "tiny". This designation is related to the low molecular weight of parvulins as compared with CYPs and FKBPs (Rahfeld et al., 1994a). Parvulin was originally discovered as a novel PPIase protein in Escherichia coli (Rahfeld et al., 1994b). Three homologs of parvulin have been identified in eukaryotes: protein interacting with the NIMA kinase (Pin1), parvulin-14 (Par14), and parvulin-17 (Par17) [START_REF] Mueller | Small family with key contacts: par14 and par17 parvulin proteins, relatives of pin1, now emerge in biomedical research[END_REF]. According to their substrate specificity, human parvulins can be subdivided into two groups. Par14 and Par17, similar to prokaryotic parvulins, exhibited the conserved isomerase activity characteristic to most PPIases. Par14 is located in the cytosol and nucleus. It might bind DNA and assist in rRNA processing for ribosome biogenesis [START_REF] Fujiyama-Nakamura | N. Parvulin (par14), a peptidyl-prolyl cis-trans isomerase, is a novel RNA processing factor that evolved in the metazoan lineage[END_REF]. Concerning Par17, there is less data about its cellular activity. However, it has been shown that Par17 promotes microtubule assembly and contains a mitochondrial targeting sequence implicated in DNA Binding [START_REF] Thiele | Parvulin 17 promotes microtubule assembly by its peptidyl-prolyl cis/trans isomerase activity[END_REF]. Pin1, the third member of the parvulin family, acts exclusively on phosphorylated Ser/Thr-Pro motifs. In addition to its C-terminal PPIase domain, Pin1 possesses an additional N-terminal domain called WW, which binds also pSer/Thr-Pro moieties but lacks isomerase activity. Pin1 is G2/M transition. However, during the G2 phase, Aurora A phosphorylates Pin1 at Ser16 to disrupt its function ability which allows the mitotic entry (Lee et al., 2013 a/b).

On the other hand, the dephosphorylation of Pin1 at Ser16 by the protein phosphatase PP2A

promotes the degradation of hBora and triggers mitotic entry. In summary, the phosphorylation of Pin1 at Ser16 has a crucial role in the Cell progression from G2 to mitosis and this pathway may explain the negatively regulator role of Pin1 in the G2/M transition.

The role of Pin1 in G1/S phase is much less known compared to its effect on the G2/M phase (Lin C. H. et al, 2015). However, several partners of Pin1, such as cyclin D1, RB, P53, and P27

are critical regulators of the cell cycle and are centrally involved in G1/S phase. In addition, E2F (section II.6.1), which enhances the expression of Pin1, is known to be a G1/S transition initiator.

II.1.2 Pin1 in gene transcription

Gene transcription is a highly regulated process that proceeds in many different steps and requires the action of DNA-binding proteins, RNA polymerases, and enzymes that modify In addition, Pin1 controls cleavage and relocation of Notch, an intercellular signaling pathway, by a direct interaction which stimulates its processing, and thus activates the nuclear, transcriptionally active form of Notch1.

Pin1 plays also a role on DNA-binding and transcriptional activity. It was shown that Pin1 regulates P53 family members (P53, P63 and P73) that play key roles in development, cell proliferation and tumor suppression. Pin1 appears to promote p53 DNA binding in stressinduced cells and the recruitment of p300 co-activator, which acetylates p53 and p73 increasing its ability to activate transcription. In the same manner, Pin1 promotes DNA binding and gene activation of Stat3, NK-kB and c-Myc transcription factors [START_REF] Mantovani | The prolyl isomerase Pin1 orchestrates p53 acetylation and dissociation from the apoptosis inhibitor iASPP[END_REF].

The most direct way by which prolyl isomerases affect gene transcription is by targeting the RNA polymerase enzyme itself. Pin1 binds directly to the carboxyl terminal domain (CTD) of Rpb1, a subunit of RNA pol II [START_REF] Xu | Pin1 modulates RNA polymerase II activity during the transcription cycle[END_REF]. The binding of Pin1 to the CTD depends on the action of the CTD kinases Cdk7, Cdk9 and Cdk12. Once bound, Pin1 might regulate the interaction of the CTD with other proteins by increasing the rate of its cis/trans isomerization.

This may involve the multimeric binding model in which the interaction of Pin1 WW domain with kinases allows the PPIase domain to isomerase the target protein (section II.5).

II.2 Pin1 related diseases

As Pin1 was shown to play a role in cell-cycle progression, apoptosis, DNA damage, and induced angiogenesis (Hoon Yu et al., 2020), it is involved in a broad variety of diseases that are correlated with unbalanced expression levels of the protein. Depending on the cell type, Pin1 protein levels can play an essential role in tumorigenesis, aging and dementia, immune response, microbial infection as well as the metabolic syndrome.

II.2.1 Pin1 overactivation in cancer

Increasing expression levels of Pin1 and its activated forms is correlated with various forms of cancer [START_REF] Wulf | Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1[END_REF]Ayla et al., 2003;[START_REF] Xu | Pin1 as an anticancer drug target[END_REF]Shi et al., 2015;[START_REF] Mcleish | Dynamic transmission of protein allostery without structural change: spatial pathways or global modes?[END_REF]. Pin1 is able to inactivate tumor suppressors and to activate up to 42 oncogene/growthpromoting regulators (Wei et al., 2015;Cheng et al., 2013). Pin1 knock out mice are highly resistant to tumorigenesis even after the over expression of oncogenes such as HER2 and HARS.

Several mechanisms have been implicated in aberrant Pin1 expression/activity in cancer. The expression of Pin1 is activated downstream of Ras and E2F, and leads to the induction of growth-promoting proteins and to the inhibition of proteins involved in cell differentiation like b-catenin, c-Jun, NOTCH1 and others (Rustighi et al, 2014). Loss of tumor suppressors such as BRCA1 is also at the origin of upregulated expression of Pin1 in cancers (Maclachlan et al, 2000). As developed in section II.6.2, the activity of Pin1 is controlled by different posttranslational mechanisms. The overexpression of some kinases, for example, is proposed to explain, the abnormal expression of Pin1 in cancers.

In the following section, the implication of Pin1 in brain and breast cancer will be detailed.

II.2.1.1 Pin1 in brain cancer

Glioblastoma is the most aggressive and the most common brain cancer in adults. The cellular origin of the glioblastoma is still unknown but recent studies suggest that astrocytes, oligodendrocyte progenitor cell, and neural stem cells could be at the origin of the cancer.

As far as Pin1 is concerned, its overexpression is clearly observed in human gliomas compared to non-cancerous brain cells. Moreover, the inhibition of Pin1 in glioblastoma cells enhances cell death response and is accompanied by increased expression of DAXX, the death-associated protein responsible for the activation of apoptotic pathways. These findings suggest that Pin1 might contribute to the survival of glioblastoma by binding to DAXX and preventing its degradation via the ubiquitin-proteasome pathway [START_REF] Ryo | A suppressive role of the prolyl isomerase Pin1 in cellular apoptosis mediated by the death-associated protein Daxx[END_REF].

Moreover, it has been shown that Pin1 is involved in glioblastoma cell migration by inducing NF-kB signaling pathway responsible for glioblastoma cell invasion [START_REF] Ryo | Regulation of NF-kappaB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA[END_REF] and in glioblastoma angiogenesis by increasing expression of vascular endothelial growth factor (VEGF). In fact, the induction of NF-kB could also activate the expression of VEFG and IL-8, another pro-angiogenic gene, in glioma cells (Xie et al., 2010).

An additional involvement of Pin1 in the brain cancer is its participation in the Warburg effect in glioblastoma. In cancer cells, the Warburg effect produced energy by glycolysis metabolism in the cytosol (fermentation) rather than by aerobic respiration pathway in mitochondria.

In fermentation, the last product of glycolysis, pyruvate, is converted into lactate (lactic acid fermentation). The binding of Pin1 to pyruvate kinase M2 (PKM2), the enzyme that catalyzes the last energy-generating step in glycolysis, promotes translocation of PKM2 to the nucleus which in turn promotes carcinogenesis at least in part by inducing c-MYC expression (Yang et al., 2012). In subsequent studies, Pin1 was shown to also play an important role in the translocation of the phosphoglycerate kinase 1 (PGK1) to mitochondria. In the mitochondria, PGK1 phosphorylates and activates pyruvate dehydrogenase kinase 1, which in turn phosphorylates and inhibits the pyruvate dehydrogenase complex responsible to promote the aerobic pyruvate metabolism pathway. This action results in a decrease of the mitochondrial pyruvate use and increase of lactate synthesis conducing to promotion of gliomagenesis [START_REF] Li | Mitochondria-Translocated PGK1 Functions as a Protein Kinase to Coordinate Glycolysis and the TCA Cycle in Tumorigenesis[END_REF].

The involvement of Pin1 in multiple oncogenic processes in glioblastoma reveals that strategies to inhibit Pin1 activity could be an avenue for anti-glioma therapies.

II.2.1.2 Pin1 in breast cancer

Pin1 plays an important role in all main cellular processes involved in breast cancer. Obvious differences have been found in the levels of expression of Pin1 in normal and human breast cancer tissues. The overexpression of Pin1 is correlated with the worse clinical outcomes, which highlights the crucial role of Pin1 in oncogenic phosphorylation-dependent pathways.

The overexpression of Pin1 might be due to activated ERBB2/HER2/NEU, RAS, and IGF signaling pathways that induce E2F, the transcription factor of Pin1 (section II.6.1). Moreover, different post-translational modifications (PTM), especially phosphorylations, have been identified that show an impact on the stability and the catalytic activity of Pin1. For example, as detailed in section II.6.2, MLK3 was shown to phosphorylate Pin1 on Ser138, increasing its activity and nuclear localization. It was observed that in breast cancer tissue, the level of pSer138-Pin1 is much higher compared to normal tissue although total protein levels were comparable. This observation suggests that the phosphorylation of Ser138 by MLK3 could be an early event in oncogenesis. Conversely, the phosphorylation of Pin1 at Ser16 by PKA resulting in a total inhibition of its catalytic activity. Accumulation of dephosphorylate Ser16 forms of Pin1 were found in human breast cancer [START_REF] Zhou | The isomerase PIN1 controls numerous cancer-driving pathways and is a unique drug target[END_REF].

Pin1 is a fundamental actor of cell proliferation; it regulates some of the proteins involved in the cell cycle, the progression and the occurrence of mitosis. One example is the Cyclin D1 which is upregulated by Pin1. The CyclinD1/CDK pathway regulates in turn the interaction of Pin1 with pRB. This interaction fosters the hyper-phosphorylated form of pRB in breast tumor cells, triggering cell proliferation. Other examples are the proteins MYC and Cyclin E that directly interacts with Pin1 and are required for the G1/S phase.

Moreover, Pin1 strengthens the degradation of FBXW7 leading to enhanced proliferative activity in breast cancer. On the other hand, Pin1 is essential for the cis/trans isomerization of the phosphorylated PTP-PEST protein. The trans conformation of PTP-PEST favors its interaction with FAK1, which lead to the dephosphorylation of FAK1 and promoting migration, invasion and metastasis (Zheng Y et al., 2011).

The implication of Pin1 in these chosen signaling pathways shows that its inhibition might be relevant for therapeutic strategies for the breast cancer.

II.2.2 Pin1 and Alzheimer's disease

Alzheimer's disease (AD) is a chronic neurodegenerative disease which is associated with massive loss of neurons, intracellular accumulation of neurofibrillary tangles, and extracellular deposits of beta-amyloid plaques in the brain. According to the World Health Organization on 2017, AD is the cause of 60-70% of cases of dementia, a broad category of brain diseases that cause deterioration in memory, thinking, behavior and the ability to perform everyday activities.

It has been shown that Pin1 plays a critical role in AD by affecting two proteins thought to be key to the disease: the amyloid precursor protein (APP) and the microtubule-binding protein Tau. In neuronal tissue, Pin1 expression increases cell differentiation and remains high throughout the lifespan. In mice, Pin1 knockout leads to a premature-age-dependent neurodegeneration similar to AD in humans, while the overexpression of Pin1 protects mature neurons against neurodegeneration induced by the overexpression of Tau [START_REF] Lim | Pin1 has opposite effects on wild-type and P301L tau stability and tauopathy[END_REF].

From these observations, it was concluded that inactivation or down-regulation of Pin1 might contribute to AD development.

Pin1 and Tau protein

Tau protein promotes the assembly and stability of microtubules that form the neuron's scaffolding. The interaction of Tau protein with microtubules is regulated by phosphorylation.

Tau is a phosphoprotein that contains about 80 potential phosphorylation sites. When Tau gets hyperphosphorylated, it becomes dysfunctional and can no longer interact with microtubules, leading to self-aggregation into insoluble filaments that form neurofibrillary tangles. It has been

shown that the binding of Pin1 at the phosphorylated Thr231-Pro motif of Tau (pT231-Tau)

promotes its cis to trans conformation. Furthermore, Pin1-mediated isomerization of phosphorylated Tau (i) restores the Tau ability to promote microtubule assembly, (ii) facilitates the Tau dephosphorylation, (iii) favors the Tau degradation, and (iv) prevents Tau aggregation in vivo and in vitro [START_REF] Lim | Pin1 has opposite effects on wild-type and P301L tau stability and tauopathy[END_REF][START_REF] Liou | Role of the prolyl isomerase Pin1 in protecting against age dependent neurodegeneration[END_REF]. In addition, Pin1 can inhibit Tau kinases such as GSK3-b to prevent Tau hyperphosphorylation [START_REF] Ma | Prolyl isomerase Pin1 promotes amyloid precursor protein (APP) turnover by inhibiting glycogen synthase kinase-3β (GSK3β) activity: novel mechanism for Pin1 to protect against Alzheimer disease[END_REF]. Thus, Pin1 appears to use different mechanisms to avoid Tau from gaining toxic function and to protect against neurodegeneration in AD.

Pin1 and APP processing

Amyloid Precursor Protein (APP) is implicated in synaptic formation and repair, neural plasticity, antimicrobial activity and iron export. APP is the precursor molecule whose proteolysis generates beta amyloid (Ab), the primary component of amyloid plaques found in the brains of Alzheimer's disease patients.

The processing of APP can be regulated by phosphorylations, mainly governed by GSK3-b.

The hyperphosphorylation of APP yields a pathogenic form of Ab leading to increased amyloid pathology. Pin1 binds APP at the threonine 668 (Thr668-Pro), accelerates its isomerization from cis to trans by over 1,000-fold, as visualized by NMR (Ramelot and Nicholson., 2001;Pastorino et al., 2006), stabilizes APP at the plasma membrane, and ultimately promotes the non-amyloidogenic processing pathway. Overexpression of Pin1 decreases Ab secretion in vitro whereas Pin1 ablation in mice promotes amyloidogenic APP processing in an age dependent manner. Indeed, at 15 months of age, but not at 6 months of age, Pin1 knockout (KO) mice exhibit increased levels of insoluble Ab42, a major toxic species (Pastorino et al., 2006;[START_REF] Ma | Prolyl isomerase Pin1 promotes amyloid precursor protein (APP) turnover by inhibiting glycogen synthase kinase-3β (GSK3β) activity: novel mechanism for Pin1 to protect against Alzheimer disease[END_REF]. Moreover, Pin1 can also directly inhibit the phosphorylation of APP by et al., 2005). This discrepancy might be due to the usage of bcleaved carboxy-terminal fragment instead of full-lenght APP and detection of the brain of mice regardless of their age (Wang L et al. 2020). Thus, under physiological conditions, sufficient levels of Pin1 protect healthy neurons against Ab pathology while the inhibition of its activity leads to increase of the amyloidogenic APP processing, resulting in Ab production an Ab plaques in the AD brain.

II.2.3 How to explain the inverse association between cancer and Alzheimer's disease?

The first evidence of an inverse association between AD and cancer came from autopsy and case control studies. A number of cohort studies achieved over the past decade, have found convincing evidence that people with prevalent cancer had lower risk of ever developing Alzheimer's disease, and those with prevalent Alzheimer's disease had lower risk of being admitted to hospital for cancer. Morris and coworkers analyzed data from a longitudinal memory cohort over a mean follow-up of 10 years. People with AD had a hazard ratio (HR) of 0,39 for the development of cancer compared to those without AD, after adjustment for age, gender and level of education. On the other hand, those with a history of cancer appeared to develop AD at a slower rate [START_REF] Roe | Cancer linked to Alzheimer disease but not vascular dementia[END_REF]. These studies showed that this "protective" association is not simply due to decreased survival in people with cancer or simply due treatment for cancer or AD, because this inverse relation between cancer and AD is seen before and after the diagnosis of each condition.

It is striking that very different levels of Pin1 expression have been found between cancer cells and neurons of AD patients. Deciphering the molecular interactions involving Pin1 in different biological mechanisms could help to explain this unusual epidemiologic dichotomy. The propensity of neurons to undergo apoptosis and that of cells to become malignant rely on very different mechanisms. Dividing cells depends on mitosis while neurons do not retain the ability to complete the mitosis once mature. Furthermore, neurons do have adequate machinery for programed cell death and will undergo apoptosis in case of overwhelming damage. Thus, genes controlling the cell cycle, cell survival and apoptosis might be differentially regulated in neurons and dividing cells.

In addition, a transcriptomic meta-analysis seeking genes that are differentially regulated in cancer and AD identified Pin1 along with Wnt, and P53 (Ibanez et al., 2014). Thus, further investigation of the genetic and metabolic overlap between cancer and AD will yield important therapeutic insights for both diseases and may confirm the importance of Pin1 level expression in cancers and AD disease.

II.3 Structure of Pin1

The first structure of human Pin1 was obtained in 1997 by X-Ray crystallography as a complex with an ala-pro dipeptide [START_REF] Ranganathan | Structural and functional analysis of the mitotic peptidyl-prolyl isomerase Pin1 suggests that substrate recognition is phosphorylation dependent[END_REF] In another report, the interaction of Pin1 variant W34A with the Cdc25 peptide was investigated. This peptide containing the pT48-P49 site of the mitotic phosphatase Cdc25C

targeted by Pin1 was found to have both reduced binding affinity and reduced activity compared to WT Pin1 (Wang et al., 2015). W34 is proposed to be significant for the binding site of WW domain specially by interacting with the proline at position +1 from the phosphorylated residue.

The contribution of each individual residue to the stability of the WW domain has been tested by random mutagenesis in full-length Pin1 [START_REF] Jäger | Structure-function-folding relationship in a WW domain[END_REF]. It was concluded that the highly conserved residues Trp11, Tyr24, Asn26, and Pro37 are essential for structural stability.

Finally, it should be noted that the WW domain interacts with both pSer-Pro peptide bonds of RNA Polymerase II CTD in their trans conformation. In addition, the NMR structures of the WW domain bound to two phosphorylated peptides, one derived from the Cdc25 phosphatase (EQPLpTPVTDL), and the other from the human tau protein (KVSVVRpTPPKSPS), showed that the bound peptides adopt the trans conformation [START_REF] Wintjens | 1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides[END_REF]. These results

clearly reveal that the WW domain selects the pT/S-P motif with the prolyl peptide bonds in the trans conformation.

II.3.2 PPIase domain

PPIase domain of human Pin1

The structure of hPin1 co-crystallized with the Ala-Pro dipeptide in cis conformation was the first structure of a parvulin member (PDB code: 1PIN, [START_REF] Ranganathan | Structural and functional analysis of the mitotic peptidyl-prolyl isomerase Pin1 suggests that substrate recognition is phosphorylation dependent[END_REF]. The Pin1

PPIase domain exhibits an overall fold composed of a 4-stranded b-sheet surrounded by 4 ahelices. The catalytic center is located in the concave side of the b-sheet topped by the long loop between b 1 and a 1 . In the X-Ray structure, the side chains of the basic residues Lys63, Arg68 and Arg69 sequester a sulfate ion located adjacent to the alanine of the ala-pro moiety (Figure 10). The presence of a sulfate ion in the basic cluster allowed to consider the potential binding to phosphorylated residues of protein partners. The catalytic selectivity of Pin1 for phosphorylated motifs was confirmed by mutating residues Arg68 and Arg69. These mutations abolished the PPIase activity as demonstrated on the phosphopeptide Cdc25 substrate (Wang et al., 2015). In addition, R68A/R69A Pin1 double mutant was unable to pull-down mitotic phosphoproteins in Hela lysate [START_REF] Innes | Non-catalytic participation of the pin1 peptidyl-prolyl isomerase domain in target binding[END_REF].

The crystal structure of human Pin1 in complex with Ala-Pro also shows a hydrophobic pocket formed by residues Phe134, Met130, and Leu122 that interact with the cyclic side chain of the

The precise role of the two highly conserved histidine residues (His59 and His157) was revealed from the characterization of their mutations on Pin1 activity and stability. A yeast complementation assay showed that the substitution of both histidines by non-hydrogen bonding residues supports viability. Trypsin degradation assays showed that the H59L mutant exhibits a ~ 5% diminished activity compared to WT Pin1, while the double mutation H59L/H157L increases the activity by ~ 15%. The activity of other H59 and H157 mutations was also reported in this study (H59F/A/N/S and H157A/N/S/F). Overall, the authors concluded that the main role of H59 and H157 is to maintain the structural integrity of the active site.

These residues may play a role in the folding of the PPIase domain, rather than being directly involved in catalysis (Baily et al., 2008).

The effect of S71 phosphorylation on Pin1 activity was also investigated. The phosphorylation of this residue located in the catalytic loop (b 1 /a 1 loop) of the PPIase domain by the DAPK1 kinase was indeed shown to inhibit Pin1 catalytic activity (Lee et al., 2011). In the full-length protein, the S71E variant (glutamate as a phosphomimetic) was found to reduce both isomerase activity and binding affinity compared to WT Pin1 (Mahoney et al., 2018). In addition, this mutation caused structural and electrostatic perturbations beyond the catalytic loop, through a network of hydrogen bonds and salt bridges spanning from the catalytic site to the opposing a2/a3 turn (Mahoney et al., 2018).

The essentials residues of the WW and PPIase domains that interact with the pSer/Thr-Pro motif The PPIase domains of Par14 and Par17 adopt virtually identical 3D structures. A significant difference between the two proteins is found in the N-terminal region, which contains 25 additional residues in the sequence of Par17. This 25-residue extension shows strong similarity with mitochondrial targeting sequences [START_REF] Lu | Function of WW domains as phosphoserine-or phosphothreonine-binding modules[END_REF].

II.3.3 Functional cooperation between the two domains: Dynamic Allostery

A high number of structural studies have attempted to understand the molecular mechanisms of Pin1 functions and how its two domains contribute to the activity and biological functions of the full-length protein in cells. The study of Pin1 by liquid-state NMR revealed inter-and intra-domain motions. In particular, it was shown that substrate binding to the WW domain changes the inter/intradomain mobility, thereby modifying the binding ability and the activity of the PPIase domain (Jacobs et al., 2003). In addition, a study by Behrsin and coworkers (Berhsin et al., 2006) showed that the WW domain is essential for the function of Pin1 catalytic activity in vivo, suggesting the existence of inter-domain communication. et al., 2005;[START_REF] Landrieu | Exploring the molecular function of PIN1 by nuclear magnetic resonance[END_REF].

Moreover, to characterize Pin1-substrate interactions, Noel et al., measured the affinity of FL Pin1 and its isolated domains towards a series of WW peptide ligands. The results showed that the Pin1 WW domain displays better affinities for these peptides when it was part of the fulllength protein rather than as an isolated domain. This suggests that a stabilization of the WW domain may be provided by the interaction with the PPIase domain. (Verdecia et al., 2000). In another study, Etzkorn and coworkers designed two peptidomimetics analogs of the Pin1 phosphoserine substrate (FFpSPR) where the peptidyl-prolyl linkage was replaced by alkene isosteres to lock the proline in either a cis or trans state. The trans-locked inhibitor was shown to bind both domains, whereas the cis-locked inhibitor was specific for the PPIase domain.

Remarkably and in contrast to WW domain properties, the PPIase domain yielded higher affinity for the cis-locked ligand when isolated rather than when belonging to the FL Pin1 (Kd PPI = 7 𝜇M, Kd Pin1 = 28 𝜇M) [START_REF] Namanja | Stereospecific gating of functional motions in Pin1[END_REF]. Despite the opposite behavior between the WW and the PPIase domains in terms of conformational preference, these results have demonstrated the existence of allosteric mechanisms in Pin1.

Further evidence of allostery was obtained by measuring the cis-trans isomerase activity of the isolated PPIase domain versus FL Pin1 by NMR. Nicholson and coworkers measured the catalyzed rate of exchange between the cis and trans isomers of the pAPP659-682 substrate, derived from the Amyloid Precursor Protein APP [START_REF] Nicholson | Dynamics of methyl groups in proteins as studied by proton-detected 13C NMR spectroscopy. Application to the leucine residues of staphylococcal nuclease[END_REF]. Interestingly, the isolated PPIase domain showed slightly greater activities (k ex = k cis® trans + k trans® cis ) than FL Pin1 (k ex = 41 ± 1 s -1 for isolated PPIase, k ex = 28 ± 1 s -1 for FL Pin1). Other NMR studies by the Peng group, in which they measured the k ex for the two phosphopeptides Cdc25 and FFpSPR, also revealed greater K ex for the isolated PPIase domain compared to FL Pin1 for both phosphopeptides [START_REF] Namanja | Stereospecific gating of functional motions in Pin1[END_REF]Wilson et al., 2013). These sparse results in vitro indicated that inter-domain contact with the WW domain is likely to alter the activity of the PPIase domain.

II.3.3.2 Evidence of allosteric effects on Pin1 dynamics

Initial NMR 15 N backbone dynamics studies of Pin1 revealed that the WW and PPIase domains undergo nearly independent rotational diffusion in the absence of substrate. This inter-domain flexibility is modulated by the interaction of phosphopeptide substrates with the loop I of the WW domain (interaction site). According to the substrate sequence, the interaction between the two domains may be alleviated or reinforced leading to different responses of the PPIase domain catalytic pocket, which suggests a connection between Pin1 flexibility and its function (Jacobs et al., 2003). The question that arises is what inter-residue interaction might sustain the allostery between the two domains of Pin1?

The classical concept of allostery involves a remote change in the protein spatial conformation which is induced by the binding of an effector molecule. These modifications resulted in increased enzymatic activity or binding affinity [START_REF] Monod | On the nature of allosteric transitions: a plausible mdoel[END_REF]Fisher et al., 2011).

Recently, a new concept of allostery, dynamic allostery, was introduced to explain the allosteric effect of ligand binding on the protein when no structural conformation change was observed [START_REF] Mcleish | Dynamic transmission of protein allostery without structural change: spatial pathways or global modes?[END_REF][START_REF] Nussinov | Allostery without a conformational change? Revisiting the paradigm[END_REF].

The dynamic allostery of Pin1 has been well studied using NMR. Indeed, an internal network of hydrophobic residues that connects the domain interface and the catalytic loop of the PPIase domain was identified [START_REF] Namanja | Substrate recognition reduces side-chain flexibility for conserved hydrophobic residues in human Pin1[END_REF] (Figure 12). The interaction of Cdc25 with the loop I of the WW domain induced a flexibility loss (stiffening) of the methyl side chains along the internal hydrophobic path, which is transmitted to the catalytic site of the PPIase domain.

This path includes residues forming the hydrophobic interface between the WW and PPIase domains, involving I28 of the b 2 /b 3 loop of the WW domain, L141, V150, and the helix a 1 of the PPIase domain, and a hydrophobic cluster, L60, L61, and V62 within the catalytic loop residues. A different behavior was observed with the FFpSPR peptide targeting the WW domain. FFpSPR induced more heterogeneous modifications that include flexibility increases or decreases on the dynamic of the side chain of the hydrophobic network described above.

allosteric regulation of the PPIase activity since the I28A mutant destabilizes the WW-PPIase interface (Wang et al., 2015). This hypothesis was supported by the binding of Cdc25 to the WW domain that decreases the inter-domain interaction and then, enhances peptidyl-prolyl isomerase activity at the PPIase catalytic loop.

Another mutation investigated on the isolated PPIase domain was S138A. The phosphorylation of S138 by mixed-lineage kinase 3 (MLK3) has been reported to increase Pin1 isomerase activity [START_REF] Wang | Dynamic allostery modulates catalytic activity by modifying the hydrogen bonding network in the catalytic site of human Pin1[END_REF]. An in vivo study comparing the activity of S138A and S139E

(phospho-mimetic) variants showed that S138E had 4 times greater catalytic activity than WT Pin1 in cellular assays, while the activity of the S138A variant was reduced by 2-fold (Rangasamy et al., 2012). S138 is located on the PPIase domain at the domain interface. The S138A mutant of the isolated PPIase domain was proposed to mimic the structural impact of the WW domain inter-domain contact. In vitro, the S138A mutant had similar affinity for the Cdc25 peptide compared to Pin1 WT but it showed ~ 20% decrease in activity. The backbone structure of the S138A mutant showed no apparent structural changes compared to Pin1 WT but this mutation significantly altered the dynamics in the catalytic site. A H/D exchange experiment revealed that S138A mutant strengthens the hydrogen bond between C113 and H59.

The stabilization of the hydrogen bond between these two residues should block the thiolate protonation of C113 which represents a pivotal step in the catalytic mechanism (see part II.4) leading to a reduced isomerization activity of the S138A mutant.

In a recent study [START_REF] Zhu | Uncorrelated Effect of Interdomain Contact on Pin1 Isomerase Activity Reveals Positive Catalytic Cooperativity[END_REF], a high number of Pin1 constructs were created with different linker lengths to evaluate their impact on the inter-domain contact and the isomerase activity while keeping the structure of the two domains intact. Linker lengths and sequences were determined based on Pin1 linkers from another species. The sequence of the ten-residue linker of human Pin1 is 40 NSSSGGKNGQG 50 . Among a series of different variants, only two deletion mutants (42DSG and 40DNSSSG) were shown to adopt a more compact conformation compared to other mutants and WT Pin1. These 2 deletion mutants showed increased catalytic activities toward the Cdc25 substrate. To verify that the increased activity was due to interdomain contact and not to direct interactions with the WW domain, the W34A/R17A mutant was investigated, W34 and R17 being precisely located at the binding site of the WW domain.

The W34A/R17A variant displayed reduced isomerase activity [START_REF] Zhu | Uncorrelated Effect of Interdomain Contact on Pin1 Isomerase Activity Reveals Positive Catalytic Cooperativity[END_REF]. When this double mutant was further modified in its linker region (40DNSSSG), an increased Pin1 activity was measured suggesting that the stabilization of the inter-domain contact can enhance the isomerase activity. These results are in disagreement with the negative allosteric regulation model of the PPIase activity by inter-domain interaction proposed by Peng et al (Wang et al., 2015).

Based on these studies, no confident conclusions can be made in regard to the accurate role of the WW domain and the inter-domain interaction on the PPIase isomerase activity and its interaction with its substrates. A more detailed study is needed in which the isomerase activity of Pin1 as well as its binding affinity will be measured in both extended and compact states of Pin1 to be able to describe the allosteric effect of WW domain on the activity of the PPIase domain.

II.4 Catalytic mechanism of Pin1

The detailed mechanism of the cis/trans isomerization catalyzed by Pin1 has not been unambiguously established, although it is now clear that PPIases operate by breaking the pbond character of the amide C-N bond. Two mechanisms have been proposed so far: a nucleophilic addition or a twisted-amide mechanism.

II.4.1 The nucleophilic-addition mechanism

The first model of the cis/trans isomerization mechanism catalyzed by Pin1 was deduced from the crystallographic structure of the complex with the Ala-Pro peptide [START_REF] Ranganathan | Structural and functional analysis of the mitotic peptidyl-prolyl isomerase Pin1 suggests that substrate recognition is phosphorylation dependent[END_REF]. The spatial arrangement of four amino acids, Cys113, His59, His157 and Ser154, discovered by Ranganathan and coworkers, suggests a covalent catalytic mechanism unique to the Pin1 class of PPIase. This mechanism involves a nucleophilic attack of the Cys113-thiolate (R-S g -H) on the carbonyl carbon atom of the peptide amide bond leading to a covalent tetrahedral intermediate between the substrate and the enzyme (Figure 13). This reaction implies the extraction of a sulfhydryl proton from Cys113 by the deprotonation imidazole nitrogen of His59. In this mechanism, the His59 would act as a catalytic base and its basic character (negative charge) would be stabilized through electrostatic interactions with the His157.

The basic cluster residues Lys63, Arg68, and Arg69 that sequester the phosphate group of the peptide represent also important anchor points for the occurring bond rotations in the isomerization step.

II.5 Proposed models for synergistic action of WW and PPIase domains in Pin1 catalysis.

In the initial investigations of human Pin1, it has been shown that the WW domain has a tenfold higher affinity in vitro for phosphopeptides than the PPIase domain [START_REF] Shen | The essential mitotic peptidyl-prolyl isomerase Pin1 binds and regulates mitosis-specific phosphoproteins[END_REF][START_REF] Smet | Regulation of Pin1 peptidylprolyl cis/trans isomerase activity by its WW binding module on a multi-phosphorylated peptide of Tau protein[END_REF]. Based on this property, it was suggested that the major role of the WW domain is to assist Pin1 in its specific substrate targeting, as well as to increase the local concentration of Pin1 substrates [START_REF] Zhou | Phosphorylation-dependent prolyl isomerization: a novel signaling regulatory mechanism[END_REF]. This concept has led to four different models for the WW/PPiase interplay in the Pin1 catalytic mechanism.

The first proposed model is the sequential binding model of Pin1 (Figure 15A). In this model, the WW domain binds a pS/T-P motif on its target protein allowing the PPIase domain to bind a second pS/T-P motif on the same substrate in the immediate vicinity (multi-sites binding). In a subtle variation of this model, the binding of a pS/T-P motif to the WW domain triggers conformation changes that allows the PPIase domain to compete with the WW domain. The pS/T-P motif can then be isomerized by the PPIase domain (single site binding). This sequential model remains widely accepted.

In the multimeric binding model (Figure 15B), the WW domain anchors Pin1 in a multiprotein complex that includes the kinase which recognizes and phosphorylates S/T-P motifs in Pin1 substrates (Jacobs et al., 2003). Following this model, the WW domain first binds and activates the kinase at pS/T-P motif (protein 1). The active kinase then phosphorylates the S/T-P motif of a nearby Pin1 substrate. Subsequently, the PPIase domain would recognize and bind the pS/T-P motif of the nearby substrate (protein 2) to exert its isomerase activity.

isomerization. The product is released with a trans conformation and subsequently, the WW domain would then bind it to prevent the reverse isomerization to occur.

Considering the fact that both domains were required for Pin1 interaction with some protein partners, Innes et al., proposed the simultaneous binding model (Figure 15D). Following a structural comparison of peptides bound to the WW and PPIase domains, they noticed that peptides with an additional Pro residue in the +1 position of the pS/T-P motif, pS/T-P-P, are targeted by the WW domain but not by the PPIase domain [START_REF] Innes | Non-catalytic participation of the pin1 peptidyl-prolyl isomerase domain in target binding[END_REF]. This observation led them to suggest that a substrate with multiple phosphorylated binding sites could allow the simultaneous binding of Pin1 to its substrate.

The role of the WW domain in Pin1 catalysis is still a matter of debate and several different models are still proposed in the literature. In addition, the collected in-vitro data still have to be validated in vivo. Phosphorylation in the PPIase domain was also shown to regulate Pin1 function. Polo-like kinase 1 (Plk1), a critical regulator of mitosis, phosphorylates Pin1 at Ser65 in the PPIase catalytic loop. Remarkably, the phosphorylation of Ser65 did not have any impact on the isomerase activity of Pin1. However, it enhanced Pin1 stability by inhibiting its ubiquitination and subsequent proteasomal degradation [START_REF] Eckerdt | Polo-like kinase 1mediated phosphorylation stabilizes Pin1 by inhibiting its ubiquitination in human cells[END_REF]. Ubiquitination is the conjugation of proteins with ubiquitin (on one or several Lys amino acid), a highly conserved 76-amino acid eukaryotic protein, and is essential for the degradation of proteins by the proteasomes (Pickart and Eddins, 2004).

II.6 Regulation of

In 2011, the Death-associated protein kinase 1 (DAPK1) has been shown to bind and phosphorylate Pin1 at Ser71. This phosphorylation inhibits the catalytic activity of Pin1 and prevents nuclear localization (Lee et al., 2011). Since Ser71 is located in the substrate binding loop of the PPIase domain, near the basic triad residues (Lys63, Arg68, and Arg69) that recognize the phosphate group of Pin1 substrates, this phosphorylation is proposed to prevent phosphorylated substrates to enter the catalytic site (Lee et al., 2011;Mahoney et al., 2018). In 2012, an increase of PPIase activity and nuclear translocation was described upon the phosphorylation of Ser138 by mixed-lineage kinase 3 (MLK3) (Rangasamy et al., 2012). These results were also observed with the S138E phosphomimetic variant of Pin1, which showed a 4 times greater catalytic activity than WT Pin1 in cellular assays (more details are provided in paragraph II.3.3.3). In cells, Pin1 phosphorylated at Ser138 promotes cell-cycle progression and cyclin D1 protein stability. All together, these studies indicate that the phosphorylation of Pin1 at multiple sites in both the WW and PPIase domains regulate its binding ability, enzymatic activity, and functions.

Pin1 function is also regulated by SUMOylation. SUMOylation consists in binding covalently a small ubiquitin-like modifier (SUMO) peptide on an acceptor lysine residue of the target protein. SUMOylation and deSUMOylation of proteins are highly dynamic processes. Their role is to regulate the biochemical properties of proteins, and to modulate in particular their activity, stability and localization. Chen and coworkers found that Pin1 is SUMOylated at Lys6 in the WW domain and at Lys63 in the PPIase domain [START_REF] Chen | SENP1 deSUMOylates and regulates Pin1 protein activity and cellular function[END_REF]. Such PTMs were shown to inhibit Pin1 catalytic activity and to reduce its binding ability for substrate proteins, abolishing its cellular function. Conversely, deSUMOylation by the SUMO protease 1 (SENP1)

after exposure to oxidative stress can restore the catalytic activity of Pin1 [START_REF] Chen | SENP1 deSUMOylates and regulates Pin1 protein activity and cellular function[END_REF].

Finally, Pin1 has been found to be oxidized in the brains of Alzheimer's disease patients. Pin1 oxidation is proposed to decrease its isomerase activity. The oxidation site in Pin1 was recently identified by mass spectrometry and X-ray crystallization [START_REF] Aluise | Peptidyl-prolyl cis/trans-isomerase A1 (Pin1) is a target for modification by lipid electrophiles[END_REF][START_REF] Chen | Pin1 cysteine-113 oxidation inhibits its catalytic activity and cellular function in Alzheimer's disease[END_REF]. Pin1 is modified by an oxidative modification on Cys113 upon hydrogen peroxide. This oxidation alters Pin1 catalytic activity and inhibits its nuclear localization, but does not affect its binding properties. Other oxidative modification sites in Pin1 have been identified at Met130

and Met146 in human neuroblastoma SH-SY5Y cells expressing Pin1 [START_REF] Ando | Tau pathology modulates Pin1 post-translational modifications and may be relevant as biomarker[END_REF],

indicating that Pin1 might have multiple oxidation sites through which its function is regulated.

Chapter 2: Pin1 inhibitors

I-Different classes of Pin1 inhibitors

As Pin1 acts on a variety of target proteins involved in multiple cellular processes, its deregulation is related to various diseases such as microbial infections, metabolic syndromes, neurodegenerative diseases, and a broad range of cancers. Pin1 has therefore become an important drug target.

In the last two decades, the development of Pin1 inhibitors has remained a challenge especially in the field of cancer studies. Indeed, a number of Pin1 inhibitors have been already developed in academic laboratories as well as in private laboratories (Pfizer Worldwide Research and Development; Vernalis R&D Ltd). For this purpose, different approaches were investigated such as low-throughput enzymatic/binding assay, high-throughput screening, virtual screening, structure-based design, combinatorial synthesis, or SAR for structure-activity relationships.

Depending on their origin, their structure, and the purpose of their development, Pin1 ligands can be classified into four different categories: inhibitors derived from natural products, small molecules inhibitors, peptide-based Pin1 inhibitors, and inhibitors to study Pin1 mechanism.

I.1 Inhibitors derived from natural products

Natural products and analogues offer a rich source of bioactive scaffolds for the development of new Pin1 drugs. To date, a dozen of different natural molecules was shown to exhibit an inhibitory activity on Pin1 with IC 50 values in the submicromolar range. Among these, the alltrans retinoic (ATRA) molecule has been used in the treatment of promyelocytic leukemia (APL).

The first described Pin1 inhibitor is the naphthoquinone Juglone that was discovered in 1998 by low-throughput PPIase screening [START_REF] Hennig | Selective Inactivation of Parvulin-Like Peptidyl-Prolyl cis/trans Isomerases by Juglone[END_REF] (Figure 17). Juglone (5-hydroxy-1,4naphthoquinone) is a reactive molecule isolated from fruit shells and leaves of walnut trees (Juglans), wish acts as a poison to many other plants [START_REF] Thomson | Naturally Occurring Quinones, But-terworths Scientific Publications[END_REF]. (Tatara et al., 2009). DTM is a putative competitor of Pin1 substrates that is able to inhibit the proliferation of colon cancer cells (HCT116). The direct interaction of DTM with Pin1 catalytic site was proposed by molecular modeling studies. In these simulations, one pentamethylene cycle was shown to make hydrophobic contacts with Leu122, Met130, and Phe134 of the PPIase domain, while the second cycle was more exposed to the solvent. The sulfur atom was not involved in the interaction through hydrogen bonds or van der Waals contacts. Interestingly, DTM did not inhibit the activity of other PPIase-containing proteins such as cyclophilin and FKBPs. However, as in the case of juglone, the structure simplicity of DTM raised the question of its specificity towards Pin1. Nevertheless, DTM can be used as a scaffold compound to develop other Pin1 inhibitors (Tatara et al., 2009).

In 2011, Dexamethasone-21-phosphate (D21P) was identified as a selective Pin1 inhibitor from a high-throughput screening of non-peptidic and non-reactive inhibitors of phosphorylation-dependent protein-protein interactions [START_REF] Graber | Selective targeting of disease-relevant protein binding domains by o-phosphorylated natural product derivatives[END_REF]. D21P is a phosphorylated derivative of the natural product cortisol (Figure 18), which is used to treat many inflammatory and autoimmune diseases. D21P selectively binds and inhibits the PPIase domain of Pin1 with a Kd » 6.8 µM and IC 50 »14.4 µM.

ATRA is an anti-cancer chemotherapy drug which is the only inhibitor used in the treatment of acute promyelocytic leukemia and breast cancer. Its biological activity could be related to the inhibition of Pin1. It facilitates the degradation of PML-RAR-a (the fusion oncoprotein promyelocytic leukemia-retinoic acid receptor a) by inhibiting the stabilizing effect of Pin1.

To date, it represents the only Pin1 inhibitor being approved by the FDA. It is important to note, however, that ATRA has a short half-life of 45 min with moderate anticancer activity against solid tumors in humans (Wei S. et al., 2015).

In 2017, Del Sal et al., identified KPT-6566, a novel selective inhibitor of Pin1 (Figure 19) also able to interact with its phosphate pocket through a carboxylate group. Mass spectrometry (MS) analysis showed that the sulfonyl acetate group of KPT-6566 was covalently bound to the sulfur atom of the Cys113 through a disulfide bridge. Consequently, a reactive quinonemimicking byproduct was released in vivo upon Pin1 binding, generating DNA damage and inducing cell death. Docking experiments indicated that KPT-6566 is stabilized in the PPIase catalytic pocket through hydrogen bonds with the side chains of Lys63 and Arg69, and hydrophobic contacts with residues involved in the recognition of the proline substrate.

Interestingly, the electrophile sulfonyl acetate moiety of the ligand directly faces the sulfur atom of the Cys113 in the docking structures. This positioning might lead to the formation of a disulfilde bond as evidenced by the MS analysis.

Interestingly, KPT-6566 does not interact with other thiol-containig PPIases. By specifically inhibiting the activity of Pin1, KPT-6566 can reduce levels and/or activity of several Pin1 partners such as Cyclin D1, NOTCH1, c-JUN, P53 [START_REF] Campaner | A covalent PIN1 inhibitor selectively targets cancer cells by a dual mechanism of action[END_REF]. Moreover, KPT-6566 was also shown to increase ROS (Reactive Oxygen Species) levels in cancer cells, resulting in the increase of DNA damage-induced cell death.

This mechanism of action makes KPT-6566 a unique ligand compared to the other natural Pin1 inhibitors. Indeed, while juglone also binds to Pin1 in a covalent manner, its relative simple structure questions its specificity. Furthermore, unlike juglone and PiB that recognize and bind other parvulins, KPT-6566 is highly specific towards Pin1. In addition, KPT-6566 has an advantage over ATRA that showed less potency towards Pin1 likely due to its non-covalent mechanism of action and its very short life time in vivo.

domains [START_REF] Urusova | Epigallocatechin-gallate suppresses tumorigenesis by directly targeting Pin1[END_REF]. The interaction of EGCG with the WW domain involves Ser16 and Arg17 of the loop1. As mentioned in chapter 1, this loop is responsible for recognition and binding of the WW domain to its substrates. Furthermore, the phosphorylation of Ser16 can inhibit Pin1 binding by preventing the WW domain to recruit the substrate. Within the PPIase domain, the gallate moiety of EGCG forms hydrogen bounds with Asp112, Ser114, and Trp73 that are localized near the active site. Furthermore, the negative charge of EGCG is proposed to increase the pKa of Cys113, which might reduce the nucleophilic character of this residue and inhibit the isomerase activity [START_REF] Bayer | Structural analysis of the mitotic regulator hPin1 in solution -Insights into domain architecture and substrate binding[END_REF]. The binding of EGCG to both Pin1 domains might explain its inhibitory activity in vivo. Indeed, Urusova et al. showed that EGCG was able to decrease the abundance of cyclin D1 and NFκB promoter activity in cells expressing Pin1, suggesting that the anti-proliferative effects of EGCG on tumor cells are directly related to Pin1 suppression [START_REF] Urusova | Epigallocatechin-gallate suppresses tumorigenesis by directly targeting Pin1[END_REF].

I.2 Small molecule Pin1 inhibitors

Another class of presumably reversible small molecule inhibitors has been reported. Within this class, various chemical scaffolds were used as starting points to develop potent Pin1 inhibitors.

The design of these new molecules was often accompanied with structural studies to confirm their direct interaction with Pin1. Most of these compounds contain a phosphate group as a strategy to target the positively charged pocket of the catalytic site, although its presence may strongly limit cell permeability. In the more recent investigations, the drug design process aimed at replacing the phosphate group by other groups exhibiting improved pharmaceutical properties.

In the next section, we describe the work of three different laboratories that developed submicromolar affinity Pin1 inhibitors, and present their strategies to improve the permeability of their compounds. These compounds will be denoted by numbers.

A structure-based approach was used by Pfizer Worldwide Research and Development to design new specific inhibitors of Pin1, starting from FKBPs ligands as scaffold molecules [START_REF] Guo | Structure-based design of novel human Pin1 inhibitors (I)[END_REF]. The binding ability of these new compounds was evaluated by molecular docking using X-Ray structures of Pin1. Compound 1, with benzothiophenyl and 3-fluorophenylalanine groups attached to a peptide pond, showed a Ki value of 6 nM. The addition of a fluorine atom at the C-3 position of the phenyl was shown to improve dramatically its inhibitory activity via its higher contribution to Pin1 binding. The X-Ray structure of the derivative of compound 1 authors suggested that the relatively spacious phosphate binding pocket of Pin1 would facilitate the rotation of the phosphate group during isomerization, preserving the electrostatic contacts with the basic side chains. As seen with small ligands in the previous section, the hydrophobic core here represented by the pipecolic ring makes interactions with the proline-binding pocket comprising Leu122, Phe125, Met130, and Phe134. These hydrophobic interactions are essential for the binding of all Pin1 ligands, regardless of their structure.

In another approach, the Pei's group employed a strategy based on cyclic peptides to identify new Pin1 inhibitors [START_REF] Liu | Membrane permeable cyclic peptidyl inhibitors against human Peptidylprolyl Isomerase Pin1[END_REF]. One advantage of cyclic peptides is to be more resistant to proteolytic degradation. Also, cyclic peptides present a more rigid conformation, which can improve their affinity for the target and enhance membrane permeability. A cyclic peptide library was designed with the following scaffold: cyclo(aX 1 X 2 X 3 X 4 X 5 a n E)BBNBRM-resin, where X 1 to X 5 represent random amino acids and "a" is D-Ala. The glutamate residue (E) serves as a convenient anchor for attachment to the solid phase and N-to-C peptide cyclization.

The invariant BBNBRM sequence was chosen to provide a flexible linker between the cyclic peptide and the resin.

The most potent peptide found, cyclo(d-Ala-Sar-D-Thr(PO3H2)-Pip-Nal-Tyr-Gln)Lys-SH (compound 15a), potently inhibited Pin1 (IC 50 ≈ 32 nM) but had no activity activity in cell assays. However, a derivative was synthesized, where an octa-arginine was linked via a disulfide bond to increase the membrane permeability (compound 15b). Treatment of cells with 100 nM of peptide 15b increased levels of negatively regulated Pin1 partners which demonstrated the intracellular inhibition of Pin1 leading to cell cycle arrest.

reduced-amide backbone leading to an entropic penalty, and less flanking residues than other potent Pin1 inhibitors.

To further understand the mechanism of Pin1 activity, cyclohexyl ketone inhibitors (compound 25) were synthesis by the Etzkorn group as electrophilic acceptors of the Cys113 thiol nucleophile located in the Pin1 active site. We have previously described two non-peptidic ligands that covalently bind to this residue and then mimic the tetrahedral intermediate binding mode (juglone and KPT6566). If Pin1 catalysis involved a nucleophilic addition mechanism, the Cys113 must be nearby to the substrate in crystal structure. Molecular modeling suggested a preference for the trans-diaxial cyclohexane conformation which would be stretched to a trans-pyrrolidine conformation after bonding with Pin1. Furthermore, the sulfur of Cys113 is too far from the ketone carbon of the substrate to allow efficient nucleophilic addition [START_REF] Xu | Cyclohexyl Ketone Inhibitors of Pin1 Dock in a Trans-Diaxial Cyclohexane Conformation[END_REF]. In addition, the C113D mutant has been shown to be catalytically active. Taken together, these data indicate that the nucleophilic addition mechanism is not a satisfactory model to describe the Pin1 catalysis mechanism.

II -Fluorinated inhibitors for Pin1

In 3) [START_REF] Bégué | Fluoroartemisinins: metabolically more stable antimalarial artemisinin derivatives[END_REF]. It is also important to note that the C-F bond strength increases as the number of geminal fluorine increases. For example, the C-F bond energy for CH 3 F is 108.3 Kcal/mol while for CH 2 F 2 is 119.5 Kcal/mol (Table 5). This observation can be explained considering the hybridization character of the C-F bonds. By increasing the number of geminal fluorine, the percentage of p-character of C-H bonds decreases, while the percentage of p-character of C-F bonds increases in CH 3 F, CH 2 F 2 , CHF 3 and CF 4 , with the resulting gradual increase of their C-F bond strength. As a consequence, the higher the hybridization index of the C-F bonds, the greater is the polarization of the C-F bonds.

II.1.2 Effect of fluorine on lipophilicity

Lipophilicity is a key parameter in medicinal chemistry. The presence of lipophilic groups on the ligand is key to enhance the binding affinity to the target protein when it presents a hydrophobic character in its active site. In addition, lipophilic groups can improve permeability across cell membrane. However, a high lipophilicity leads to undesirable properties such as reduced solubility in aqueous solvents or entrapment in cell membranes. One of the challenges for medicinal chemists is to establish the right balance between the required lipophilicity and a minimal overall polarity.

Lipophilicity is commonly expressed as octanol/water distribution coefficient, log P, where log P = log(c oct /c eau ). Lipophilic compounds are mostly partitioned in the octanol layer and display high log P values (up to ca 6-8 for traded drugs), whereas the least lipophilic compounds have low negative log P values (ca -5 for some hydrophilic drugs).

A common mistake is to consider that fluorination would systematically increase lipophilicity. This is not the case for fluorinated alkyl groups in which the presence of a single fluorine atom tends to decrease the overall lipophilicity (Smart B.E. et al., 2001) (Table 6).

Log P (octanol-water)

CH 3 CH 3 CH 3 CH 2 F CH 3 (CH 2 ) 3 CH 3 CH 3 (CH 2 ) 3 CH 2 F
1.81 0.75 3.11 By contrast, lipophilicity increases after aromatic fluorination, polyfluorination, or fluorination adjacent to atoms with p-bond. In these cases, the excellent overlap between the fluorine 2s and 2p orbitals with the carbon orbitals contributes to increase lipophilicity (Smart B.E. et al., 2001). This increase in lipophilicity is particularly appreciated in medicinal chemistry and it has been advantageously used for the development of receptor inhibitors. Kusner et al. used the fluorine substitution to develop leukotriene receptor inhibitors that reduced bronchoconstriction in asthma sufferers. For this specific application, they showed that these fluorinated inhibitors were more potent than compounds incorporating aliphatic chains to achieve comparable increases in lipophilicity.

2.33

II.1.3 Effect of fluorine on molecular conformation

The fluorine van der Waals radius is 1.47 Å falling between that of oxygen (1.57 Å) and hydrogen (1.20 Å). While substitutions of a hydrogen or oxygen by a fluorine is common, such modifications lead to a minor steric effect at the interaction sites. However, the volume of a trifluoromethyl group is approximately twice that of a methyl group and is equivalent to an isopropyl group. So, the introduction of a trifluoromethyl group may impose more steric protein-ligand interactions, which should result in improved binding affinities. As an example, a series of compounds were tested for binding to thrombin, and the fluorinated ligands were on average five times more potent than non-fluorinated compounds due to favorable polar interactions between the fluorine atom and the interaction site of thrombin [START_REF] Olsen | A fluorine scan of thrombin inhibitors to map the fluorophilicity/fluorophobicity of an enzyme active site: evidence for C-F...C=O interactions[END_REF].

II.1.4.2 The role of fluorine on biological absorption

Absorption corresponds to all phenomena involved in the transfer of an active medicinal substance from its site of administration to the biological target. Depending on its administration site, a drug needs to cross distinct barriers, like the intestinal barrier for oral administration, the blood-brain barrier if the target is in the brain, and ultimately the cell membranes. Several physicochemical parameters are known to interfere in the absorption process: lipophilicity, pKa, solubility, and molecular weight.

Fluorine is expected to influence the pKa by increasing the acidity of the neighboring protons.

Accordingly, Böhm et al., incorporated fluorine to decrease the basicity of an amidrazone function in thrombin inhibitors, which resulted in an efficient increase of inhibitor concentration in blood after oral administration of the compound in rats (Lee K et al., 1999).

II.1.4.3 The role of fluorine in metabolic stability

Oxidative metabolism

A common problem in the drug discovery process is the low metabolic stability of the developed compounds. This problem is mostly due to in vivo oxidation, which is typically mediated by cytochrome P450 enzymes, a superfamily of heme-thiolate enzymes found mainly in the liver. This problem could be overcome in some cases by the introduction of fluorine.

Indeed, the resistance of fluoro-compounds to oxidation can be explained by the C-F bond strength combined to the weak stability of the O-F bond. Chauret et al. proposed fluorine substitutions to enhance the stability of aromatic methoxy groups that are present in phosphodiesterases (PDEs) inhibitors. Difluoromethoxy groups were introduced and allowed an efficient stabilization of these drugs against oxidation in vivo, which led to a new generation of potent PDE inhibitors [START_REF] Chauret | Improving metabolic stability of phosphodiesterase-4 inhibitors containing a substituted catechol: prevention of reactive intermediate formation and covalent binding[END_REF].

Hydrolytic metabolism

Fluorination is also used to enhance the hydrolytic stability of drugs. As an example, the monofluorinated prostacyclin (PGI 2 ), a potent vasodilator and inhibitor of platelet aggregation, showed improvement of its half-life time (greater than one month) in slightly basic medium (pH » 7.4) compared to the non-fluorinated PGI 2 (half-life time of 10 minutes) (Bannai et al., 1983). Furthermore, the difluorinated analogue revealed even higher chemical stability with a half-life time of 90 days. Indeed, placing a fluorine atom on the alkene carbon of the enol ether function lowers the electron density of the enol ether thanks to the high electronegative character of fluorine, thereby inhibiting drug hydrolysis [START_REF] Chang | 7,7-Difluoroprotacyclin derivative, AFP-07, a highly selective and potent agonist for the prostacyclin receptor[END_REF].

II.2 Why fluorinated peptide inhibitors for Pin1?

All Pin1 inhibitors described previously showed either low cell permeability, low specificity, or reduced potency. In the first section of this chapter, we have introduced some well-described Pin1 inhibitors, some of which contained fluorine atoms (D21P, shikimic acid derivative, benzothiophenyl derivative: compounds 1, 2 and 3). Again, the purpose of introducing fluorine atoms on Pin1 ligands was to improve their lipophily and biodisponibility, as well as their affinity/selectivity towards Pin1. In the resolved X-Ray structures, fluorine atoms often played important roles in the stability of Pin1 complexes. In the present work, we seek to determine whether the stereoelectronic properties of fluorine may be used to design new transition state analogues of Pin1.

II.2.1 Characteristics of the CF 3 -pseudoproline residue.

Mutter et al. have developed for the first time pseudoprolines (YPro) in the middle of 90s (Dumy et al., 1997;Keller et al., 1998). YPro comprise oxazolidines or thiazolidines motifs obtained by condensation reactions of serine, threonine, or cysteine with aldehydes, ketones, or their acetals (Figure 22). Their 5-membered cyclic nature makes them very good proline mimics.

coupling reaction at the N-terminus is more challenging. In contrast to other amino acids, the use of activated esters, symmetrical anhydrides, or mixed anhydrides has been found to be totally ineffective. The coupling reactions carried out in the absence of base reagent from a diastereoisomeric mixture of fluorinated pseudoprolines were shown to be diastereoselective, and resulted in the formation of dipeptides comprising a pseudoproline of configuration (R) at Cd position. Fmoc-protected dipeptides were also obtained with good yields and the reaction can be transposed on a large scale. Thus, the coupling reactions can be performed from a diastereoisomeric mixture of fluorinated pseudoprolines. Due to a dynamic kinetic resolution process, the coupling reaction systematically gives rise to dipeptides where the pseudoproline possesses the (R) configuration at the Cd position.

Theoretical and experimental studies have been carried out to highlight the impact of CF 3 group on the structural and thermodynamically properties of fluorinated pseudoprolines. For this aim, the N-terminus and C-terminus of CF3 YPro were respectively N-acetylated and amidated in order to mimic the environment of a CF3 YPro within a peptide chain. Miclet et al., have shown that the incorporation of the electronegative CF 3 group at the Cd position induces an electronwithdrawing effect which leads to the extension of the Cd-N bond due to a decrease of the bond overlap between the Cd and the N atoms [START_REF] Feytens | Local control of the cis-trans isomerization and backbone dihedral angles in peptides using trifluoromethylated pseudoprolines[END_REF]. On the other hand, the electronic energy of the transition state in Cd-CF3 YPro peptide is about 4 kcal/mol lower compared to Cd-YPro, resulting in a weaker energy barrier in Cd-CF3 YPro comparing to proline.

Another consequence of the CF 3 substitution is the puckering stabilization of the 5-membered cycle. This stabilization depends on the Cd configuration. Indeed, Cd-(R)-CF3 YPro are uppuckered while Cd-(S)-CF3 YPro are down-puckered. This puckering preference may be due to favored electrostatic interactions between the fluorine atom of the CF 3 group and the H atom of the CbH 2 group, and to the overlaps of the Ce-F antibonding orbital (s*) with the Cd-H binding orbital (s) and lone pair orbitals of the ring oxygen. In fact, the combination of the steric and electronic effect of CF 3 group contribute in decreasing the rotational barrier of (2R)-CF 3 -Oxa residue.

II.2.2 Conformational properties of peptides incorporating CF 3 -YPro residue

In peptides, the Xaa-Pro peptide bonds undergo spontaneous cis/trans isomerization. However, the trans conformation is preferred in most cases as detailed in chapter 1 (part I.1). Miclet et al.

showed that the use of oxazolidine-based pseudoprolines (ΨPro) substituted by a trifluoromethyl group at the Cd position modifies significantly this cis/trans ratio in Xaa-ΨPro dipeptides [START_REF] Chaume | Homochiral versus Heterochiral Trifluoromethylated Pseudoproline Containing Dipeptides: A Powerful Tool to Switch the Prolyl-Amide Bond Conformation[END_REF], which is strongly influenced by the ΨPro C δ and the Xaa C α configurations. This CF3 YPro residue appears very valuable for the design of new Pin1 inhibitors since it will allow (i) the control of the peptide bond geometry, (ii) the stabilization of the cis-trans transition state, and (iii) a significant enhancement of hydrophobicity.

II.2.3 Mimicking the phosphate group with difluorophosphonate

Pin1 inhibitors possessing charged phosphate group showed chemical and/or metabolic instability and are not very effective in crossing cell membranes, limiting their inhibitory activity [START_REF] Duncan | Discovery and characterization of a nonphosphorylated cyclic peptide inhibitor of the peptidylprolyl isomerase, Pin1[END_REF]. To overcome these drawbacks, the replacement of the phosphate group by an isostere (as carboxylate groups) was investigated to improve pharmaceutical properties. Unfortunately, in most cases, these analogues led to less-potent Pin1 inhibitors.

A new strategy is being considered by our collaborator at Cergy-Pontoise which consists in replacing the phosphate group by a a,a-difluorophosphonate (pCF 2 ). pCF 2 is an 'isosteric and isopolar' non-hydrolyzable mimic of the phosphate moiety that still preserves its electron withdrawing effect and geometrical features. This replacement should improve considerably peptides metabolic stability. The negative charge of the pCF 2 group may be counterbalanced by the increase in lipophilicity provided by the CF 2 group to facilitate membrane crossing. Note that the slight decrease of the pKa value due to the presence of the pCF 2 may alter the interaction with the Pin1 phosphate pocket [START_REF] Hamilton | Synthesis of a potent inhibitor of HIV reverse transcriptase[END_REF]. Another strategy could be adopted, using a monofluorophosphonate group in order to maintain a pKa similar to the phosphate [START_REF] Panigrahi | Combining a Clostridial enzyme exhibiting unusual active site plasticity with a remarkably facile sigmatropic rearrangement: rapid, stereocontrolled entry into densely functionalized fluorinated phosphonates for chemical biology[END_REF].

II.2.4 Design of a peptide library

The use of fluorine in the design of drugs and drug candidates is continuously expanding (79% of drugs designed in the period 2010-2019 contain fluorine), a brand that is being driven by a deeper understanding of its effects on molecular properties and biological activities. As detailed in section II.1, the introduction of fluorine should enable major improvements in the pharmacologic profiles of the substrates such as protein affinity, metabolic stability, lipophilicity, and others. Furthermore, the stereoelectronic properties of fluorine play a role in stabilizing the conformation of transition state analogues. As described in section II.2.1, the incorporation of a CF 3 on the oxazolidines (at the Cd carbon) can lower the rotational energy barrier of the cis/trans peptide bond isomerization, strengthen a key H-bond within the peptide, and enhance the cis population.

We choose to take advantage of the overall properties imparted by the fluorine to develop fluorinated peptides as a new class of Pin1 inhibitors.

Based on the specificity of Pin1 against the pSer-Thr-Pro motifs, we have selected small templates of four residues comprising the motif pSer-Pro in position 2 and 3, and determined the amino acid preference of Pin1 at positions 1 (N-ter) and 4 (C-ter) of these templates.

At the N-ter, we have tried different moieties from simpler ones like the acetyl group to more sophisticated ones like Fmoc, Phenylalanine, and others. At the C-ter, a tryptamine was incorporated, inspired by ligands described by the Etzkorn's laboratory (Namanja et al., 2011, section I.4.4). Ligands with peudoproline instead of proline at position 3 were also synthesized in which we incorporated the CF3 group at the Cd carbon. The replacement of the pSer by a

Ser or an Asp has been attempted to facilitate the synthesis, although it is expected to reduce binding affinity. Peptides containing a C-terminal Phe instead of tryptamine were also designed to simplify the synthesis process (Phe being compatible with solid phase peptide synthesis).

Beside the design of these new fluorinated peptides, several well-studied Pin1 inhibitors were also integrated in the study as a reference of their binding properties. The structure of the ligands and their interaction with Pin1 is detailed in chapter 4.

Chapter III: Production of Pin1 samples and NMR assignments I-Expression and purification of recombinant Pin1 proteins

The production of (His) 6 -tagged Pin1 as a soluble protein in E. coli has been previously reported in a high number of studies (Fiebg et al., 2003;[START_REF] Namanja | Substrate recognition reduces side-chain flexibility for conserved hydrophobic residues in human Pin1[END_REF][START_REF] Zhu | Uncorrelated Effect of Interdomain Contact on Pin1 Isomerase Activity Reveals Positive Catalytic Cooperativity[END_REF]. We thus designed pET15b expression vectors that encode fusion proteins comprising an N-terminal (His) 6 -tag followed by a PreScission protease cleavage site, and a Pin1 fragment (WW domain,

PPIase or full-length protein). Each ampicillin-resistant plasmid was transformed into an E. coli BL21 (DE3) Gold pLysS expression strain (Agilent) that harbors a chloramphenicol resistant gene and an IPTG-inducible T7 promotor.

I.1 Expression of Pin1 proteins in LB medium

BL21 Gold cells containing a pET15b{Pin1} vector were cultured at 37°C under agitation in LB medium supplemented with 100 µg/mL ampicillin and 30 µg/mL chloramphenicol. When the OD measured at 600 nm (OD 600 ) was around 0.7-0.8, protein expression was induced by addition of 500 µM IPTG, and the cultures were further grown overnight at 18°C. Cells were pelleted by centrifugation (3000xg for 20 minutes), resuspended in a phosphate buffer containing 25 mM phosphate buffer pH 7.8, 500 mM NaCl, 30 mM imidazole, 1 mM DTT, and 0.5% (v/v) Triton X-100, and frozen at -80°C.

I.2 Expression of Pin1 proteins in M9 medium

The investigation of structural and dynamical properties of 20-kDa proteins by NMR requires the preparation of isotopically labeled 15 N and 15 N/ 13 C protein samples. For this aim, we employed the Marley protocol [START_REF] Lu | A method for efficient isotopic labeling of recombinant proteins[END_REF] commonly used for preparing high yields of uniformly labeled proteins in M9 medium. The method consists in first growing bacteria in a rich LB medium up to an OD 600 of 0.6, and then concentrating them in an M9 medium lacking nitrogen and carbon sources. In the absence of NH 4 Cl and glucose, the residual LB medium containing unlabeled nitrogen and carbon sources is expected to be consumed within 40 minutes at 37°C under agitation [START_REF] Lu | A method for efficient isotopic labeling of recombinant proteins[END_REF]. 15 NH 4 Cl and 13 C/ 12 C-glucose are then introduced into the cultures, and protein expression is induced with IPTG at 18°C overnight.

SDS-page analysis of the total bacterial fractions before and after IPTG induction showed highintensity bands with apparent molecular weights that correspond to the molecular weights of was complete even in the presence of 220 mM imidazole, indicating that the enzyme does not require buffer exchange to be active in the elution fractions collected during the first Ni-NTA purification step. Based on these results, we decided to remove the dialysis step from the purification procedure to fully avoid protein loss. Since a significant reduction of imidazole concentration is however required before the second Ni-NTA purification step, protein samples were diluted twice with equilibration buffer after PreScission cleavage.

I.4.2 Optimization of Pin1 concentration

Centrifugal filter devices are widely used in the NMR community for protein concentration as they generally provide fast ultrafiltration and high concentration factors. We thus used an Amicon centrifugal device with a 3-kDa membrane cutoff (Millipore) as a first approach to concentrate FL Pin1 after the gel filtration column. However, our attempts to concentrate FL Pin1 using centrifugal devices were unsuccessful or resulted in the loss of 40% to 60% of the protein, although we could not observe protein precipitation. Such behavior was observed regardless of the buffer composition (Tris-HCl or phosphate) and NaCl concentration (50 mM to 200 mM) of the protein samples. To understand the source of this protein loss, we probed the influence of the membrane cutoff (3 kDa or 10 kDa) and the rotation speed (1500xg or 3000Xg) on the protein concentration performance. Surprisingly, a constant amount of FL Pin1 corresponding to 0.3 mg/mL was shown to pass through the concentration membrane regardless of the molecular weight cutoff, the rotation speed, and the initial protein concentration (Table 1). As a result, highly diluted Pin1 samples containing less than 0.3 mg/mL could not be concentrated with centrifugal devices. The detection of FL Pin1 (18,7 kDa) in the flow through when using a 3-kDa cutoff membrane is highly unexpected, and protein retention higher than 95% is usually observed in the lab when concentrating 10-kDa proteins using 3-kDa cutoff centrifugal devices. Interestingly, such loss of Pin1 protein was also observed using Vivaspin centrifugal devices with a 3-kDa cutoff membrane (Sartorius), indicating that this behavior is likely protein-specific and may be explained by the modular organization of FL Pin1 as two independent domains of 12.7 kDa and 5.1 kDa.

I.5 Purification of Pin1 proteins using HPLC instead of gel filtration

Since we could not fully avoid protein loss during concentration of FL Pin1 using centrifugal or pressure-based devices, we attempted to replace gel filtration by reverse phase HPLC (High-Performance Liquid Chromatography) in the last step of the purification process. The main advantage of HPLC is that the protein is separated from all compounds including buffer components, and can be recovered after lyophilization at the desired concentration in the desired buffer. However, proteins are likely to become unfolded during the HPLC process or can even precipitate because of the acidic conditions and the presence of high concentration of acetonitrile. It is thus necessary to check if the protein can be properly refolded after lyophilization.

To test purification of FL Pin1 by HPLC, the fusion protein was first purified on a His-Trap column and incubated overnight with PreScission to cleave the 6xHis tag. The cleavage solution was loaded on a C4 column and FL Pin1 was further purified using a 10-100% acetonitrile gradient, with and without 0.1% TFA. Protein elution was monitored by real time measurement of UV absorbance at 220 and 280 nm. With 0,1% TFA, Pin1 was eluted at 13.55 minutes and a protein loss of ~10% was quantified. Without TFA, elution of FL Pin1 was not detected, indicating that the presence of 0.1% TFA is necessary to elute the protein. After purification by HPLC with TFA, the protein was frozen, lyophilized, and then resuspended with the NMR buffer. Despite the presence of 30 mM Tris pH 7.50 in the NMR buffer, the measured pH of Pin1 sample was very acidic (pH ranged between 2 and 4), likely because of the presence of a remaining fraction of TFA in the sample which was not sublimated during lyophilization.

Unfortunately, our attempts to correct pH value by dialysis or by directly adding NaOH in the protein solution resulted in the full precipitation of FL Pin1.

A 1 H-15 N-HSQC spectrum was recorded on the protein sample obtained after HPLC and subsequent lyophilization to check the folding state of FL Pin1 in acidic conditions. As shown in Figure 31, the HSQC spectrum is very different from that obtained after gel filtration and protein concentration. The weak dispersion of amide cross-peaks in the 1 H dimension clearly indicates that FL Pin1 is unfolded after HPLC in acidic conditions. By contrast, amide crosspeaks are well dispersed in the HSQC spectrum obtained at pH 7.5 after gel filtration, suggesting that the protein is well-folded under these conditions. Overall, our results indicate that FL Pin1 cannot be further purified by HPLC after a first purification step on a nickel

II-Optimization of FL Pin1 stability using Differential Scanning Fluorimetry

To optimize the composition of NMR buffers before recording NMR experiments, we have evaluated the stability of FL Pin1 in different buffers using Differential Scanning Fluorimetry (DSF). DSF is a widely used technic for investigating both protein folding and protein thermal stability. It consists in monitoring thermally induced protein denaturation by measuring fluorescence emission of a dye. Fluorescence emission intensity is indeed expected to increase with the concentration of fluorescent dye bound to hydrophobic patches of unfolded proteins.

By monitoring fluorescence emission at regular intervals as a function of temperature, it is then possible to estimate the apparent melting temperature (Tm) of a protein under a given condition.

Proteins exhibiting high Tm values in a given buffer are expected to be more stable. One of the most used fluorescent dyes to optimize protein buffer composition by DSF is SYPRO Orange (Boivin et al., 2013). It is relatively sensitive and its emission and excitation wavelengths are respectively 569 nm and 470 nm. The main advantage of DSF is that the method can be implemented on a real-time PCR instrument, which allows the screening of a large number of conditions through the use of 96-wells plates (25 µL volume per well). The method is also highly sensitive and protein concentration around 2.5 µM is usually sufficient to determine melting temperatures.

We first determined melting temperatures of FL Pin1 for 5 different buffers (pH ranging from 6 to 7,5) in the presence of varying concentrations of NaCl (50 mM, 200 mM, and 400 mM).

While increasing ionic force is known to improve stability of many proteins, our results show that the melting temperature of Pin1 decreases significantly with the concentration of NaCl in all tested buffers, excepted for Tris-HCl at pH 7.5 (Figure 34). This indicates a destabilizing effect of high salt concentration on FL Pin1. This result is an advantage because a high ionic force highly reduces sensitivity of the NMR signal, in particular when using a spectrometer equipped with a cryoprobe. The phosphoramidite method was employed by our collaborators at Cergy-Pontoise to incorporate protected phosphate groups on serine residues. However, this phosphorylation step or the subsequent deprotection of the phosphate group resulted in medium to low yields. For some ligands, serine phosphorylation could not be achieved, whereas in a last peptide, these chemical steps resulted in the complete hydrogenation of unsaturations, leading to the CZ115 compound.

For CB06, CB07, CB08, CZ279, CZ282, CZ40, CZ55, CZ178, and CZ181, the position P3, which corresponds to the third residue after the serine, was a proline. For the other residues CZ280, CZ281, GG84, GG82, CZ34, CZ52, and CZ115, the proline residue was replaced by Binding properties of the synthetized ligands have been measured by NMR as detailed in the following section. NMR-based Kd values obtained for FL Pin1 and its isolated domains are presented in Table 9 and will be discussed hereafter.

II. Interaction of Pin1 with ligands

To define ligand binding modes, we measured amide 15 N-1 H chemical shift perturbations Trp34, and Glu35 with an average Kd value of » 32 ± 5 µM (Figure 46A). This value is approximately 15 times better than the affinity obtained with CB06 and CB08 showing that a tryptamine was much more favorable at the C-ter than the two-natural aromatic amino acids, phenylalanine or tryptophan, capped by carboxamide groups.

Two residues of the PPIase domain catalytic site were also perturbed in a significant manner:

Gly123 and Met130. However, the binding curves of these two residues were characteristic of very weak interactions with Kd values in the millimolar range, suggesting non-specific interactions. As a result, CZ55 proved more specificity to the WW domain compared to CB06 and CB08.

Interestingly, significant CSPs were also observed at the C-terminal end of helix a4 in the PPIase domain, including Phe139 and Ala140. Helix a4 forms a significant part of the domain interface between the WW and the PPIase domains. This indicates that the binding of CZ55 to the WW domain affects inter-domain interactions.

To demonstrate the specificity of CZ55 towards the WW domain, we titrated the isolated WW and PPIase domains with CZ55, and calculated CSPs and binding affinities for each independent domain (Figure 46B). The CSPs values of the WW region are remarkably similar in FL Pin1 and the isolated WW domain. However, residues Tyr23 and Gln33 were less perturbed in the isolated WW domain. In addition, the Kd of Trp34 obtained with the isolated WW domain was 4.5-fold higher compared to the value obtained with the full-length protein.

W34 plays a significant role in the interaction with the ligand proline suggesting that the binding site for the proline of CZ55 was affected by the absence of the PPIase domain. Kd determination for other residues of the WW region showed on average twofold higher values: 69 ± 9 µM for the isolated WW domain versus 32 ± 5 µM for FL Pin1. This rather small but meaningful difference in affinity may be due to some allosteric effects of the PPIase domain on the structure and/or dynamic of the WW domain that, in the end, affect its binding properties toward CZ55.

The titration of the isolated PPIase by CZ55 did not show any perturbation for residues of the catalytic site (data not shown), which confirmed the specificity of CZ55 for the WW domain and the modulation of the PPIase/WW interface contacts upon CZ55 binding to the WW domain.

CZ52 is the analogue of CZ55 with a CF 3 group at the Cd carbon of Y H,CF3 Pro. This fluorinated mimic of proline promotes a cis conformation as confirmed by the NMR structural analysis in As CZ55, CZ40 which bears a regular Pro residue at the P3 position, mainly adopts a trans conformation of the pSer-Pro peptide bond in water (80%). The presence of 8 molar equivalents of CZ40 produced higher numbers of residues with significant CSPs in the WW domain, as compared to CZ55. Residues located in the canonical WW binding site (Met15, Gly20, Phe25, Trp34, Glu35) were the most affected with an average Kd of 26.2 ± 6 µM (Figure 48A). In addition, a second region of the WW domain (His27 and Asn30) showed significant CSPs.

These residues belong to the b2-b3 loop (Loop II) located at the WW-PPIase domain interface.

This suggests that these perturbations are not due to a direct contact with the substrate but to a structural/dynamical modification of the domain interface. Calculating Kd values for these two residues, we found 40.9 µM and 27.3 µM for His27 and Asn30 respectively, which are in the same affinity range as the Kd obtained for the residues defining the binding site of the WW domain. We can conclude that the binding of CZ40 caused an allosteric effect on the Pin1 structure. CZ40 also produced CSPs in the catalytic site of the PPIase domain (Gly123, Met130, Gln131, and Gly155). Such perturbations were also observed for CZ55 although they were less intense and concerned only two residues G123 and Met130. Surprisingly, no significant perturbations were observed for the PPIase residues delineating the WW-PPIase interface as it was observed for CZ55 (perturbations of the helix a4). The average Kd value obtained for catalytic residues of the PPIase domain showing significant CSPs was 438 ± 40 µM. This is an order of magnitude higher compared to the Kd determined for the WW domain and it is unlikely that these CSPs observed on the PPIase domain are due to an allosteric effect of the WW domain binding. These perturbations may be due to the binding of the minor population of CZ40 (20%) which adopts the cis peptide bond conformation. Indeed, the presence of the Fmoc group in CZ40 slightly increased the affinity for Pin1 compared to CZ55 which bears an acetyl group at the N-terminus.

Interaction of Pin1 with CZ115

More important modifications were introduced to CZ115. First, an additional amino acid was inserted at the P1 position. Furthermore, in the time course of its synthesis, this ligand underwent a complete hydrogenation of carbon-carbon unsaturations which leads to a cyclohexane at P1 (instead of the phenyl group of the phenylalanine) and a reduced tryptamine.

Unfortunately, this reduction results in the creation of three asymmetric carbons whose stereochemistry is not controlled. Therefore, CZ115 consists in a mixture of several diastereoiomers (more details in chapter 5).

CZ115 possesses the CF 3 -YPro in P3 and showed more than 80% of the pSer-CF 3 -YPro in the cis conformation. Compared to the other ligands, the interaction of CZ115 with Pin1 showed the most extended perturbations among the residues of the PPIase domain. As shown in Figure 50A, three sites of the PPIase domain are mainly affected by the presence of CZ115: (i) the proline-binding pocket including Gly123, Gln129, Met130, and Glu135, (ii) residues surrounding the peptidyprolyl bond including Asp112, Cys113, Ser154, and G155, and (iii) residues next to the triad of basic amino acids (Lys63, Arg68 and Arg69) including Lys63, His64, and Gln66. In addition, CZ115 produced chemical shift changes for residues away from the catalytic site but that are located on the same side of the Pin1 molecular surface (Ser71, Ser72, Lys82, Ala116, and Phe125). This may indicate a large mobility of CZ115 around the catalytic site and/or that the binding is associated with Pin1 conformational changes.

Another observation we made from titration of Pin1 with CZ115 is the strong homogeneity of the Kd values determined on each disturbed residue. The average was 28 ± 6 µM (figure 50B).

In addition, we did not detect any significant perturbation within the WW domain in FL Pin1, which makes CZ115 a PPIase domain-specific ligand. This is why we decided to further analyze this interesting ligand, including 19 F, Exsy and relaxations experiments (see chapter 5). As a first step, we studied its interaction with the isolated domains.

reveal a specific mode of interaction with the WW domain, where the peptide adopts a cis peptide bond, and the Fmoc group is a pivot point.

III.2 Hints of allostery from binding

Previous structural studies [START_REF] Smet | Regulation of Pin1 peptidylprolyl cis/trans isomerase activity by its WW binding module on a multi-phosphorylated peptide of Tau protein[END_REF] showed that phospho-peptide substrates had slightly different binding affinities for the isolated domains versus FL Pin1, suggesting the modulation of domain binding affinity by inter-domain interactions. Therefore, we produced the isolated WW and PPIase domains and performed independent titrations experiments (FL Pin1 versus truncated domains) to gain insights into putative cross allosteric effects on binding between the two domains.

Four ligands were studied with the isolated domains: CZ55, CZ52, CZ115, and CZ181. The choice of these ligands was based on their affinities and/or their specificity against one domain over the other. The interaction of CZ55 with the isolated WW domain showed a twofold lower affinity in comparison with the FL protein and slight differences in CSP. Residues that directly interact with the Proline of the ligand were less affected by the binding event when the WW domain was isolated suggesting looser contacts. In this example, the presence of the PPIase domain assists the structure and or dynamics of WW for a tighter binding.

Another evidence of an allostery mechanism can be obtained from the careful analysis of the CSP data upon CZ55 binding. By binding to the WW domain of the PL Pin1, CZ55 was able to disturb residues at the domain interface of the PPIase domain. These perturbations were clearly due to an indirect effect of binding to the WW site, located on the other side of the protein, since CZ55 had no affinity for the PPIase domain, as attested by titration experiments on the isolated PPIase domain. The differences in Kd suggested that the inter-domain interaction affected the binding of CZ55 on the WW domain. Conversely, previous studies showed that the inter-domain flexibility and the extent of interaction between the two domains depends on the ligand type (Jacobs et al., 2003). One substrate was able to reduce the interdomain interaction, while a second could enhance the inter-domain contacts. These interdomains cross-talks have focused our interests and will be the subject of further developments in the next chapter.

The presence or absence of the WW domain did not affect the interaction of CZ52 and CZ115

with the PPIase domain. Similar Kd values were obtained with FL Pin1 and the isolated PPIase domain. In contrast, for CZ181 the Kd value was 3 times lower for the isolated PPIase domain than for the FL Pin1. Peng et al., [START_REF] Namanja | Stereospecific gating of functional motions in Pin1[END_REF] also found a greater affinity of a cis-locked analog of the FFpSPR inhibitor for the isolated PPIase domain compared to FL Pin1.

These results led us to further understand the allostery between the two domains of Pin1. For this purpose, we performed further experiments with apo Pin1 or in the presence of ligands.

These experiments and results will be detailed in the next chapter.

Chapter 5: Fluorinated inhibitors to characterize the allosteric regulation of Pin1

While the crystal structure of FL Pin1 indicates a compact organization with close contact between the two domains [START_REF] Ranganathan | Structural and functional analysis of the mitotic peptidyl-prolyl isomerase Pin1 suggests that substrate recognition is phosphorylation dependent[END_REF], the solution structure (PDB code: 1NMV)

of FL Pin1 revealed a more extended organization with only transient interactions between the two domains. These transient interactions were deduced from the absence of inter-domain NOEs in the NOESY spectra, and the short lifetime (in the range of milliseconds) calculated for the intramolecular complex (Matena et al., 2013). Interestingly, the extended state was described as an ensemble of several fast exchanging states rather than one single conformer.

Indeed, the linker connecting the two domains was disordered, which allows a substantial range of domain motions (Bouchard et al., 2018).

Numerous studies have demonstrated the ability of Pin1 to bind many different protein partners, most of them have in common the pS/T-P motif. In these published studies, only a few authors highlighted that the equilibrium between the extended and compact states was altered upon binding [START_REF] Zhu | Uncorrelated Effect of Interdomain Contact on Pin1 Isomerase Activity Reveals Positive Catalytic Cooperativity[END_REF]Peng et al., 2015). Depending on the sequence of the substrate bound to the loop 1 of the WW domain, the interaction between the two domains can be modified, either reinforced or weakened. In the compact state, the two domains interact via the loop 2 of the WW domain (residues 27-32) and the end of the helix a4 (137-141) and a small region of the strand b6 (148-149) of the PPIase domain.

Two families of Pin1 WW domain substrates can be defined in the literature: one family of substrates, such as the peptide Cdc25 (EQPLpTPVTDL), that favors the extended state of Pin1 and a second family, including the Pintide peptide (WFYpSPR) or others (FFpSPR and YpSPTpSPS), that enhances the inter-domain contacts and stabilizes a compact state of Pin1.

Importantly, this ligand-dependent inter-domain interaction triggers differential response in the dynamic of the PPIase domain as well as its catalytic activity (Jacobs et al., 2003;Wilson et al., 2013;Peng et al., 2015).

Various structural studies have been undertaken to understand the dynamics and the allosteric regulation governed by the WW domain by looking at many different ligands.

During my thesis, we decided to take advantage of the domain binding preference of some of our fluorinated ligands to better understand the activity mechanism of Pin1. In particular, we investigated the effect of the PPIase domain binding on the WW domain. To compare our results with those already published in the literature, we selected two well-studied peptides, Pintide and Cdc25, known to selectively bind the WW domain of Pin1.

Pintide (WFYpSPR) is an artificial peptide derived from a screening assay of pSer-Pro containing peptide libraries. It corresponds to the minimal sequence with optimal catalytic activity efficiency (k cat /K m ) (Yaffe et al., 1997). In some studies, Pintide was replaced by FFpSPR for enhanced solubility properties. Pintide preferentially binds the Pin1 WW domain with µM affinity.

Cdc25 is a natural peptide of ten residues (EQPLpTPVTDL) corresponding to the pT48-P49 region of the protein CDC25C, an essential mitotic phosphatase targeted by Pin1. Full-length CDC25C is a single-subunit enzyme with a complex N-terminal regulatory domain. It belongs to the dual-specificity phosphatases family (DSP) that can hydrolyze the phosphate of phosphorylated tyrosine or serine/threonine residues. Specifically, CDC25C dephosphorylates and activates the Cdk/cyclin complex, thereby playing a fundamental role in transition between cell cycle phases (Trunnell et al. 2011).

The affinity, dynamics, and activity of FL Pin1 and the isolated domains were well studied in the presence of these two peptides (Jacobs et al., 2003;[START_REF] Namanja | Substrate recognition reduces side-chain flexibility for conserved hydrophobic residues in human Pin1[END_REF][START_REF] Namanja | Stereospecific gating of functional motions in Pin1[END_REF]Wilson et al., 2013;Wang et al., 2015;[START_REF] Zhu | Uncorrelated Effect of Interdomain Contact on Pin1 Isomerase Activity Reveals Positive Catalytic Cooperativity[END_REF]Strotz et al., 2020). However, the experimental conditions such as Pin1 concentration, buffer composition, and pH were not the same in all these studies, resulting in significantly different Kd values, correlation time tc values, or cis/trans isomerase activity. As a benchmark, we performed titration, relaxation experiments as well as activity assays using these two peptides. These studies will allow us to compare the properties of our own ligands to these well described Pin1 substrates.

I. Specific ligands for the WW domain I.1 Interaction of FL Pin1 with Pintide and Cdc25

The interaction of FL Pin1 with Pintide and Cdc25 mostly perturbed residues in the binding site of the WW domain. This included residues located in the β-hairpin loop 1 (Met 15, Ser16, Gly20, and Arg21) and residues Trp34 and its neighbors Gln33 and Glu35. Average Kd values the same downfield shifts in the 15 N and 1 H dimension for both ligands. Accordingly, we can interpret these results as an indicator of inter-domain contacts. In agreement with the description found in the literature for the Pintide ligand, CZ55 may favor a close state of the FL Pin1 conformation upon binding of the WW domain. However, it is important to point out that the correlation peaks corresponding to residues at the WW/PPIase interface undergo smaller frequency shifts for CZ55 in comparison with Pintide, while the correlation peaks of residues at the WW binding site show similar shift magnitude. Furthermore, no residues in the loop 2 of the WW domain (27-32) were disturbed upon binding to CZ55 in the WW domain.

These results are similar to those obtained with the peptide FFpSPR. FFpSPR is close in sequence to Pintide (WFYpSPR) but is slightly more soluble in aqueous buffer. It promotes contacts between the two domains but not as much as Pintide. Taken together, CZ55 may restrict the flexibility of Pin1 but with a lower magnitude compared to Pintide.

These results led to an extensive study of the impact of the ligands on the dynamic of FL Pin1.

To this end, series of 15 N R1 and R2 relaxation experiments of FL Pin1 were recorded in the absence and presence of ligands. Nuclear relaxation is a fundamental aspect of NMR that includes all the phenomena of energy exchange which drive spins back to the thermal equilibrium after a perturbation. The transversal and the longitudinal relaxations provide unique information about the amplitude and timescales of motions of proteins in the pico-to-nanosecond range.

In a magnetic field B 0 , the vector sum of all magnetic moments creates a net magnetization M 0 at the thermal equilibrium, which can be defined as magnetization along the z axis (M z ). Each individual magnetic moments precess with a random phase about the applied filed, such that there is no global magnetization in the xy plane (M x = M y = 0). When the system is perturbed by an external radio-frequency RF pulse of field B 1 perpendicular to B 0 , possessing a frequency v 0 sufficiently close to the Larmor precession frequency of the nuclear spins, the energy is absorbed by the system. As a result, the spin populations a and b can be equalized (for a given duration of the pulse), leading to the cancellation of the M z component. However, the RF pulse the WW domain as well as residues in the helix a4 and in the b6 strand of the PPIase domain.

Finally, a loss of flexibility was detected for residues of the whole helix a1. The helix a1 forms a wall on the concave site of the inter-domain interaction and creates a significant part of the interior surface of the cavity opposite to the WW domain's hydrophobic pocket. These dynamic modifications of FL Pin1 upon Pintide binding are in agreement with those observed with FFpSPR. Both peptides increase rigidity in different regions of FL Pin1 compared to the apo Pin1. This is in line with the fact that both peptides can stabilize the inter-domain interaction.

Calculation of the average 15 N R2/R1 ratios gave an approximation of t c [START_REF] Namanja | Stereospecific gating of functional motions in Pin1[END_REF]LE K et al., 1989). Values of 11 ns and 12 ns were obtained for the WW and PPIase domain, respectively. This result confirms that Pintide binding in the WW domain increased the overall rotational correlation time relative to the apo state. Furthermore, the difference between the t c of WW domain and PPIase is lower than in the apo Pin1 revealing a limited rotation of each domain by strengthening the inter-domain interaction.

By contrast to Pintide, Cdc25 binding was accompanied by an increase in flexibility for residues in the inter-domain interface with t c = 6.6 and 8.3ns for the WW and PPIase domains, respectively. These values are significantly lower than those obtained with apo FL Pin1. Figure 5A and C showed a significant decrease of the R2 and R2/R1 values in the loop2 of the WW domain as well as in the C-terminus of the strand b6 of the PPIase domain. The results obtained in our study are slightly different than those obtained by Peng et al., [START_REF] Namanja | Substrate recognition reduces side-chain flexibility for conserved hydrophobic residues in human Pin1[END_REF].

While in our study, the C-terminus of the helix a1 and the helix a2 showed slight loosening, in the other study, they exhibit a significant loss of flexibility. However, the study of the FL Pin1/Cdc25 complex performed by Peng was based on the analysis of the side chain dynamics (combination of 2 D and 13 C relaxation experiments), whereas we observed the 15 N relaxation on the backbone amide groups. As far as the catalytic site of the PPIase domain was concerned, both our studies and the one published by Peng et al. revealed that the catalytic loop experienced an increase in dynamics upon Cdc25 binding in the WW domain. This a proof that a dynamics allostery occurs between the WW and PPIase domain in FL Pin1. Again, it is worth to stress that Pintide had an opposite effect since many regions of the PPIase domain were more constrained upon complexation of the WW domain.

The 19 F NMR spectra of CZ115 (Figure 63) showed two groups of signals centered at -78.0 and -78.8 ppm. These two ensembles were assigned to the two states, trans and cis of CZ115. This assignment was based on the 1 H NMR spectra where only two populations in slow exchange where observed with a 80:20 ratio. Based on the analysis of 1 H-1 H correlations in the NOESY spectra, these two populations were specifically assigned to the cis and trans conformers of the pSer-YPro peptide bond. The integration of the 19 F resonances centered at -78.0 and -78.8 ppm were 23% and 77% respectively, which matched the trans and cis populations.

Interestingly, upon addition of increasing concentrations of Pin1, we observed a coalescence of the upfield resonances corresponding to the cis conformations as well as a significant increase of their linewidths and a slight shift towards higher frequencies. In contrast, the signals of the trans conformations were not affected by the addition of Pin1.

Both the line broadening and the frequency shifts detected for the peaks of the cis conformation proved an interaction with Pin1, in the intermediate to fast exchange regime. The large line broadening observed is likely to be the consequence of a significant increase of the ligand correlation time between the free and the bound states. These data unambiguously show that only the cis conformation of CZ115 binds to FL Pin1.

How can we explain that several peaks are observed for the both conformers of CZ115? To answer this question, we undertook a careful NMR analysis including three different experiments.

First, we recorded a series of 19 F spectra of CZ115 at temperatures ranging from 278 K to 384 K. This experiment aimed to accelerate the putative conformational exchange between the different populations (Figure 64). In the case of interconverting conformers, the exchange may become fast enough at high temperature to observe a single peak between the two exchanging populations. This will not be the case if the NMR peaks correspond to different stereoisomers.

As shown on Figure 64, all NMR signals broaden between 288K and 308K, to such an extent that the minor resonances at -78 ppm almost disappeared. The major peaks between -78.5 and -79 ppm are no longer resolved due to the increase in linewidth but do not appear to coalesce together. Rather, the slight downfield shift indicates that they are in exchange with the cis conformers.

II.3 Relaxation experiments of Pin1 complexes with CZ181 and CZ115

In the same way as for WW ligands, we measured R1 and R2 relaxation experiments on FL Pin1 in the presence of saturating amounts of CZ115 and CZ181. Binding of CZ115 to FL Pin1 led to an increase of the protein flexibility in the inter-domain surface of the PPIase domain (Figure 67) comprising residues in the helix a1 (Glu87-Lys97) which delineates the interaction surface between the PPIase and WW domains. Interestingly, CZ181 caused a loss of flexibility for the residues in this region (Figure 67). Thus, the binding of these two ligands had opposite effects in the regions mediating the interaction between the WW and PPIase domains. Looking at the residues of the WW domain reinforced this observation. The interaction of CZ115 with the PPIase domain slightly increases the dynamics of the WW domain, especially for the residues located on the loop1 of the WW domain as well as in the strand b3 (residues Gln33, Trp34, and Glu35). In contrast, CZ181 induced a loss of flexibility for these residues. For CZ115, the estimation of the t c values based on the R2/R1 ratio gave 6.5ns and 8.2ns for the WW and PPIase domains, respectively. These values were slightly lower compared to those of apo Pin1 (t cWW = 6.9ns, t cPPIase = 9.3ns). On the contrary, CZ181 binding increased the t c values to 8. WW domain on the dynamics of the PPIase domain, which may be relevant for tuning its activity.

In addition to the stabilization of a compact structure of FL Pin1, CZ181 induced an overall stiffening of the catalytic site of the PPIase domain including the catalytic loop, the peptidylprolyl bond pocket as well as the proline binding pocket.

III. Measuring the inhibitory activity of the ligands.

III.1 Measuring the inhibitory activity of CZ55 and CZ115 by fluorometric test

In addition to the affinity measurements, we were interested in measuring the inhibitory activity of the ligands. Several methods have been developed for these measurements. One of them assesses the activity of Pin1 in the presence of ligands using a commercial fluorometric test (coupled assay) which is commonly used in publications describing new Pin1 inhibitors. as a novel class of enzymes highlighting their important biological activity (Fischer et al., 1989). For the rational design of PPIases inhibitors, it is important to determine the steady-state kinetic parameters.

For the first time, they developed a spectrophotometric method, a chymotrypsin-coupled enzymatic assay, to probe the catalytic activities of peptidyl prolyl cis/trans isomerases. The proteolytic enzyme chymotrypsin has a very high specificity for substrates of the Xaa-Pro-Phe type, only when the Xaa-Pro peptide bond is in a trans state. The coupled assay of Fischer exploits the conformational selectivity of chymotrypsin toward chromogenic substrates with pnitroanilide at C-terminus of the peptide (Xaa-Pro-Phe-pNA) (Schema 1).

In aqueous solution, the equilibrium population of the cis and trans Xaa-Pro conformers of this peptide substrate represent approximately 10% and 90%, respectively. Adding chymotrypsin results in the rapid cleavage of about 90% of the peptide, while peptides with cis proline bonds are cleaved at a rate limited by the uncatalyzed proline isomerization. The addition of a prolyl isomerase will accelerate this latter reaction, allowing indirect monitoring of the PPIasecatalyzed cis-to-trans isomerization.

These inherent limitations have led Kofron et al., (Kofron et al., 1991) to develop an improved method for monitoring the peptidyl prolyl cis/trans isomerase activity. These improvements consisted in improving the experimental conditions. First, the substrate has been dissolved in a

LiCL/TFE medium. This salt/solvent system shifts the cis population from » 10% to 40-60% and in some cases to 70% depending on the substrate sequences. Then, the p-nitroaniline was monitored at varied wavelengths, away from the absorption maximum (l max 390 nm). These experimental modifications allow a large increase (×30) in the useful concentration of the chymotrypsin substrate (Suc-AA-cis-PF-pNA). Furthermore, in order to suppress the uncatalyzed thermal isomerization, the experiment was conducted at very low temperature (0°C).

Despite these improvements, some limitations remain. When the goal is to identify potent Pin1 inhibitors, they must be stable to digestion by the protease chymotrypsin and not interfere with its proteolytic activity. Furthermore, it is not possible to detect the two directions of the reversible prolyl isomerization, only the cis-to-trans isomerization is investigated.

III.1.2 Setting up the test conditions

In our studies, we used the commercial test SensoLyte® Green Pin1 Activity Assay (AnaSpec)

to investigate the catalytic activity of Pin1 in the presence of putative inhibitors. The commercial test contained five components: the fluorogenic substrate, a solution containing the Pin1 protein, a solution called "developer" containing the proteolytic enzyme, the assay buffer, and a solution of tannic acid as a positive control (known Pin1 inhibitor). The sequence of the fluorogenic substrate was not specified, nor was the concentration of the chymotrypsin (or chymotrypsin like) in the 'developer' solution. Consequently, we applied the procedures provided in the commercial test, partly as a black box.

Before starting the experiments, we had to determine the concentration of Pin1 that we needed as well as the quantity of ligands that we were going to add.

We decided to use the Pin1 protein overexpressed in our laboratory since it was provided in limited quantities in the commercial assay. -A Preparation period generally consisting of RF pulses and possibly single delays.

After this period, the magnetic moments of all nuclei summed vectorially to a magnetization M lying along the magnetic field at equilibrium will be perturbed in "nonequilibrum" spin state (away from its equilibrium state).

-An Evolution period of length t 1 (corresponding to the indirect dimension). It is an incrementable delay for mapping chemical shifts or coupling constants that are allowed to evolve at their appropriate frequencies.

-A Mixing time t m during which through-bond or through space couplings are allowed to interact. The mixing period can comprise further RF pulses and delays. Note that not all 2D NMR experiments require a mixing period.

-A Detection period of length t 2 (corresponding to the direct dimension) during which the FID is acquired.

-A relaxation period is required at the end of the experiment to ensure that the spin is at equilibrium before the next cycle of acquisition.

The 2D frequency correlation spectra is obtained after a double Fourier transformation of the time domain function M (t 1 , t 2 ).

The spectra will be a function of the two-frequency variable w 1 and w 2 .

S (w 1 , w 2 ) = ∬ M (t 1 , t 2 ) 𝑒 -iw 1 t 1 𝑒 -iw 2 t 2 dt 1 dt 2

In the case of chemical exchange between two states A and B (characterized by precession frequencies w A and w B ) during the evolution period t 1, the state A will contribute to a component Adding Pin1 to the sample did not accelerate significantly the k ex value. A slightly lower value (k ex = 46 s -1 ) was even found in the presence of 50 µM of Pin1, reflecting that CZ115 was not a substrate for Pin1 although it selectively binds the PPIase domain with a Kd of 28 µM. Indeed, if CZ115 was a substrate, an increase of k ex should be observed with unchanged cis and trans populations.

In a second assay, we evaluated the putative inhibitory activity of CZ115. For this purpose, we used Cdc25 as a Pin1 model substrate and recorded three different EXSY experiments:

-Cdc25 alone

-Cdc25 + Pin1 -Cdc25 + Pin1 + CZ115
As expected, for Cdc25 alone, no exchange peaks were observed in the EXSY spectra due to too slow cis/trans isomerization rate of the pT-P peptide bond. Adding Pin1 with a 1/40 ratio (Pin1/Cdc25) resulted in the apparition of several exchange cross peaks. The methyl protons of the pT residue were sufficiently isolated and used for the integration of the cis and trans forms of Cdc25.

By adding CZ115 to the Pin1/Cdc25 mixture (sample conditions: 0.2 mM CZ115, 2 mM Cdc25, and 0.05 mM Pin1 at 298K), a similar value was determined which informed that CZ115 possessed no inhibitory effect.

This result is surprising given the affinity of CZ115 for the PPIase domain (Kd of 28 µM).

There is also a discrepancy with the results obtained with the fluorogenic assay described in the previous section, where CZ115 did activate the PPIase activity. This highlights the difficulty of establishing a reliable assay to measure PPIase activity. Note also that the Cdc25 ligand is both a substrate for the PPIase domain of Pin1 and a ligand of the WW domain. In our studies (and others), no Kd was determined for the binding of Cdc25 to the PPIase domain because of its too low affinity for this domain. The mechanism of action of Pin1 inhibitors is probably as complex as the substrate processing.

IV. Discussion

IV.1 Overview of the binding properties of the tested ligands

In this chapter, we detailed the study of three of our ligands, CZ55, CZ115, and CZ181 that showed a selective binding for one of the Pin1 domain. In addition, we were interested in two known peptide substrates of Pin1, Pintide and Cdc25. These analyses were aimed at better understanding the FL Pin1 mechanism. Cdc25 has been extensively used for structural, dynamics, and functional studies of the protein Pin1. Pintide has also been used in several published studies, although its analogue FFpSPR with improved solubility properties has often been preferred.

Cdc25 and Pintide preferentially bind the loop 1 of the WW domain with Kd values of 9.8 ± 2 and 11.5 ± 3 µM, respectively. Furthermore, both peptides were able to induce chemical shift perturbations in residues of the domain interface (loop 2 of the WW domain and the a4 /b6 region of the PPIase domain). However, for these residues, the CSPs showed different The binding of CZ55 to the loop 1 of the WW domain also induced perturbations at the WW/PPiase interface. The monitoring of the CSPs showed great similarity with those induced by Pintide. Furthermore, the Kd value of CZ55 measured on the isolated WW domain was ~2 times higher than that measured with FL Pin1. This increase in Kd was also observed with Pintide and it was concluded that the PPiase domain had a positive allosteric effect on the ability of the WW domain to bind Pintide. (Verdicia et al., 2000).

R2 values showed increased stiffness for the Pin1/CZ55 complex compared with the apo protein, with greater t c for both the WW and the PPIase domains. In details, the a4 /b6 region corresponding to the inter-domain interface of the PPIase domain was the most affected by the binding of CZ55. In addition, a slight loss of flexibility was also observed in the helix a1 that forms the wall of the concave surface of the inter-domain interface. Interestingly, the presence of the helix a1 is only found in the PPIase domains of proteins that have a WW domain. This helix may play a functional role in the regulation the communication between the two domains [START_REF] Ranganathan | Structural and functional analysis of the mitotic peptidyl-prolyl isomerase Pin1 suggests that substrate recognition is phosphorylation dependent[END_REF].

Compared to the results obtained on the Pin1/Pintide complex, the pattern of R 2 measured for the Pin1/CZ55 complex was similar. However, the loss of flexibility caused by Pintide was more pronounced, especially for the helix a1 and the catalytic loop of the PPIase domain.

On the contrary, the calculated t c values for the Pin1/Cdc25 complex were consistent with weaker inter-domain contacts. This is in agreement with the CSPs observed at the domain interface which where "opposite" to those observed for pintide and CZ55. Further evidence for the lack of cooperativity between the two domains upon Cdc25 binding is the similarity of the Kd values obtained for the isolated WW domain and the FL Pin1 (Vogeli, Molecules 2020).

Such difference in the inter-domain contacts were also observed with the ligands specific to the PPIase domain. CZ115 and CZ181 displayed differences in CSPs, which is primarily due to their different structures. CZ115 contains a Ser (Y H, CF3 Pro) oxazolidine residue and a central peptide bond adopting mainly a cis conformation (~80%). In contrast, CZ181 possesses a reduced-amide bond which gives rotational flexibility around the peptidyl-prolyl bond mimic.

Consequently, their mode of interaction differs significantly and may also explain the difference in Pin1 dynamics.

Referring to the literature, only two examples of structural and dynamics studies are available, which investigate specific ligands of the PPIase domain of Pin1. This includes the reducedamide inhibitor (Ac-pSer-Y[CH 2 N]-Pro-tryptamine) [START_REF] Xu | Reduced-Amide Inhibitor of Pin1 Binds in a Conformation Resembling a Twisted-Amide Transition State[END_REF], an analogue of CZ181, and a derivative of FFpSPR with a cis alkene isostere instead of the pSer-Pro peptide bond [START_REF] Namanja | Stereospecific gating of functional motions in Pin1[END_REF]. The reduced-amide inhibitor and the cis-locked inhibitor only bind to the PPIase domain with Kd of 12 µM and of 28 µM, respectively.

The X-Ray structure of R14APin1/reduced-amide complex showed that the reduced amide inhibitor binds the catalytic region by two different sites as shown in chapter 2, section I.4: the basic residue triad and the prolyl bond pocket. No direct hydrogen bonding was detected between the residues of the active site (including His59, Ser154, His157 and Cys113) and the prolyl nitrogen although these residues were all in its direct vicinity (<5Ǻ). In the other study, the cis-locked inhibitor displayed CSPs for the phosphate and the prolyl pocket, as well as for the residues defining the catalytic loop and interacting with the peptidyl-polyl peptide bond.

In our hands, the NMR titration of CZ181 showed significant CSPs for the residues interacting with the peptidyl-prolyl bond (active site) while the catalytic loop including the basic residue triad was less disturbed. The binding of this peculiar ligand is inherently unusual as it mimics the transition state of the substrate. Compared to CZ181, the main differences observed for CZ115 in terms of CSP were the intensities more pronounced in the PPIase domain, especially in the catalytic loop, and to a lesser extent, in the region corresponding to the helix a1.

However, CZ181 caused marked allosteric perturbations for the important residues of the WW domain, CZ115 having virtually no effects on this regulatory domain.

It is interesting to relate this observation to the CZ115 affinity properties. The absence of the WW domain had only a limited impact on the Kd value of the PPIase domain for the CZ115 ligand (28 µM for the FL Pin1 and 20 µM for the isolated PPIase domain). This feature is reminiscent of Cdc25 (vide supra) that was also not influenced by the other domain (the PPIase domain in that case), in terms of dynamics and affinity.

On the contrary, inter-domain contacts were clearly identified for CZ181. The truncation of the WW domain allowed an increase in affinity (threefold smaller Kd values for the truncated Pin1-PPI over FL Pin1). Similar behavior was described by Namanja et al. [START_REF] Namanja | Stereospecific gating of functional motions in Pin1[END_REF] who found a higher affinity (up to fourfold) for the cis-locked inhibitor on the isolated PPIase domain.

In summary, CZ181 (an analogue of the reduce-amide inhibitor described by Xu G et al.) showed similarities to the cis-locked inhibitor in terms of CSP, affinities and WW domain dependence. CZ115 binding exhibited a different CSP profile and was almost insensitive to the presence or the absence of the WW domain.

IV.2 Consequence of ligand binding to PPIase binding on Pin1 dynamics

To further explain our observations, we performed relaxation experiments on Pin1 complexed with CZ181 or CZ115. It has been well described that, depending on the type of ligand, binding to the WW domain could either enhance or limit the dynamics of the FL Pin1 protein. The question these arises whether ligands of the PPIase domain could have the same differential effects.

Interestingly, CZ115 and CZ181 had different dynamic response upon binding to the PPIase domain. Differences mainly occurred in two regions, the catalytic site and the domain interface.

CZ115 produced loosening of both regions, while CZ181 induced a loss of flexibility. Such a stiffening of the protein dynamics was already described by A.T. Namanja et al. upon binding of their cis-locked inhibitor. A thorough analysis of the side chain dynamics showed that the binding of this ligand caused an extensive stiffening along an internal conduit connecting the catalytic site with the domain interface.

But so far, to the best of our knowledge, no peptide ligands have been described that are specific for the PPIase domain and induce an increase in the overall Pin1 dynamics. Our analysis demonstrated that, when interacting with Pin1, CZ115 increased the R2/R1 ratio for many residues of the PPIase/WW interface. As a result, the t c values increased for both the PPIase domain and the WW domain, as it was observed upon binding of Cdc25 to the WW domain of Pin1.

Importance of the WW domain for the activity of Pin1

The impact of the WW domain on the activity of the PPIase domain has been studied using different substrates (Wang et al., 2015;[START_REF] Zhu | Uncorrelated Effect of Interdomain Contact on Pin1 Isomerase Activity Reveals Positive Catalytic Cooperativity[END_REF]. With Cdc25, that promoted an extended conformation of Pin1, the k ex value was found 1.2-fold higher for the isolated PPIase domain (k ex = 41 s -1 ) (Wang et al., 2015). We can hypothesize that, in this case, the presence of residual interactions between the WW and the PPIase domain in FL Pin1 is detrimental to the catalytic activity. However, cautions must be taken because the WW domain has a high affinity for Cdc25 which could bias activity measurements on the PPIase domain. But a similar conclusion was made using the I28A mutant of the FL Pin1. Ile28 is a key residue in the interdomain interface and its mutation strongly destabilizes the interaction between the two domains (Wang et al., 2015). This mutant exhibited increased isomerase activity (2.2-fold) suggesting again that allosteric interactions with the WW domain had a negative impact on the catalytic activity of Pin1. However, such a result was not observed for FFpSPR. Using this substrate, similar k ex (k ex values are 97 and 91 for FL Pin1 and the isolated domain respectively) (Wang et al., 2015) were measured with FL Pin1 and the isolated PPIase. As for Pintide, this peptide binds the WW domain and promotes a compact conformation of Pin1. These two examples showed different implications of the WW domain in the regulation of Pin1 activity.

Interestingly, this antagonism was associated to different conformations of the complex. Cdc25 induced and preferred an open state for optimal activity while FFpSPR induced a compact state and was not affected by the presence of the WW domain in terms of activity.

In the case of our ligands, we have conducted both fluorogenic assays and EXSY measurements. At low concentrations of CZ52, CZ55 and CZ115, we have measured increased activities on a fluorogenic Pin1 substrate, but we consider these results with a lot of caution (see part III.1.3). EXSY measurements have proven that CZ115 was not a substrate of Pin1, despite the fact that it binds the PPIase domain. Finally, the Pin1 activity measured on the model substrate Cdc25 was not changed by the presence of 0.1 equivalent of CZ115, which contrasts with the results obtained using the commercial fluorogenic test.

Nevertheless, a k ex of 60.7 ± 3.2 s -1 was calculated for CZ115 alone at 298K, which is in the same range of magnitude than those obtained with Cdc25 and FFpSPR in the presence 1/40 ratio of Pin1 (Wang et al., 2015). This result highlights the impact of the incorporation of a CF 3 group at the C d position of the oxazolidine on the rate of the uncatalyzed isomerization reaction.

V. WW domain modulated the binding affinities of the PPIase domain

The To test whether the origin of the decrease in affinity of CZ115 against the PPIase domain was due to the allosteric effect induced by Pintide, we measured the affinity of CZ115 in the presence of CZ55, which, as Pintide and Cdc25, selectively binds the WW domain. CZ55 did not induce any CSPs in the FL Pin1 PPIase domain and it was strictly unable to bind to the isolated catalytic domain. This makes it a very selective WW domain ligand, even more specific than Pintide (Kd_PPIase/Kd_WW > 1000/32 for CZ55 and 100/11.5 for Pintide). Furthermore, the relaxation experiment of FL Pin1 with CZ55 showed that CZ55 was able to strengthen the inter-domain interaction. The titration of FL Pin1 with CZ115 in the presence of CZ55 produced an identical CSPs profile than in the presence of Pintide (data not shown). The Kd value obtained for CZ115 in the presence of CZ55 was in the same order of magnitude (95.6 ± 8 µM)

than with Pintide confirming that the decrease in affinity of CZ115 against the PPIase domain was mostly due to the stabilization of the close conformation of Pin1.

V.2 CSPs of WW-complexed FL Pin1 upon CZ181 binding

We previously showed in chapter 4 that CZ181 selectively recognizes the PPIase domain of Pin1. The titration of FL Pin1/Cdc25 complex with CZ181 showed a CSP profile quite similar to that obtained in the absence of Cdc25 (Figure 74). However, two residues showed very weak CSPs in the presence of Cdc25 compared to the first titration. These two residues are of prime importance for Pin1 activity. The first is located in the catalytic loop (S71) and the second belongs to the proline pocket (C113). These observations thus revealed a lower propensity of the PPIase domain to bind CZ181. The Kd value of CZ181 with FL Pin1/Cdc25 complex was » 67 ± 8 µM, slightly higher than with FL Pin1 (44 ± 6 µM), which confirmed our qualitative observations. This loss of affinity could be due to the remoteness of the WW domain following the Cdc25 binding. In contrast, the titration of CZ181 into the Pin1/Pintide complex restored the high CSP value for residues S71 and C113 and gave a Kd 7 times lower (9.5 ± 3 µM), or

V.3. Comparisons with published results

Matena et al., used a PEG molecule to stabilize the closed state of Pin1 (Matena et al., 2013).

Then they measured the affinity of the PPIase domain using the peptide Suc-ApSPF-pNA by fluorescence anisotropy and titration NMR experiments. In both cases, they showed that the affinity was enhanced compared to Pin1 alone. We observed such a result but only for the CZ181 ligand. In contrast, the stabilization of the WW/PPIase interface was harmful for CZ115

binding.

In a recent study, Zhu et al., modified the inter-domain linker, while keeping the two structured domains intact, to study its impact on inter-domain contact and on Pin1 activity [START_REF] Zhu | Uncorrelated Effect of Interdomain Contact on Pin1 Isomerase Activity Reveals Positive Catalytic Cooperativity[END_REF]. By shortening the WW-PPIase linker, these authors were able to strongly stabilize a compact conformation of Pin1 for which they obtained the highest activity. This is apparently

in disagreement with the model proposed by Peng et al., where the WW domain has negative role on the Pin1 activity. (Wang et al., 2015). In a more recent publication, [START_REF] Zhu | Uncorrelated Effect of Interdomain Contact on Pin1 Isomerase Activity Reveals Positive Catalytic Cooperativity[END_REF] demonstrated that, in fact, the inter-domain contacts were not sufficient to explain the increase or decrease in Pin1 activity. They claimed that inter-domain contacts and isomerase activity were not correlated.

From our perspective, this may actually depend on the type of substrate used for the activity test. We observed that CZ181 and CZ115, that both bind specifically the PPIase domain, imposed opposite effects on Pin1 dynamics (decreased dynamics with CZ181, increased dynamics with CZ115). In return, the PPIase affinity for CZ181 was positively impacted by the WW domain whereas the PPIase affinity for CZ115 was negatively impacted by the presence of the WW domain. Cautions should be taken when comparing activity and affinity, which are not directly related. As an example, the Cdc25 peptide has significantly better affinity for the PPIase domains in the context of FL Pin1 but the best isomerase activity is obtained with the isolated PPIAse domain and not with FL Pin1. However, so far, we have not been able to measure PPIase activity on our ligand which could be Pin1 ligands without being Pin1 substrates.

Chapter 6: A new proposed model for the interaction between the WW domain and the PPIase domain.

Four different models (sequential, multimeric, catalysis-first and simultaneous binding) have been proposed to explain how the WW domain can assist the catalytic mechanism of the PPIase domain (see section II.3 and the figure 13 in chapter 1).

The sequential model proposes that first, the WW domain binds to the pS/T-P motif and then gives way to the PPIase domain. The second model (multimeric-binding model) suggests that Pin1 interacts with a multimeric complex through its WW domain, allowing the PPIase domain to recognize and bind to other motifs in the complex. In the catalysis-first binding model, the PPIase domain first catalyzes its substrate from cis to trans conformation, creating a WW domain binding site. In this case, the PPIase domain would not be able to catalyze the reverse isomerization. Finally, the simultaneous binding model proposes that both domains can bind simultaneously to substrates containing multiple pS/T-P motifs, each with low affinities (more details in chapter 1, paragraph II.3).

For half of these models, only one phosphorylated site of the Pin1 substrate is recognized (sequential binding and catalysis first). This implies that the WW and PPIase binding sites are close to each other, so that the domain exchange (WW by PPIase for the sequential binding or

PPIase by WW for the catalysis first) is feasible. It is difficult to determine the precise distance between the two binding sites since the linker between them is highly flexible. However, the loop 1 of the WW domain and the catalytic loop of the PPIase are located on two different faces in the compact conformation of the FL Pin1. In the five X-Ray structures inspected (PDB codes: 6VAJ, 4U86, 3OOB, 4QIB, 3NTP), typical distances between these two loops were above 30 Ǻ. A direct translocation of the ligand from one domain to the other would then require a significant rearrangement of the two domains. Such a change in the relative position of the two domains has not been identified so far.

The Sequential and multimeric binding models postulate that the binding to the WW domain acts as an anchor and that the PPIase domain can then explore the neighborhood to bind a second site. This second site can be on the same protein or on another one. The latter models seem very attractive considering the large number of phosphorylated sites found on average on Pin1 substrates and/or the many Pin1 partners capable of forming oligomers.

However, as developed in the following, we considered an alternative model in which only one phosphorylated site is isomerized. Based on the oligomerization properties of FL Pin1, this new model would allow spatial proximity between the WW and PPIase domains.

I. MD insights into a new model of Pin1 activity mechanism.

With our collaborator B. Iorga (ICSN, Gif-sur-Yvette), we observed that several crystallographic structures of Pin1 present a dimer in their asymmetric units (PDB code: 1PIN, 1F8A, 2Q5A, 3NTP), with an average surface contact of 400 Ǻ 2 . Looking at these dimers in crystal, we were struck by the close proximity of the WWbinding loop of the first protein and the PPIase catalytic loop of the second protein chain (Figure 75). B. Iorga performed several MD simulations (GROMACS force field, in explicit solvent) to assess the stability of the dimer without any ligand or with CZ55.

The 2Q5A pdb structure served as an initial structure for the dynamics calculations. The protocol of the MD simulations as well as the detailed analysis of the trajectories will not be presented here since this work has been carried out by our collaborator. The main observations are the following:

-The dimer was stable in explicit water during the complete simulations (500 ns to 1 µs long)

The interface between the two monomers was defined by the helix a2 and the a2/a3 loop of the PPIase domain of the monomer A, and the strand b1 and the b2/b3 loop of the WW domain of the monomer B. The presence of the CZ55 ligand reinforced this interface.

-The catalytic loop (b4/a1 loop) of the monomer A was kept close to the loop 1 (binding site) of the WW domain during the time course of the simulation. A 4-5 Ǻ long groove, mainly delineated by hydrophobic residues, connected the catalytic loop in monomer A and the WW binding site in monomer B.

-It was possible to simulate the migration of the ligand from one binding site to the other without any modification of the force field (passage from the PPIase domain of the monomer A to the WW domain of the monomer B and vice-versa).

attractive interactions, l<1 for repulsive interactions) and f is the volume fraction occupied by the molecule (Tokuyama and Oppenheim;1994).

This equation allowed us to determine the expected diffusion coefficient Ds, for each f value, which can be calculated from the protein specific density (value fixed at 1.44 according to Fischer H et al., 2004). The factor l was considered as equal to 1.1, based on the study of Bax et al., (Chou J. J. et al., 2004).

The linear regression of the experimental data gave us the D 0 value, where f = 0. Theoretical hydrodynamic radii of Pin1 were then deduced from theoretical Ds values, following the Stockes-Einstein equation (Figure 76A, orange dots).

Finally, the difference between the two curves (R h (DLS) -R h (th)) showed a linear increase up to 0.5 mM and reached a plateau corresponding to a hydrodynamic radius difference of 0,37 nm a higher concentration (Figure 76B).

We were then interested in the difference of R h expected upon dimerization. Taking into account the well-characterized Hen Egg White Lysosyme as a reference (M L = 14.3 KDa, R h = 16 Ǻ, V L = 17 nm 3 according to Chou J. J. et al., 2004) and considering a constant density for proteins (Tokuyama and Oppenheim; 1994), we can calculate the theoretical volumes V M and V D for the monomeric and dimeric Pin1, respectively: Then, by considering that the monomer and dimer have spherical shapes (strong approximation):

M L /V L = M M /V M = M D /V D V M = V L ×M M /
V M = 4/3 p R h,M 3 and V D = 4/3 p R h,D 3
Hence: R h,M = 1.75 nm and R h,M = 2.20 nm These calculations are very basic and do not take into account the evolution of the protein shape upon dimerization nor the change of the solvation sphere. However, this provides an order of magnitude of the expected increase in R h (DR h = 0.45 nm) if dimerization occurs. This value is in the range of our experimental observations and may confirm a dimerization of the Pin1 protein at high concentration. This dimerization could be a first step towards further oligomerization, as observed at 2mM. However, these results are preliminary and supplemental experiments would be needed to calculate RMSD values and error bars for these DLS data.

For the monomeric Pin1, we were particularly concerned by the discrepancy between the theoretical hydrodynamic radius (1.75 nm) derived from reference values (from Lysozyme) and the measured experimental values (2.4 nm). The non-spherical shape of the protein, the dynamics of the two domains, the presence of a large solvation sphere, systematic biases between the two techniques (reference values on Lysozyme obtained by NMR, experimental data measured by DLS) are some examples of factors that could explain these differences.

To confirm the results obtained by DLS, we used NMR spectroscopy as a second technique.

We measured the translational diffusion coefficient of Pin1 at different concentrations by the acquisition of DOSY experiments.

I.2 DOSY experiments

The Diffusion-Ordered SpectroscopY (DOSY), applied to the analysis of complex mixtures, the presence of a dimer at higher concentration, although species of very high molecular weights were identified at 2 mM. Their formation followed a slow process and was reversible.

By DOSY NMR, the calculated hydrodynamic radius at 2 mM would be compatible with Pin1 tetramers. However, this measurement corresponds to an average between several interconverting species, some of which could have a very high molecular size as shown by DLS. Our relaxation measurements were in very good agreement with both standard protein references and already published data on Pin1. At concentrations higher than » 1 mM, the t c values reflect an equilibrium between a monomeric form of Pin1 and oligomers. At 2 mM, we obtained t c values that could perfectly correspond to a Pin1 dimer. However, the t c curve did not reach a plateau at this concentration and it is likely that higher t c values could be measured at higher Pin1 concentrations. The existence of a dimer in solution was therefore likely at Pin1 concentration ~1mM. Such a dimer can be found in crystal structures with an interaction surface of ~ 400 Ǻ.

Interestingly, the isolated PPIase domain was unable to form dimeric species at high concentrations which revealed that the WW domain contributes to the dimerization of Pin1 in solution. Although other explanations have been proposed, this result might be related to the fact that the isolated PPIase domain is inactive in cells highlighting the essential role of the WW for the function of Pin1 in vivo (Berhsin et al., 2006).

These experiments would be compatible with new roles for the WW domain in vivo: the WW domain could stabilize a Pin1 dimer in solution and facilitate the ligand exchange between the two domains. Of course, protein partners/substrates could assist the dimerization of Pin1.

In the X-Ray structures, the binding site of the WW domain is in close vicinity to the catalytic site of the PPIase domain of the other monomer. This may facilitate the passage of the ligands between the two domains. MD calculations have proven that in this model, the two binding sites were connected by a hydrophobic groove of 4-5 Ǻ long and that peptide ligands could exchange between the two sites in the time course of the simulation. More experiments will be necessary to study the interaction of both domains with their substrates in the dimer as well as the catalytic activity of the PPIase domain. Indeed, our collaborator at Cergy Pontoise University are considering the development of bidentate substrates that could simultaneously bind the WW domain of one monomer and the catalytic site of the PPIase domain of the other monomer. The synthesis of such ligands will allow us to study the interaction of the dimer with its substrates by NMR. Studies of the dimer dynamics in the presence of substrates will be necessary. Indeed, the relaxation experiments of the dimer/substrate complex could give us some insights into the interplay of the WW domain with the PPIase domain of the other monomer. Activity tests on the dimer will be also important to consider.

One obstacle that could affect the progress of the project is the high concentration that we need to stabilize the dimer state of Pin1 in solution. In the absence of bidentate ligand, a mutation will be required to stabilize the dimer at low concentrations. Our collaborators at ICSN are analyzing the dimeric interface to find a position in which a disulfide bridge could be introduced to stabilize the dimer. The dimer is not symmetrical, so the mutation of a single residue is not sufficient to establish such a disulfide bridge. So far, they suggested the mutation of two residues, Gln 109 in the helix a 2 and His 27 in the b 2 /b 3 loop into Cysteine. This possibility will be assessed in the next future.

General discussion

The unique and singular role of Pin1 in the regulation of phosphorylated clients function has made this PPIase a priority in recent drug discovery efforts. Thus, the development of potent Pin1 inhibitors has been an attractive topic and a large panel of Pin1 inhibitors has been identified. Tens of academic laboratories as well as private laboratories were involved in the development of different families of potent Pin1 inhibitors which exhibited, in some cases, submicromolar affinity and inhibitory activity in the nanomolar range. However, in most of these series, the presence of a phosphate group was a major drawback in the cell permeability of the compounds. Among the new Pin1 inhibitors, those in which the phosphate group was replaced by other groups, such as carboxylic acid, appeared to be the most effective, because they balanced the binding affinity of Pin1 with the need for membrane permeability. By introducing a fluorine atom in our ligands, we have succeeded in presenting some originality compared to all the ligands described in the literature. Indeed, the fluorination of ligands at the

I.2 Expression of 15N-and 15 N, 13 C-FL Pin1 and the isolated domains

For the expression of the labelled proteins, we applied Marley's protocol [START_REF] Lu | A method for efficient isotopic labeling of recombinant proteins[END_REF].

First, the bacterial cells were incubated at 37°C with shaking for three to four hours in a rich medium. At an optical density A 600 » 0.6, the cells were centrifuged over 15 min, 3000 rpm at room temperature. The LB medium is removed and the pellet is resuspended in the M9 medium that not contain the nitrogen and carbon sources at this step. An incubation at 37 ° C with

I.3 Purification of Pin1 and isolated domains

After adding 1 µg/ml of leupeptin, pepstatin and 1 mM of PMSF, the cells obtained from the culture are lysate by sonication at 4°C (ten times 15s followed by ten times 20s with a 60s pause between each sonication, amplitude: 40%). The soluble extract is then isolated by centrifugation at 30,000 xg, 4 ° C for 30 min then filtered through a 0.22µm PVDF filter.

The lysate is loaded on a pre-equilibrated Nickel Nitrilotriacetic acid column (Ni-NTA) on an Akta Pure with 30 mM potassium phosphate buffer pH 7.8, 500 mM NaCl, 30 mM imidazole and 1mM dithiolathreitol. The column is then rinsed until the OD at 280 nm decreased to zero and get stable. After that, the protein is eluted with 30 mM potassium phosphate buffer pH 7.8, 500 mM NaCl, 500 mM imidazole and 1mM dithiolathreitol. The interest fractions are combined. The digestion of the His-tag was achieved by adding the PreScission Protease directly to the protein and both were incubated overnight at 7°C. 1 mM of EDTA in final concentration is added to the mix to prevent further degradation of the protein of interest by proteolysis.

The next day, the PreScission/protein mixture is loaded again onto the NiNTA column. This step allows the elimination of the fragments after digestion as well as the PreScission enzyme which also contain a His-tag. On the other hand, the protein of interest will not bind to the column and it will be eluted directly.

The Superdex GE healthcare size exclusion column of 120 mL was equilibrated with the Tris buffer 30 mM, 50 mM NaCl and 2 mM DTT, pH 7.00. The fractions of the second affinity purification step are combined and injected directly from the elution buffer of the Nickel column purification step. The protein is eluted in the equilibration buffer described above at the elution time of approximately 70 minutes.

For the NMR experiments, the protein was concentrate by an amicon cell (pressure concentration system) until reached the desired concentrations. The concentration was measured at 280 nm wavelength using a NanoDrop 2000 UV/Vis spectrometer (Thermo Scientific) and calculated based on the predicted extinction coefficient of 20.87 M/cm.

HPLC is used as the second WW domain purification step after digestion of his-tag with the PreScission enzyme and thus makes it possible to remove the fragments after digestion as well as PreScission. Obtaining protein in lyophilized form also has the advantage of facilitating and prolonging storage. The elution fractions digested or not with PreScission are injected into a C4-300 nm 10-250 nm column previously equilibrated in an aqueous solution containing 0.1% TFA (v/v). The elution is carried out with an acetonitrile gradient at 6 mL/min from 10 to 100% acetonitrile in 30 minutes. The elution time of the isolated domain was estimated to be around 7 min.

The collected fractions are checked by MALDI-TOF mass spectrometry and on SDS PAGE denaturing gel at 15% acrylamide.

After purification by HPLC, the fractions containing the pure WW domain are combined and lyophilized.

II. Differential Scanning Fluorimetry DSF.

Different buffers at pH varying from 6 to 7.5 are prepared at 500 mM. Except for the Tris buffer, the preparation of buffers is carried out by mixing the acid and the conjugate base. For the Tris buffer, the powder is dissolved directly and the pH is adjusted using a 1 M sodium hydroxide or hydrochloric acid solution. Each solution is filtered through a 0.22 µm PVDF filter before use.

SYPRO® Orange 5000x diluted in DMSO is used as thermofluoride. For a volume of 200 µL, 2.5 µL of SYPRO® Orange are added to 197.5 µL of filtered milliQ water (concentration of the stock solution of 62.5x).

A 96-well PCR plate (Dominique Dutscher, Brumath, France) is filled (V total = 25 µL for a well) while respecting the conditions set. The plans of the plates are given in appendix (). The plate is filled on ice and in the dark in order to limit the denaturation of the protein and by following this order: 1) water, 2) NaCl, 3) buffer with 50 mM final concentration, 4) additives, 5)

unlabeled Pin1 at the final concentration of 0.05 mg/mL and 6) SYPRO Orange 5x final. A film is placed on the plate to prevent any evaporation during the experiment. The acquisitions are carried out on a real-time PCR apparatus (RT-PCR, LightCycler 480 II, Roche, Basel, Switzerland). The fluorescence in each well is measured at regular intervals with a temperature gradient of 0.01 °C/s for a temperature range of 20°C to 95°C. The interaction of the fluorophore with the protein manifests as a sigmoid. The temperature Tm corresponds to half of denatured protein (inflection point of the slope). Thus, the Tm is the maximum of the first derivative of the sigmoid. Data is analyzed using Mathematica software.

III. NMR experiments

III.1 NMR resonance assignments:

The 15 N, 13 C-labeled apo Pin1 sample contained 0.6 mM Pin1 in 30 mM tris buffer (or sodium phosphate buffer) at pH 7.00, 50 mM NaCl, 2mM DTT, and 8% D 2 O in a 5mm NMR tube.

NMR experiments were performed at 298K on a triple resonance Bruker Avance 500 MHz spectrometer equipped with cryogenic probe.

Experiments 1 H, 15 N-HSQC (8 scans), HNCO (8 scans), HN(CA)CO (32 scans), HNCA (32 scans), CBCANH (32 scans), and CBCA(CO)NH (32 scans) are recorded. A 1 H, 15 N-HSQC experiment as well as a 1D 1 H experiment is performed after each 3D experiment to check the stability of the sample.

All spectra were processed with NMRPipe (Delagio et al., 1995) and analyzed using NMRFAM-Sparky.

III.2 Chemical shift perturbations and binding affinity calculations

The R 1 and R 2 parameters were measured at 500 MHz, 298K with established pulse schemes (Kay et al., 1989;Dayie and Wagner, 1994). In the complexed samples, Pin1 concentration was 0.6 to 0.7 mM with 8-fold excess of ligands. For the apo protein, five different concentrations of Pin1 were used: 0.25, 0.7, 1.0, 1.5 and 2.0 mM.

The same relaxation delays were used for apo and complexed samples. R 1 ( 15 N) delays included T = 2, 20, 100 (x2), 200, 300, 500, 750, 1000, 3000, 4000, and 5000 ms. The R 2 ( 15 N) delays were T = 16.96 (x2), 33.92, 50.88, 67.84, 84.8, 101.76, 135.68, 169.6, 203.52, 254.4 and 305.28 ms. All spectra were acquired with 64 x 2084 complex points.

Backbone amide 15 N R 1 and R 2 relaxation rates were determined by fitting peak heights as functions of relaxation decay times to single-exponential decay functions. The spectra were processed and analyzed with NMRPipe by using python scripts.

III.4 EXchange SpectroscopY experiments:

Cis/trans isomerase rates of CZ115 and Cdc25 were measured in the absence and presence of Pin1 at 298K at 500 MHz, by following cis/trans exchange of the longitudinal magnetization of pseudoproline H d nuclei and the H d methyls of the pT5, respectively in 2D 1 H-1 H EXSY spectra (Jeener et al., 1979). The concentration of CZ115 and Cdc25 was 2 mM in which we added 50 µM of fresh unlabeled Pin1. EXSY spectra of Pin1/Cdc25 complex in the presence of 200 µM of CZ115 were also recorded.

Exchange mixing times were: 0.5, 1, 5, 10, 25, 50, 75, 100; 150, 200, 300, 1000, 2000 and 5000 ms. The exchange rate constants, k EXSY , were estimated by fitting the ratios of trans-to-cis crosspeaks (or cis-to-trans) over the trans (or cis) diagonal peaks as a function of the exchange mixing time to the two-state function (Ernst et al., 1987) in collaboration with Dr. Guillaume Bouvignies.

III.5 DOSY experiments

A series of 1D 1 H DOSY experiments was recorded at 500 MHZ and 298K for unlabeled Pin1 with a concentration range of 0.2 to 2 mM.

Diffusion experiments were performed using a Pulse Field Gradient Stimulated Echo sequence (PFG-STE).

To determine the Pin1 self-diffusion coefficient at different concentrations, a series of 1D experiments were recorded with identical diffusion delays of 0.55 s and field gradient pulses of 1.2 ms duration and intensity ranging from 2 G cm -1 to 47.5 G cm -1 (4 to 95% of the maximum 10 A amplifier power). Each experiment was derived from the accumulation of 544 scans (4 K complex points). The diffusion coefficient of Pin1 was obtained from the decay of peak intensity of the aliphatic protons by using NMRPipe. The corresponding hydrodynamic radius Rh was deduced by applying the Stockes-Einstein equation (see chapter 6).

IV. Pin1 activity assay

The activity of Pin1 was assayed in presence of Pin1 Green substrate and different concentrations of the ligands: CZ55 and CZ115 (0.05, 0.5, 50, 500 and 5000 µM). A stock solution of 1 mg/mL of fresh Pin1 was prepared. The Pin1 Green substrate was diluted in the assay buffer to have a concentration of 1 µM. The chymotrypsin was also diluted 100x in the assay buffer. All the stock solutions were kept in ice.

A 96-well black plate (Sigma Aldrich, St. Louis, USA) is filled (V total = 100 µL for a well) while respecting the conditions set. The plans of the plates are given in appendix (). The plate is filled on ice in order to slow down the reaction and by following this order: 1) 30 µL of chymotrypsin 2) 10 µL of Pin1 3) 10 µL of ligands 4) 50 µL of green substrate. The plate was recover with a transparent film to prevent evaporation during the manipe.

The fluorescence of Pin1 Green substrate is measured after cleavage by the chymotrypsin using a Fluostar Optima Plate Reader (BMG Labtech, Ortenberg, Germany) capable of detecting emission at 520 nm with excitation at 490 nm. The plate was shacked before each measurement and the fluorescence intensity recorded every 10 min, for 12h.

V. Dynamic light scattering DLS

The hydrodynamic ratio (Rh) of Pin1 was measured at concentration ranging from 0.05 mM to 1.5 mM (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.5). Before each experiment, the solution containing Pin1 was filtered through a 0.22 µm PVDF filter to eliminate the aggregation and the concentration of Pin1 was then checked by measuring the OD at 280 nm.

The device used is the DynaPro instrument (ProteinSolutions, Charlottesville, VA), providing a wavelength of 830 nm. The samples were prepared in a 12 µl quartz cuvette at 20°C (20 µL of protein). Autocorrelation curves were obtained for an angle of 90 ° with an acquisition time of four hours and analyzed using the Dynamics software, version 6.

Chapter 5 :

 5 Jeanne and Emma ... I.4.2 Optimization of Pin1 concentration I.5 Purification of Pin1 proteins using HPLC instead of gel filtration I.6 Overview of selected methods to prepare Pin1 protein samples II. Optimization of FL Pin1 stability using Differentiel Scanning Fluorimetry III-NMR assignment of FL Pin1 and its isolated WW domain and PPIase domains III.1 Backbone assignment of FL Pin1 in phosphate buffer and Tris buffer III.2 Backbone assignment of Pin1 WW domain and PPIase domain IV.3 Dynamic properties of FL Pin1 and its isolated domains Chapter 4: A new generation of Pin1 fluorinated inhibitors I. Description of synthetized ligands II. Interaction of Pin1 with ligands III. Discussion III.1 Looking for a rationale for the design of Pin1 peptide ligands III.2 Hints of allostery from binding Fluorinated inhibitors to characterize the allosteric regulation of Pin1 I. Specific ligands for the WW domain I.1 Interaction of FL Pin1 with Pintide and Cdc25 I.2 CSPs of FL Pin1: CZ55 vs Pintide and Cdc25 I.3 Cdc25 and Pintide induced different dynamic impact with FL Pin1 I.3.1 Overview of the origin of the relaxation phenomena in NMR I.3.1 Cdc25 and Pintide induced different dymacis in FL Pin1 I.3.3 Relaxation experiments Of FL Pin1 in the presence of CZ55 II. Specific ligands for the PPIase domain II.1 CSPs induced by CZ181 and CZ115 on Pin1 II.2 Structural features from 1D 19 F NMR experiments II.3 Relaxation experiments of Pin1 complexes with CZ181 and CZ115 III. Measuring the inhibitory activity of the ligands III.1 Measuring the inhibitory activity of CZ55 and CZ115 by fluorometric test III.1.1 Generality about the coupled assay test III.1.2 Setting up the test conditions III.1.3 The inhibitory activity of CZ55 against Pin1 III.2 Activity measurements by NMR III.2.1 Basic principal of EXSY` III.2.2 Pin1 activity in the presence of CZ115 IV. Discussion IV.1 Overview of the binding properties of the tested ligands IV.2 Consequence of ligand binding to PPIase on Pin1 dynamics V. WW domain modulated the binding affinities of the PPIase domain V.1 CSPs of WW-complexed FL Pin1 upon CZ115 binding V.2 CSPs of WW-complexed FL Pin1 upon CZ181 binding V.3 Comparaisons with published results Chapter 6: A new proposed model of the interaction between the WW domain and the PPIase domain I. MD insights into a new model of Pin1 activity mechanism I.1 DLS experiments of Pin1 I.2 DOSY experiments I.3 Tc of Pin1 from the relaxation parameters II. Discussion

  ont montré que les substrats phospho-peptidiques avaient des affinités légèrement différentes pour les domaines isolés par rapport à FL Pin1, suggérant que les interactions transitoires entre les deux domaines peuvent affecter leurs interactions avec leurs substrats. Nous avons donc testé les ligands présentant les meilleures affinités (Kd < 45 µM) et montrant des spécificités de liaison pour le domaine WW (CZ55) ou PPIase (CZ52, CZ115 et CZ181). Les mesures d'affinité ont été conduites sur la protéine entière (FL Pin1) et sur les domaines isolés. L'affinité de CZ55 est deux fois plus faible en absence du domaine PPIase par comparaison à Pin1 entière. De plus, des différences des CSPs ont été détectées surtout au niveau des résidus qui interagissent avec la proline des ligands, mettant ainsi en évidence le rôle stabilisant du domaine PPIase sur l'interaction du domaine WW avec ces substrats. D'autre part, la comparaison des valeurs de Kd obtenues pour le domaine PPIase isolé avec celles obtenues sur Pin1 FL a permis de mettre en évidence des phénomènes d'allostérie entre les deux domaines ainsi qu'une influence du domaine WW sur l'affinité du domaine PPIase vis-à-vis de ses substrats. Les premiers résultats présentés semblent indiquer que l'effet allostérique du domaine WW dépend de la nature du ligand lié au domaine PPIase. Oon n'observe aucun effet du domaine WW sur l'affinité des peptides CZ52 et CZ115 alors que pour le peptide CZ181, les valeurs de Kd ont été divisées par 3 en retirant le domaine WW. Ces résultats nous ont conduit à étudier en détail les effets allostériques qui existent entre les deux domaines de Pin1 pour mieux comprendre la régulation de son activité. Les ligands fluorés que nous développons permettent de cibler un domaine spécifique de Pin1 et constituent en cela des outils essentiels pour cette étude. Dans cet objectif, des expériences de relaxation de FL Pin1 en absence et en présence des ligands (CZ55, CZ115 et CZ181) ont été enregistrées. De façon intéressante, chaque ligand a eu un impact différent sur la dynamique de FL Pin1. En liant le domaine WW, CZ55 cause une diminution de la flexibilité de la région à l'interface entre les deux domaines ainsi qu'au niveau du site catalytique du domaine PPIase. Parmi les études disponibles dans la littérature, de telles modifications ont été observées avec le peptide Pintide. De plus, en se liant à CZ55, Pin1 montre des valeurs du temps de corrélation t c pour chaque domaine légèrement supérieur à celles obtenues sur Pin1 apo. Ainsi, nous avons montré que CZ55 renforçait les contacts entre les deux domaines de Pin1. Concernant les ligands se liant au domaine PPIase, CZ115 induit une flexibilité dans deux régions de Pin1: au niveau du site catalytique et à l'interface entre les deux domaines. Ce changement de dynamique est caractérisé par des valeurs de t c plus faibles que celle trouvées pour Pin1 apo. En revanche, un comportement opposé a été trouvé pour CZ181 qui conduit à une perte de flexibilité pour les mêmes régions. Les de t c mesurées sont plus importantes pour ce complexe. Ces résultats confirment que la nature du ligand peptidique auquel Pin1 se lie, peut stabiliser ou perturber les interactions transitoires entre ses deux domaines, qu'il s'agisse de ligand spécifique du domaine WW ou du domaine PPIase. Ces phénomènes de liaisons sont accompagnés par des changements de la dynamique globale de Pin1. Pour comprendre comment l'interaction entre les deux domaines pouvait affecter l'activité cis/trans isomérase du domaine PPIase, nous avons utilisé deux n approches : un test d'activité en présence d'un substrat fluororescent et des expériences RMN d'EXchange SpectroscopY ( 1 H-1 H EXSY). Ces techniques ont été appliquées sur trois de nos ligands, CZ55 qui lie le domaine WW et CZ115 et CZ52 qui lient le domaine PPIase. Concernant les tests d'activité par fluorescence, nous avons trouvé qu'à faible concentration CZ55 induisait une augmentation de l'activité de Pin1, mais qu'à partir de concentrations élevées, il avait un effet inhibiteur. On pourrait interpréter ces résultats, en accord avec les expériences de relaxation, en concluant que le renforcement de l'interaction entre les deux domaines provoqués par CZ55 a un effet positif sur l'activité de Pin1. Ce résultat est toutefois en contradiction avec le modèle proposé par Peng et al. qui attribue une action inhibitrice au domaine WW. Nous retrouvons une action inhibitrice de CZ55 à concentration élevée Cette observation pourrait être liée à un effet de compétition de CZ55 au niveau du site actif, car, malgré sa mauvaise affinité pour le domaine PPIase, il pourrait agir comme inhibiteur compétitif à très haute concentration. Cependant, un profil d'activation/inhibition a également été observé pour CZ52, ligand de mauvaise affinité mais spécifique du domaine PPIase. Pour sa part, d'après les tests par fluorescence, CZ115 a montré uniquement un effet activateur de la fonction isomérase de Pin1 ce qui nous a également paru difficile à interpréter pour ce ligand spécifique du domaine PPIase. Nous nous sommes interrogé sur la stabilité des ligands et de la protéine Pin1 dans les conditions du test (présence de chemotrypsine). La dégradation partielle de Pin1 observée nous a conduit à ne plus considérer les résultats obtenus aves ce test fluorimétrique commercial. Nous avons enregistré une série des expériences EXSY en présence de Pin1 et son substrat Cdc25. L'activité de Pin1 a bien été mise en évidence par cette approche RMN mais les mesures de vitesses d'interconversion cis-trans en présence de CZ115 n'ont pas permis de mettre en évidence une activité inhibitrice de ce ligand. Des efforts supplémentaires seront engagés pour compléter et améliorer les tests d'activité de la protéine Pin1. Dans un second temps, pour évaluer l'effet du domaine WW sur l'affinité du domaine PPIase vis-à-vis de ses substrats, nous avons réalisé une série d'expériences dans lesquelles nous avons stabilisé l'état ouvert ou fermé de Pin1. Pour cela, nous avons saturé le domaine WW de Pin1 FL avec les deux peptides Cdc25 et Pintide connus pour leur effet antagoniste sur Pin1. Ainsi, l'interaction de Cdc25 avec le domaine WW peut déstabiliser l'interaction entre les deux domaines alors que le Pintide la renforce. Nous avons ensuite titré les complexes Pin1-Cdc25 et Pin1-Pintide avec CZ115 et CZ181, deux ligands qui lient spécifiquement le domaine PPIase. Dans le complexe Pin1-Cdc25 (état ouvert est stabilisé), l'affinité de CZ115 n'a pas été affectée tandis que dans l'état fermé (Pin1-Pintide), l'affinité à baissée d'un facteur trois. Pour ce qui concerne CZ181, la stabilisation de l'état fermé a pu améliorer son affinité vis-àvis du domaine PPIase avec une valeur de Kd quatre fois plus faible. Ainsi, avec CZ115 (dont la ligation induisait spontanément une ouverture de la charnière WW/PPIase), le renforcement de l'interaction entre les deux domaines conduit à une perte d'affinité alors qu'elle l'améliore dans le cas de de CZ181. La liaison du ligand CZ181 au domaine PPIase provoquait à l'opposé une fermeture de la charnière WW/PPIase) Ces résultats confirment donc que le domaine WW peut améliorer ou perturber l'interaction du domaine PPIase vis-à-vis de ces substrats. L'ensemble de nos données permet d'apporter un éclairage nouveau sur cette régulation allostérique : la nature précise du ligand du domaine WW (qui renforce ou déstabilise les contacts inter-domaines) ne permet pas de prévoir à elle seule si l'effet sera bénéfique ou délétère pour la liaison du domaine PPIase à un deuxième ligand. Tout dépend du type de ligand PPIase. Certains ligands de ce domaine semblent présenter une préférence pour un état fermé, d'autres pour un état ouvert. Cette préférence pourrait être prédite sur la base d'études de dynamique du ligand PPiase en en complexe avec FL Pin1. Durant ma thèse, nous nous sommes aussi intéressés aux états oligomériques de la protéine à Pin1. L'existence d'état dimère pourrait amener à imaginer un mécanisme catalytique nouveau pour cette protéine. Jusqu'à présent, quatre modèles différents ont été proposés pour expliquer comment le domaine WW pouvait moduler l'activité PPIase dans la fonction cellulaire de Pin1 : le modèle séquentiel, multimérique, catalyse d'abord et simultané. Le modèle séquentiel propose que dans une première étape, le domaine WW se lie au motif pS/T-P, puis, soit s'échappe et permet au domaine PPIase de se lier et de remplir son rôle catalytique soit le domaine PPiase se lie dans un deuxième temps à un site pS/T-P voisin sur la même protéine. Le deuxième modèle (modèle de liaison multimère) suggère que Pin1 interagit avec un complexe multimérique : son domaine WW se lie avec le motif pS/T-P d'un premier substrat tandis que le domaine PPIase reconnait un site sur une autre protéine qui formerait un heterodimère avec la première. Dans le modèle de liaison catalyse d'abord, le domaine PPIase est le premier à intervenir. Il se fixe à une protéine substrat présentant le motif pS/T-P en cis, isomérise cette liaison en trans, créant un site de liaison au domaine WW. Dans ce modèle, la liaison du domaine WW sur la liaison peptidique isomérisée en trans empêche le domaine PPIase de se lier à nouveau et de catalyser l'isomérisation inverse. Enfin, le modèle de liaison simultanée propose que les deux domaines se lient simultanément à des substrats contenant plusieurs motifs pS/T-P. Le gain entropique de cette double liaison permet de lier des motifs pS/T-P présentant individuellement de faibles affinités.En se basant sur différentes structures cristallographiques de Pin1 montrant Pin1 sous forme dimérique au sein de l'unité asymétrique, nos collaborateurs (ICSN, Gif-sur-yvette), ont lancé des expériences de dynamique moléculaire sur Pin1. Grace à ces expériences, nous avons montré que Pin1 était stable sous forme dimérique à l'échelle de la simulation. L'assemblage dimérique est asymétrique, et de manière intéressante le site catalytique du domaine PPIase d'un des deux monomères est proche dans l'espace du domaine de liaison WW de l'autre domaine (Figure3). Ces deux sites sont reliés par un canal hydrophobe de 4 à 5 Ǻ de long. Des expériences de dynamique moléculaire ont montré que l'échange d'un ligand peptidique était possible entre ces deux sites au sein du dimère.

  DNA and its associated chromatin. It has been shown that Pin1 is implicated at different stages in the regulation of gene transcription. Pin1 is reported to regulate the nuclear localization of a number of transcription factors. For example, Pin1 promotes nuclear localization of cyclinD, b-catenin, and NK-kB, while it prevents nuclear localization for NF-AT, FOXO4 and CRTC2.

  inhibiting GSK3-b activity. The suppression of Pin1 causes GSK3-b activation, leading to increased levels of T668-phophoryated APP and amyloidogenic APP processing. Moreover, Pin1 promotes APP protein degradation by binding to the Thr330-Pro motif of GSK3-b (Ma et al., 2012; Xiong et al., 2013) suggesting a novel role of Pin1 against Ab toxicity by using multiple mechanisms. Contrasting results founded by Uchida et al., showed that in the Pin1 KO mice, soluble and insoluble Ab levels are lower compared with WT mice and promoted Ab production in vitro (Akiyama

  . It consists of 163 amino acids that form two distinct domains, the N-terminal WW domain spanning the first 39 amino acids and the PPIase domain spanning residues 52-163. The two domains are connected by a 15-residue flexible linker between Pro37 and Pro52. The WW domain consists of a three-stranded antiparallel b-sheet and the PPIase domain exhibits a four-stranded anti-parallel b-sheet surrounded by four alpha helices (Figure 7). Both domains recognize and bind a phosphorylated serine or threonine when preceding a proline residue (pS/T-P motifs) but only the PPIase domain displays a catalytic activity. The binding regions involve the b 1 '/b 2 ' loop I (residues Ser16-Arg21) in the WW domain and the long catalytic loop b 1 /a 1 in the PPIase domain (residues His64-Arg80).

  The nature and spatial distribution of amino acids within the catalytic center are however conserved between Par14 and Pin1. In the catalytic center of both proteins, the hydrophobic cavity is formed by residues Leu44/Leu61, Leu82/Leu122, Met90/Met130, Phe94/Phe134 and Ile125/Ile159 (as referred to Pin1/Par14 sequences). The specific arrangement of theses residues in Pin1 is important to accommodate the substrate proline moiety. The Asp74 residue in Par14 replaces the catalytic residue Cys113 of Pin1. The basic triad residues (Lys63, Arg68, and Arg69) of Pin1, that confer preferential binding to phosphorylated substrates and immobilize the side chain of the Ser/Thr residue preceding the proline, are absent in the Par14 sequence. Therefore, Pin1 and Par14 exhibit distinct binding specificities. While Pin1 recognizes only phosphorylated Serine or Threonine preceding a Proline, Par14 is considered as a non-phosphate specific parvulins with high activity toward substrates with Arginine or Leucine preceding the Proline (Hoppstock et al., 2016). Another notable structural difference between Pin1 and Par14 is the presence of the WW domain in the N-terminal region of Pin1 while the N-terminal region of Par14 is intrinsically disordered and subjected to a high degree of post-translational modifications (Van der lee et al., 2014).

II. 3

 3 .3.1 Evidence of allostery mechanisms from binding and activity measurements NMR studies have been conducted on complexes between the Pin1 protein and peptides derived from the Tau protein. These peptides can contain up to three phosphorylated pS/T-P motifs in their sequence. Upon binding to the WW domain, they affected the catalytic activity and the binding affinity of the PPIase domain. These modifications depend on the sequence of peptides as well as the number of pS/T-P sites: Tau peptides with a single phosphorylation site increased the efficiently of isomerase activity while multiple binding sites inhibited it. This demonstrated that the PPIase activity depends on the phosphorylation motif bound by the WW domain (Smet

  Pin1 function at multiple levels II.6.1 Transcriptional and post-transcriptional regulation Pin1 expression is correlated with cell proliferative potential in normal human tissues, and Pin1 levels gradually decrease with ageing. The regulation of Pin1 expression is mediated by the E2F family of transcription factors (E2F1, E2F2and E2F3). These transcription factors interact on the E2F binding site located in the Pin1 promoter to increase Pin1 protein levels downstream of oncogenic growth factor signaling[START_REF] Wulf | Role of Pin1 in the regulation of p53 stability and p21 transactivation, and cell cycle checkpoints in response to DNA damage[END_REF]. Furthermore, the intracellular domain of the transmembrane receptor NOTCH1 directly induces the transcription of Pin1, which in turns acts on NOTCH1, thereby generating a positive feedback loop to promote tumorigenesis. On the contrary, p53 and the AP4 act as transcriptional repressors and reduce Pin1 transcription(Mitchell and Smith, 1988; Jeong et al, 2014). Xbp1 induces the transcription of p53 via HEPN1 and represses E2F1 via NF-kB activation, resulting in reduced Pin1 transcription. Furthermore, BRCA1, a tumor suppressor gene, repressed the transcription of Pin1(MacLachlan et al., 2000). BRCA1 control DNA damage by interacting with different proteins. The mutation of BRCA1 led to accumulate DNA damage in cells(Mersch et al., 2015).

  Fisher et al., have found that juglone irreversibly inhibits the enzymatic activity of several parvulins, like the E. coli Par10, the yeast Ess1 (see Chapter I, table 1), and human Pin1 (IC 50 = 0,64 µM). It directly targets and covalently modifies thiol group of the active cysteine Cys113 in the catalytic center ability of PiB to inhibit activity of both PPIases has been corroborated with a molecular modeling study which predicted the binding of PiB into the PPIase catalytic site[START_REF] Uchida | Pin1 and Par14 peptidyl prolyl isomerase inhibitors block cell proliferation[END_REF]. However, although inhibitory effects were observed both in vitro and in vivo, there are few evidences of direct PiB binding to Pin1 in the cellular context.Two years later, a new class of Pin1 inhibitors was developed from a combinatorial synthesis program to find cell-active analogs of pepticinnamin C, a farnesyl transferase inhibitor synthesized by actinomycetes that is able to induce apoptosis. Among the apoptosis-inducing compounds that failed to inhibit farnesyl transferase, three of them were shown to possess an inhibitory potency against Pin1, including the pepticinnamin C analog (figure17) with IC 50 = 0.6 µM. The reason why these compounds were tested for Pin1 inhibition was due to its role in signaling pathways downstream of Ras. In 2009, Uchidia et al., have used a PPIase activity screening to identify a new potent inhibitor of Pin1, the Dipentamethylene Thiuram Monosulfide (DTM) (IC 50 = 4 µM) (Figure17)

  this thesis project, we were interested in the development of fluorinated peptides as new inhibitors of Pin1. In particular, we took advantage of the know-how of our collaborators at the LCB of Cergy Pontoise in the fluorine chemistry for the conception of fluorinated peptidomimetic molecules. As detailed in the following sections, fluorine has many advantages in medicinal chemistry.II.1 Interest of fluorine in medicinal chemistry: generality about fluorineII.1.1 Physico-chemical properties of fluorineFluorine atom is the chemical element with atomic number 9. It is the thirteenth element in order of abundance in the earth's crust (0.028%), and is the first halogen. Fluorine is the smallest element of the periodic table after hydrogen atom. With a van der Waals radius of 1.47 Å, covalently bound fluorine occupies a smaller volume than a methyl, amino, or hydroxyl group (Table

Fluorine, being the

  most electronegative element with 3.98 on the Pauling electronegativity scale (compared to 2.20 for hydrogen, and 3.44 for oxygen), provides highly polarized C-F bonds which present a strong dipolar moment (µ C-F = 1.41 D) and a low-lying C-F σ* orbital available for hyperconjugative donation. The combination of these effects give the C-F bond the strongest energy of dissociation compared to any other C-X (X: any atom) bond (

  changes. For example, methoxybenzenes adopt a planar conformation with a C-C-O-C dihedral angle around 90°, while the OCF 3 group is out of the aromatic plane in trifluoromethoxybenzenes (Massa M. A. et al. 2001) II.1.4 Influence of fluorine on biological properties There are no endogenous fluorinated molecules in humans. In contrast, fluorinated compounds are present in most of the major therapeutic classes (for examples: the anti-cancer Faslodex by Astrazeneca, the antidepressant Fluoxine by Eli Lilly, the antibiotic Flurithromycin by Pharmacia, and the anti-HIV Efavirenz by Bristol-Myers Squibb).

  Buffers with pH < 7.0 (citric acid and Bis-Tris) appear to destabilize FL Pin1, as shown by the reduced Tm values obtained regardless of the NaCl concentration, when compared to buffers with pH ≥ 7.0. The highest Tm value (55°C) was obtained with phosphate buffer pH 7.0 in the presence of 50 mM NaCl (Figure34). Based on this first series, we selected phosphate and Tris buffers with pH ranging from 6.70 to 7.50 for additional DSF experiments, although pH values Ala137, Ala140, Leu141, Thr143). These data confirm the allosteric impact of the WW domain which presence affects the dynamic properties of the PPIase domain, in particular in the b4/a1 catalytic loop. Conversely, the presence of the PPIase domain also affects the dynamic properties of the WW domain.Chapter 4: A new generation of Pin1 fluorinated inhibitors I. Description of synthetized ligandsBased on the literature, we have selected versatile templates which comprise a minimal peptide backbone length of 2 or 3 residues and different N-and C-terminal extremities. At the Nterminus, acetyl, trifluoroacetyl, or fluorenylmethoxycarbonyl (Fmoc) protecting groups were chosen, whereas tryptamine or carboxamide capped the C-terminal extremity. In position P2, which corresponds to the second residue from the N-terminus, we inserted either phosphorylated serine (ligands CB06, CB08, CZ40, CZ34, CZ55, CZ52, CZ115, CZ178, and CZ181) or unphosphorylated serine (ligands CB07, CZ279, CZ280, CZ281, CZ282, GG84, and GG82) to determine if the negative charge is required for the binding to Pin1. The serine residue was preferred over the threonine based on a study published by Etzkorn et al., in which the inhibition of Pin1 catalytic activity was 5-fold higher with compounds incorporating the pSer-Pro motif than equivalent compounds incorporating the pThr-Pro motif[START_REF] Xu | Reduced-Amide Inhibitor of Pin1 Binds in a Conformation Resembling a Twisted-Amide Transition State[END_REF]. In addition, the substitution of pThr for pSer was shown to increase the k cat /K m of the isomerase activity.As mentioned in chapter 2, the phosphate group of phosphorylated Pin1 inhibitors suffers of chemical and/or metabolic instability and is detrimental to cell permeability, impeding in vivo inhibitory activity. Different strategies have been developed to overcome these drawbacks, such as the replacement of the phosphate by a carboxylate group. However, these analogues led to less-potent Pin1 inhibitors. The cases where the ligands (without the phosphate group) still have a good affinity are cyclic peptides with high molecular weight or non-peptidic molecules (see chapter 2, section I.2 and I.3)

  the measurement of binding parameters, NOESY spectra were recorded to determine the population of cis and trans conformers for each ligand. In the case of trans conformers, a correlation peak was identified between the Ha of the Serine and the Ha of the proline while in the case of cis conformer, a correlation peak was identified between the Ha of the Serine residue and the Hd of the Proline. The percentage of the conformers was calculated by a simple integration of the proton signals in the 1D 1 H spectra.

(

  CSPs) in FL Pin1 and the isolated domains. Chemical Shift Perturbation is a powerful technique for studying interaction between protein-protein or protein-ligand at the amino acid level. It consists of titrating an unlabeled ligand into a 15 N-labelled protein and monitoring amide chemical shift changes of the protein at each stage of the titration by acquiring 2D 15 N, 1 H-HSQC spectra. The chemical shift of a given nucleus is a parameter sensitive to the chemical environment, which value is influenced by neighboring atoms being either covalently bonded or close in space. As such, the formation of a non-covalent biomolecular complex is expected to affect chemical shifts of nuclei localized at the interface of the interaction.Monitoring the frequency shifts of each 15 N/ 1 H correlation throughout the titration allows to determine protein binding surfaces by mapping amide CSPs onto the protein structure. It also allows to identify protein regions far from the interacting surfaces but which undergo a structural rearrangement induced by ligand binding. The quantitative analysis of the amide CSPs also informs about ligand affinity through the determination of apparent Kd values at the residue level.

  Average Kd estimated from 1 H-15 N NMR titration experiments of FL Pin1 or its isolated domains. ND = not determine

  of the origin of the relaxation phenomena in NMR

  5 ns and 11.3 ns. The relaxation parameters measured separately on the WW and on the PPIase domains were in perfect agreement and indicated that upon binding to the PPIase domain, CZ115 favored an open conformation of the FL Pin1, whereas CZ181 stabilized the interface and promoted a close state of the protein. This result is reminiscent of the properties induced by the WW domain ligands Cdc25 and Pintide, respectively. It is also worth to stress that some CSPs displayed opposite frequency shifts upon titrations by the two PPIase ligands, as previously observed for the WW ligands.

III. 1 . 1

 11 Generality about the coupled assay test Fischer et al. were the first to discover the Peptidyl Prolyl cis/trans Isomerase family (PPIases)

  First, we measured its activity at different concentrations (0.1, 0.5, 2, and 10 µg in 100 µL) in the presence of the substrate and chymotrypsin, and we compared it to the activity of the commercial Pin1 from the kit at 0.5 µg/100 µL (270 nM, recommended concentration in the standard protocol). The fluorescence monitoring for 60 minutes is represented in the figure 68. From the curve slopes, we haveIII.2 Pin1 Activity measurements by NMRIII.2.1 Basic principle of EXSYWe used the 1 H-1 H EXchange SpectroscopY (EXSY) to quantify the inhibitory activity of CZ115. For the simple two states system describing the Pin1 substrate (cis and trans conformation of the pSer-Pro peptide bond), the experiment consists in quantifying and fitting the cross peaks cis-to-trans and trans-to-cis as well as the corresponding diagonal peaks in a set of NOESY experiments acquired with many different mixing times (Figure70A and B). Jeener et al. were the first to propose the use of 2D NMR experiments to measure chemical kinetics. The pulse sequence is shown in Figure 70C. In general, 2D NMR experiments contain several distinct elements:

  directions and magnitude. These results were in line with our relaxation measurements and with already published analyses that highlighted their different allosteric behavior. Cdc25 can stabilize an open state of PL Pin1 where the WW and PPIase domains are distant from each other while Pintide promotes a closed state where the WW and PPIase domains are directly interacting.

  binding of CZ115 and CZ181 to the PPIase domain induced different conformational preferences for FL Pin1. While CZ115 favored the extended state, CZ181 stabilized a compact state of Pin1.We decided to take advantage of these two specific ligands of the PPIase domain to gain more insights into the role of the WW domain in the Pin1 binding features. For this purpose, we stabilized the open and closed state of Pin1 by binding Cdc25 and Pintide to the WW domain, respectively, and we calculated again the affinities of CZ181 and CZ115.V.1 CSPs of WW-complexed FL Pin1 upon CZ115 bindingAfter adding saturated amounts of Cdc25, we titrated FL Pin1 with CZ55. The CSPs map of FL Pin1 is represented in Figure73. Adding increasing concentration of CZ115 did not produce any chemical shift perturbations in the WW domain, showing that the binding of CZ115 on the PPIase domain did not affect the interaction of Cdc25 with the WW domain. This confirmed that Pin1 can bind simultaneously two ligands, one within the WW domain and a second within the PPIase domain[START_REF] Namanja | Stereospecific gating of functional motions in Pin1[END_REF].The CSPs measured on the PPIase domain of the FL Pin1/Cdc25 complex were quite similar to those obtained with FL Pin1 in the absence of Cdc25 and with the isolated PPIase domain. No modifications were observed on either the nature of the residues perturbed by CZ115 or CSP intensities. This result reflects that the binding mode of CZ115 within the PPIase domain was not affected by a further decrease in the interaction between the PPIase domain and the WW domain (caused by Cdc25) or even by the complete removal of the WW domain. Indeed, the Kd value of CZ115 in the presence of Cdc25 was equal to 32 ± 5,9 𝜇M, which is in the same 123, 125 and 130 belong to the proline binding pocket of Pin1 substrates. These modifications of CSPs probably reflected changes in the binding mode of CZ115 when the closed conformation of Pin1 is stabilized. This alternative binding mode is also reflected by the Kd value, about three times greater than without Pintide. Hence, for CZ115, stabilizing the close state of Pin1 was detrimental for its affinity, i.e. the WW domain had a negative impact on its binding to PPIase.

  M L and V D = V L ×M D / M L With M M = 18.75 KDa and M D = 2×M M = 37.5 KDa V M = 22.2 nm 3 and V D = 44.5 nm 3

  allows the separation of NMR signals of different components according to their diffusion coefficient. It is achieved by the acquisition of Pulse Field Gradient Spin Echo (PFG-SE) experiments (Figure77A). After a 90° pulse, a Pulse Field Gradient (PFG) of length d leads to a phase shift of the magnetization. This shift depends on the spatial position of the spins along the axis of the PFG (spatial encoding). Then, a 180 ° pulse is introduced which refocuses the chemical shifts. Finally, after a diffusion delay D, a second PFG (identical to the first) decodes the spatially encoded phases (spatial decoding) before the FID acquisition. In the case of a stationary spin system, the effects of the first and the second PFD exactly cancels out. However, the molecules in aqueous solution undergo translational diffusion Brownian movements during the diffusion delay D. As a result, the position of the spins along the gradient axis changes between the two PFGs, causing an incomplete phase refocusing. This leads to an attenuation in intensity of the spin echo. correlation time by 15 N relaxation measurements. These three methods are based on different physical principles and provide independent data. The measurement of the rotational correlation time by relaxation techniques involves the spins dynamics, it depends on the modulation of the magnetic interactions within the protein in solution. The measurement of the translational diffusion coefficient is based on the Brownian molecular movements. This may explain the differences in the results we obtained with these different techniques. Indeed, the nominal values of the diffusion coefficient differed significantly for the monomeric Pin1. DLS revealed

C

  d position of pseudoproline residues allowed us to generate selective PPIase domain ligands. To date, there are very few examples in the literature of acyclic peptide ligands that are selective for the PPIase domain. Unfortunately, we have not been able to obtain clear results on the inhibitory activity of our ligands. Nevertheless, the incorporation of fluorine in Pin1 inhibitors may generate important modifications in the absorption process, recognition, metabolism, or elimination of the molecules. In a next project, we aim to replace the phosphate group by a difluorophosphonate (pCF 2 ), which should significantly improve the metabolic stability of the peptides. Pin1 has been studied extensively over the past 20 years, with most structural work aimed at understanding the dynamics and allostery of this two-domain protein. Various studies have been performed looking at Pin1 binding to many different peptides including Cdc25 and FFpSPR. It was shown that upon binding to the WW domain, these peptides exhibit different allosteric pathways in Pin1 by weakening or strengthening the inter-domain contacts respectively.Interestingly, both peptides had different behavior towards the isolated WW domain. While Cdc25 has similar affinity compared to FL Pin1, FFpSPR showed lower affinity for the isolated WW domain compared to FL Pin1. This showed the differential impact of the PPIase domain on the interaction of the WW domain with its substrates. Similar results were observed with our ligands by studying the dynamics of FL Pin1 complexes which showed that one subset of WW ligands was able to reduce the overall dynamics of the Pin1 protein while other a second subset increased it. Interestingly, two of the fluorinated ligands that bind selectively the PPIase domain revealed also different impacts on the interdomain contacts. Furthermore, an allosteric mechanism was demonstrated where modification of Pin1 protein dynamics by ligation of a WW ligand correlated with an increase in affinity for the first subset of the PPIase domain ligands, while the affinity of the second subset was decreased. This revealed that, depending on the sequence of the PPIase ligands, WW domain can enhance or disturb the interaction of the PPIase to its substrates.Measurements ofPin1 activity in the presence of different ligands after stabilization of the open and closed states of Pin1 would be necessary for a comprehensive understanding of the allostery that exists between the PPIase domain and the WW domain. In this work, we envisioned a new model for the catalytic mechanism of Pin1 and the interplay of its two domains. This model consists of a Pin1 dimer where the WW domain of a monomer is in close proximity with the PPIase domain of the other monomer. Based on several crystallographic structures of Pin1 which present a dimer in their asymmetric units and MD simulations that assessed the stability of the dimer with or without ligands, we were interested to demonstrate the oligomerization properties of Pin1 in solution. Several techniques have been employed and have shown that, at concentrations higher than 1 mM, Pin1 is present in oligomeric forms in solution. However, additional experiments are needed to confirm the existence and the functional implications of this putative Pin1 oligomers. Design of new bidentate ligands as well as the production of covalent Pin1 dimer are in progress.

  shaking (210 rpm) for 40 min allows the bacteria to consume the residual LB medium. The isotopic sources, 1 g / L of 15 NH 4 Cl and 4 g / L of 12 C or 13 C glucose (depending on the desired labeling) are then introduced into the cultures. A new incubation of 30 min at 37 ° C is carried out so that the labeled molecules are incorporated into the metabolism of the bacteria. The induction of the cells is performed by adding 0.5 mM of IPTG in final concentration. The cells are incubated overnight at 18°C. In the morning, they are collected by centrifugation (6000 RPM -30 min -4 ° C) and resuspended in an extraction buffer (80 mL of extraction buffer: 25 mM phosphate buffer, 500 mM NaCl, 30 mM imidazole, 1 mM DTT for 1L of M9 culture medium). The suspension is then frozen at -80°C until the purification step.

  Nous avons utilisé la DSF dans le but de déterminer les conditions optimales de stabilité de Pin1 en solution. Dans un premier temps, nous avons déterminé les températures de fusion Tm de Pin1 pour chaque type de tampon (50 mM) en présence d'une concentration variable de Tous les ligands comportant la liaison pSer-Pro (CB06, CB08, CZ55 et CZ34), adoptent majoritairement une conformation trans, et ont montré une nette préférence de liaison pour le domaine WW. Cependant, l'affinité de ces ligands a été très impactée par la nature des extrémités N-et C-terminales. La meilleure affinité a été obtenue avec le ligand CZ55 qui comporte un groupe acétyle en N-ter et une tryptamine en C-ter (» 32 ± 5 µM).Le greffage du groupe CF 3 sur le carbone Cd de l'oxazolidine Ser(Y H , CF3 Pro) augmente radicalement le ratio de population cis:trans (> 80% dans l'eau). Le domaine catalytique montre une forte préférence pour le substrat en conformation cis. Ainsi, en synthétisant des ligands principalement en conformation cis, nous avons pu générer des ligands sélectifs du domaine Elle consisterait à remplacer le groupement phosphate par un groupe a,adifluorophosphonate (pCF 2 ). pCF 2 est un très bon mime « isostérique et isopolaire » non hydrolysable du groupe phosphate mais n'a jamais encore été utilisé dans ce contexte. Ce remplacement devrait améliorer considérablement la stabilité métabolique des peptides.

	Pour les ligands CZ52, CZ34 et CZ115, la proline a été remplacée par son analogue CF 3 -YPro.
	Malheureusement, tous les ligands sans groupement phosphate ont présenté un manque de
	spécificité vis-à-vis des deux domaines de Pin1 ainsi que des très faibles valeurs d'affinité (>
	1mM).

NaCl (de 50 mM à 400 mM) (fig.

3

, gauche). Les résultats montrent que Pin1 est plus stable dans le tampon phosphate à pH 7. Ainsi, les valeurs de Tm montrent un effet déstabilisant du sel sur notre protéine. Dans un second temps, nous avons ajouté aux tampons plusieurs additifs connus dans la littérature pour leur effet stabilisant potentiel sur les protéines. Dans notre cas, la présence d'additifs combinés avec 50 mM NaCl n'a entraîné aucune variation significative de Tm (fig. 3, droite). Sur la base de ces résultats, nous avons finalement affiné le choix du tampon phosphate pour aboutir à la composition suivante : 30mM à pH 7, 50 mM NaCl et 2mM DTT. Avant d'étudier l'interaction de Pin1 avec les ligands, une étape d'attribution du spectre 15 N-HSQC de Pin1 entière était nécessaire. L'attribution des déplacements chimiques des noyaux PPIase. C'est la principale originalité de notre nouvelle génération de ligands avec CF 3 -YPro en position P3 par rapport aux nombreux ligands Pin1 qui ont été rapportés dans la littérature. La valeur Kd la plus faible a été obtenue avec le ligand CZ115 (28,6 µM) qui comporte une phénylalanine réduite en N-ter et une tryptamine réduite en C-ter.

Un autre ligand capable de cibler le domaine PPIase a été synthétisé par nos collaborateurs : CZ181. Ce ligand est l'analogue de l'inhibiteur à amide réduit conçu par Etzkorn et al

(Xu GG et al., 2011)

pour imiter « the twisted amide transition state » du mécanisme catalytique de Pin1. Dans CZ181, le groupe CF 3 a été incorporé sur l'extrémité N-ter pour servir de sonde RMN 19 F. Les inhibiteurs de Pin1 comportant un groupe phosphate chargé sur la serine ou la thréonine souffrent d'instabilité chimique et/ou métabolique. Sa présence est aussi préjudiciable à la perméabilité cellulaire et entrave l'activité inhibitrice in vivo. De plus, lors la synthèse de nos ligands, l'étape de phosphorylation a entrainé des pertes de rendement considérables. En général, la phosphorylation sur des composés peptidiques n'est pas une « réaction facile ». Nous avons donc voulu tester des ligands sans groupement phosphate au niveau du résidu Sérine et de compenser éventuellement son absence par un résidu glutamate. Dans la littérature, le remplacement de groupement phosphate par un groupe isostère a aussi été étudié mais ces analogues ont conduit à des inhibiteurs de Pin1 moins puissants. Une nouvelle stratégie est envisagée en collaboration avec nos collaborateurs (LCB, Cergy Pontoise).

Table 2 :

 2 Essential

residues of the WW domain and the PPIase domain that interact with the pSer/Thr-Pro motif of Pin1 substrates

Table 3 :

 3 Van der Waals radius R vdw (Å) and Pauling electronegativity scale (c p ) for different atoms of the periodic table.

		H	F	O	N	C	Cl
	R vdw (Å)	1.20	1.47	1.52	1.55	1.70	1.75
	c p	2.20	3.978	3.44	3.04	2.55	3.16

Table 4 )

 4 .The C-F bond strength in monofluorinated compounds is relatively greater than that of the C-H bond by 4 Kcal/mole, and is stronger than the other carbon-halogen bond by over 25 Kcal/mol.

		Bond lenght (Å)	Energy of dissociation (Kcal/mole)
	CH 3 -H	1.09	104.3
	CH 3 -F	1.39	108.3
	CH 3 -Cl		82,9
		1.77
	CH 3 -Br		69.6

Table 4 :

 4 comparison of the physical properties of some C-X bonds.

Table 5 :

 5 Energy of dissociation for the C-F bonds.

Table 6 :

 6 The effect of fluorine substitution on lipophilicity by comparing Log P.

H N , C𝛼, C𝛽, C' et N de Pin1 a été obtenue à partir des spectres hétéronucléaires 15 N-HSQC, HNCO, HNCACO, HNCA, HNCACB et CBCACONH. Toutes les résonnances amides non prolines de Pin1 ont été attribuées, à l'exception des résidus Glu4, Arg17, Ser18, Ser19 et Ser114. Les résonnances d'amide des résidus du linker étaient absentes dans les spectres enregistrés sur Pin1. Nos résultats sont en accord avec l'attribution disponible de Pin1 sur la BMRB (code 5305). Afin de déterminer le site d'interaction de nos ligands avec Pin1 et avec ses deux domaines isolés et afin de calculer leur affinité pour ces trois constructions, nous avons mesuré les perturbations des déplacement chimiques (CSPs) par RMN causées par l'ajout de concentrations croissantes des ligands. Ainsi, il a été possible d'identifier finement quels résidus sont impactés par la liaison du ligand et de déterminer son ou ses sites de liaison.

Acknowledgments

Foremost, I would like to thank the members of the jury: Pr. Isabelle Milazzo, Dr. Serge BouAziz, Dr. Karine Loth and Dr. Nadia Izadi-Pruneyre who have accepted to evaluate the scientific relevance of my thesis. I am most grateful for their time and contributions. I am particulary grateful to Dr. Nadia Izadi-Pruneyre and Dr. Karine Loth, my former M2 internship supervisors at Instiut Pasteur-Paris and Centre de Biophysique Moléculaire (LBM)-Orleans, who introduced me to NMR of biomolecules and for their support and recommendation in my research of my thesis. This work was realized at the Laboratoire des Biomolécules (LBM) of Sorbonne Université, directed by Dr. Sandrine Sagan, and by Dr. Olivier Lequin, to whom I extend my thanks for having welcomed me to the research unit. To Dr. Emeric Miclet and Dr. Ludovic Carlier, my PhD supervisors, I express my utmost gratitude, foremost, for having given me the unique opportunity to accomplish this project. I must shed light on their availability and their enthusiasm which, apparently, never leaves them. I thank them for having opened the doors of the NMR laboratory to me, for their confidence, and guidance, for the knowledge in NMR and chemistry they continuously shared with me and the patience with which they did, it was an honor and pleasure to be their student. I would like to express my gratitude to our collaborators at Cergy-Pontoise University, Laboratoire de Chimie Biologique (LCB): Pr. Thierry Brigaud, Dr. Grégory Chaume, Dr. Maud Larregola and Dr. Chiara Zanato for synthesizing the ligands that I tested along my thesis, for the scientific discussions that took place during our meetings and their individual contributions and suggestions throughout my study; especially Maud for the time and the energy she dedicated to my project. But also to our collaborators at ICSN, Gif-sur-Yvette, Dr. Bogdan Iorga and Dr. Nawel Mele for the MD experiments on the Dimer form of Pin1. I would also like to extend my thanks to Dr. Jean-Jacques Lacapère and Dr. Stéphanie Finet for their help in running the DLS experiments and for the scientific discussons. I would like to thank Pr. Olivier Lequin, Dr. Patrick Fuchs and Isabelle Correira, for their valuable and constructive recommendations during this research work.

Abbreviations

Results and Discussion

According to literature, aromatic residues were preferred at positions P1 and P4. Therefore, as a first step, a tryptophan and a phenylalanine were chosen at the C-terminus (CB06 and CB08), while the N-terminus was simply capped by an acetyl group for both ligands. The titration experiments undertook with CB06 and CB07 revealed very week affinities of Pin1 for these two ligands and a lack of specificity against the WW domain. CZ55, which contained a tryptamine at the C-terminus, showed a Kd value of » 32 ± 5 µM, 15 times better than the values obtained with CB06 and CB08. The indole present in this capping group appeared to be a good mimic of tryptophane at the P4 position while the aminoethyl group of tryptamine provided beneficial flexibility to better fit the WW binding site. Then, we decided to keep the tryptamine at this position for the next ligands. Replacing the acetyl group with the Fmoc protecting group at the N-terminus further improved the affinity against the WW domain (CZ40), in good agreement with the aromatic preference at the P1 position for Pin1 ligands.

Several CSPs were also detected at the catalytic site of the PPIase domain revealing lack of specificity of CZ40 compared to CZ55, with however a 20-fold weaker affinity for this domain.

For CZ52, CZ34 and CZ115, the proline was replaced by a CF 3 -YPro. The incorporation of a CF 3 group at the Cd carbon of oxazolidines (y CF3,H Pro) radically enhanced the cis population (> 80% in water) allowing to target the PPIase domain. As largely described in the chapter 1, the catalytic domain shows a strong preference for substrates adopting the cis conformation.

Therefore, by stabilizing the cis conformation in these CF 3 -YPro containing ligands, we were actually able to generate selective PPIase domain ligands. To date, there are very few examples in the literature of acyclic peptide ligands that are selective for the PPIase domain.

The lowest Kd value was found for CZ115 (28 ± 6 µM) which was highly specific for the PPiase domain (no binding detected on the WW domain). Almost all the residues within the catalytic site, but also adjacent residues were affected suggesting that CZ115 may diffuse in a large area close to the active site and/or may introduce local conformational changes in the PPIase domain.

For CZ52, the Kd value was 490 ± 50 µM, much lower compared to CZ115, which showed the importance of having an aromatic residue at the N-terminus to enhance the affinity of ligands against PPIase domain. Finally, the PPIase affinity was restored for CZ34 that possessed a Fmoc group at the N-terminus (41 ± 8 µM), but significant perturbations were also detected on the WW domain. Kd determination even gave a better affinity for the latter domain (7 ± 4 µM).

We have observed for CZ40 that the Fmoc group increased affinity (compared to CZ55) but also degraded the specificity of interaction. The unusual CSP pattern observed for CZ34 may The determined populations and rate contants were the following (Figure 72):

p trans = 23.4 ± 0.2 % ; p cis = 76.6 ± 0.2 % k cis⟶trans = 14.2 ± 0.7 s -1 ; k trans⟶cis = 46.5 ± 2.5 s -1 ; k ex = 60.7 ± 3.2 s -1

Material and Methods

I. Expression and purification of unlabeled FL Pin1 and the isolated domains

For the expression of FL Pin1 and the isolated domains, we used the E.Coli BL21-Gold(DE3)pLysS expression strain and pET 15b vector.

The expression vector pET 15b was used to clone the ADNc of the genes encoding the FL Pin1 protein and the isolated domains. The vector encodes recombinant proteins fused to His-tag sequence in the N-terminal position and carries a cleavage site for the PreScission protease. The His-tag allows the purification of the proteins by the Nickel-NTA agarose resin column. The vector had also a resistance gene to ampicillin as well as gene encoding T7 RNA polymerase.

The transformed E. coli bacteria with the plasmid containing the gene expressing human Pin1

and the isolated domains are pre-cultured at 37°C overnight in 50 mL of LB containing 100 µg/mL ampicillin and 40 µg/mL chloramphenicol under agitation (250 rpm). The precultures initiate the growth of bacteria previously stored at -80°C in the presence of 20% glycerol.

The Erlenmeyer flasks used are four times larger than the final volume of LB to allow a good oxygenation of the medium under agitation (210 rpm).

Eight 2 L Erlenmeyer flasks are used for the culture of Pin1 and the isolated domains. Bacterial growth in the culture media is monitored by turbidity by measuring the optical density (OD) at 600 nm, using an OD600 DiluPhotometerTM (IMPLEN, Munich, Germany).

Isotopes 14 N or 15 NH 4 Cl and 12 C or 13 C-glucose were obtained from Cambridge Isotope Laboratories (Tewksbury, USA).

I.1 Expression of Pin1 and the isolated domains in LB medium

The LB media are inoculated by dilution of the preculture in a way to have a starting OD measuring at 600nm around 0.05. Then, cultures were grown shaking at 37°C for three to four hours until an optical density A 600 » 0.8. The induction of the cells is performed with 0.5 mM IPTG in final concentration from a 1M solution. The media are incubated at 37°C for four hours. The cells are then collected by centrifugation (6000 rpm, 30 min at 4°C) and resuspended in 40 mL of extraction buffer (25 mM phosphate buffer, 500 mM NaCl, 30 mM imidazole, 1 mM DTT) for 1 to 2 L of culture and then frozen at -80°C.