L’identification de molécules organiques capables de moduler des interactions protéine-protéine (PPIs) est longtemps restée un domaine peu exploité par la recherche pharmaceutique privée comme académique. Cependant, le développement de méthodologies innovantes pour l’étude des PPIs et la validation récente de ce type d’inhibiteurs dans des essais précliniques, démontrent que les PPIs constituent une nouvelle source de cibles importantes. Les composés capables de moduler ces interactions représentent une nouvelle classe d’outils prometteurs, tant en recherche fondamentale qu’en thérapeutique. Elles peuvent aider à différencier les multiples fonctions portées par une même protéine, à replacer la protéine dans une cascade de réactions, ainsi qu’à disséquer et reconstituer des réseaux de signalisations protéiques. Ces molécules permettront également de faire émerger de nouvelles familles d’agents pharmacologiques actifs dans diverses pathologies.Mon travail de thèse s'est projeté dans l'avenir de la recherche biomédicale, en ciblant les interactions protéine-protéine. J’ai pu durant mon doctorat mettre en œuvre plusieurs méthodologies pour étudier et caractériser des interactions protéiques afin de développer des inhibiteurs de ces interactions. J’ai ainsi pu travailler sur l’optimisation d’un composé inhibiteur de l’interaction de la protéine virale Nef VIH-1 avec les domaines SH3 des Src kinases, le composé DLC27. J’ai également pu mettre en évidence la pertinence biologique de ce composé, qui cible un mode d’interaction unique, ou très rare, au niveau cellulaire en étudiant l’interaction avec les domaines SH3 de deux protéines, ALIX (ALG2-Interacting Protein X) et la sous-unité p85 de la PI3K (phosphatidylinositol 3-kinase).J’ai également pu caractériser la surface et le mode d’interaction de protéines virales impliquées dans le complexe de réplication du virus du SRAS (Syndrome Respiratoire Aigu Sévère). Cette étude tend à montrer que la protéine virale nsp10 agit comme une plateforme de reconnaissance pour ses partenaires, les protéines virales nsp14 et nsp16. Ces interactions permettent l’activation ou l’augmentation des activités respectives de nsp16 et nsp14 et jouent un rôle au niveau de la réplication virale. Suite à l’identification d’un ‘point chaud’ d’interaction, le résidu Tyr96 à la surface de nsp10, nous avons mis en évidence la première famille de molécules inhibitrices du complexe nsp10-nsp14 en couplant des méthodes informatiques (in silico) à des criblages expérimentaux. Ces molécules pourraient être utilisées comme antiviraux ou servir d’outils pour la recherche, en permettant par exemple de mieux comprendre et d’élucider les mécanismes moléculaires impliqués dans la réplication du virus du SRAS et des coronavirus en général. / Protein-protein interactions (PPIs) participate in and regulate almost all essential cellular functions. As a consequence, they are frequently involved in various pathologies (going from cancer development to viral replication and host cell infection) but their study remains a challenge.Thus understanding those interactions as well as finding small drug candidates able to modulate them, a field of research not currently fully developed, appear as the future of the healthcare industry.In this context, I chose to learn different techniques to study PPIs that are usually employed in academic (IMR laboratory, CNRS, France) or corporate environments (Genentech, USA). Moreover, I also worked on the development of small organic inhibitors of PPIs coupling in silico methodologies (chemo-informatics, Drug Design) to biological and structural validations.During my PhD, I could manage and work on different projects involving the study of PPIs involved in cancer signaling pathways as well as the development of potent antiviral drugs targeting the HIV and SARS viruses.My organizational, personal and scientific skills as well as the practical experience I developed on various techniques (from cell biology to biophysics, structural biochemistry and Drug Design), make me feel confident on the management of PPIs drug discovery projects.I am thus able to efficiently work on, and manage, the study of protein-protein interactions in various pathologies as well as the development of potent PPIs inhibitors, that will be a major breakthrough for Biotech/Pharma companies in the coming years.

Cytochromes P450 are diverse group of heme enzymes found in most species on Earth. In humans they are involved in metabolism of foreign compounds or steroids, bacteria employ cytochromes P450 for utilization of various hydrophobic substrates. General reaction catalyzed by cytochromes P450 is monooxygenation, when one atom of oxygen molecule is introduced into the substrate, while the other is reduced producing water. NADPH:cytochrome P450 oxidoreductase or cytochrome b5 usually serves as an electron donor providing electrons needed for activation of oxygen in eukaryotic organisms, in bacteria small FeS proteins or flavoproteins are these electron donors. It was shown earlier that bacterial electron donor flavodoxin could also interact with human cytochromes P450 in vitro. This thesis employs molecular modeling techniques to support a hypothesis that flavodoxin is responsible for reduction of human (1A2, 2A6, 2A13, 2C9, 2C19, 3A4) and bacterial (101A1 a 176A1) cytochromes P450 heterologously expressed in Escherichia coli. An initial guess of possible mutual orientations of cytochrome P450 and flavodoxin was predicted using information-driven protein-protein docking. The stability of these complexes was examined by directed dissociation method. The most stable orientation for each cytochrome P450 was further...

Le paludisme, première endémie parasitaire mondiale ayant engendré près d’un demi-million de morts en 2017 (d’après l’OMS), est due à une infection par un parasite du genre Plasmodium. Cet apicomplexe infecte, au cours de son cycle de vie, un hôte définitif, un moustique femelle du genre Anopheles, et un hôte intermédiaire homéotherme (l’Homme pour au moins 6 espèces). Chez ce dernier, après une phase de développement hépatique, le parasite envahit puis lyse les érythrocytes. L’accroissement exponentiel de la parasitémie engendre les symptômes du paludisme et permet la production de formes sexuées (gamétocytes) qui seront transmises au vecteur arthropode, permettant ainsi la complétion du cycle de vie du parasite.Plasmodium a co-évolué avec ses hôtes et mis en place divers modes de régulation de l’expression de ses gènes. La phosphorylation est l’une des modifications post-traductionnelles majeures et rapides qu’il utilise pour répondre aux changements environnementaux auxquels il est confronté au cours de son cycle de vie. Nombre de ses kinases et phosphatases jouent un rôle essentiel dans l’invasion de cellules hôtes, la croissance et la division cellulaires, ainsi que la motilité de certains stades. En revanche, le rôle des cinq pseudokinases de Plasmodium dans son développement n’a jusqu’ici pas été exploré.Durant ma thèse, j’ai caractérisé l’unique pseudo-Tyrosine Kinase-like (pTKL) de Plasmodium et étudié son rôle au cours du cycle intra-érythrocytaire du parasite.L’annotation de la pTKL de P. falciparum (PfpTKL) m’a permis d’identifier différents domaines et motifs, et notamment un domaine SAM (Sterile Alpha Motif), deux motifs RVxF (connus pour leur capacité d’interaction avec la Protéine Phosphatase de type 1, PP1) et un pseudo-domaine kinase appartenant à la famille des Tyrosine Kinases-like (TKL). Nous avons montré que ce pseudo-domaine kinase est capable de lier l’ATP de manière cation-indépendante, mais est dépourvu d’activité enzymatique. Des études d’interaction in vitro couplées à l’utilisation de modèles hétérologues (Levure, ovocytes de Xénope) m’ont permis d’identifier deux protéines parasitaires partenaires de PfpTKL : le domaine SAM de PfpTKL interagit directement avec la pseudo-protéase PfSERA5 (SErine Repeat Antigen 5), alors que les deux régions de la protéine contenant les motifs RVxF de PfpTKL interagissent avec PfPP1c (phosphatase majeure de Plasmodium). De façon intéressante, le deuxième motif RVxF est directement impliqué dans l’interaction avec PP1c et serait capable de moduler l’activité de cette dernière de manière allostérique.La localisation de la pTKL de P. berghei (PbpTKL) a ensuite été étudiée par immunofluorescence et confirmée par des expériences de fractionnement cellulaire. Nous avons ainsi observé que PbpTKL est exportée dans l’érythrocyte infecté au stade trophozoïte, puis retenue dans le parasite et la vacuole parasitophore au stade schizonte. L’étude de l’interactome de PbpTKL par IP/MS au stade trophozoïte a montré que PbpTKL s’associe à diverses protéines impliquées dans l’organisation du cytosquelette de l’érythrocyte, ainsi que dans l’érythropoïèse et l’homéostasie cellulaire. Ces observations suggèrent que pTKL joue un rôle, direct ou via ses partenaires, à l’interface entre le parasite et sa cellule hôte.Enfin, afin d’approcher la fonction de pTKL chez le parasite, nous avons généré différentes lignées génétiquement modifiées. L’étude phénotypique des souches de P. berghei KO et iKD pour pTKL a montré qu’elle était dispensable pour la complétion du cycle intra-érythrocytaire, l’expression des gamétocytes ainsi que l’activation des gamétocytes mâles. Ces données suggèrent que pTKL est dispensable pour ces stades de développement ou que l’expression de gènes redondants compense son absence. Quoi qu’il en soit, il est important de poursuivre les recherches sur le rôle de cette protéine aux autres stades de développement du parasite, notamment du zygote aux stades hépatiques. / Malaria is the first endemic parasitic disease in the world with nearly half million deaths in 2017 according to the WHO. This disease is the result of an infection by an agent belonging to the Plasmodium genus. This apicomplexan parasite infects two hosts over its complex life cycle: a definitive one – a mosquito belonging to the Anopheles genus – and a homoeothermic intermediate host. At least six Plasmodium species can infect humans. In its intermediate host, Plasmodium first replicates in hepatocytes before releasing erythrocyte-infectious stages in the bloodstream. Once there, parasites invade and replicate within erythrocytes, before lysing them to release other infectious stages. This triggers an exponential rise in the parasitemia, as well as malaria symptoms. Sexual stages, called gametocytes, are produced over this intra-erythrocytic cycle to be transmitted to the arthropod vector, thus allowing the completion of the parasite life cycle.Plasmodium co-evolved with its hosts and set up diverse gene expression regulation pathways accordingly. Phosphorylation is one of the major and fastest post-translational modifications used by the parasite to respond to environmental changes. Many of its kinases and phosphatases play key roles in host cell invasion, cellular growth and division, as well as motility of specific developmental stages. However, the role of the five pseudo-kinases expressed by Plasmodium has not been explored yet.During my PhD project, I have performed the characterization of the unique Plasmodium pseudo-Tyrosine Kinase-like (pTKL) and explored its role over the parasite intra-erythrocytic cycle.P. falciparum pTKL (PfpTKL) in silico annotation allowed the delineation of the protein domains. Notably, a SAM (Sterile Alpha Motif) domain, two RVxF motifs (known for their binding potential with the major protein phosphatase type 1, PP1) and a pseudo-kinase domain belonging to Tyrosine Kinase-like (TKL) family were found. This pseudo-kinase domain was found to be able to bind ATP in a cation-independent way although devoid of kinase activity. Two parasite protein partners of PfpTKL have been identified using in vitro protein-protein interaction studies together with heterologous models (yeast, Xenopus ovocytes). First, PfSERA5 (SErine Repeat Antigen 5) specifically and strongly interacts with PfpTKL SAM domain and second, PfPP1c binds the two RVxF-containing regions of PfpTKL. Interestingly, the second RVxF motif, which is located within the pseudo-kinase domain, directly binds PfPP1c and seems to be involved in the allosteric regulation of the phosphatase activity. The subcellular localization of P. berghei pTKL (PbpTKL) was studied by IFA as well as sequential lysis of erythrocytes followed by immunoprecipitation assays. PbpTKL was shown to be exported to the host cell cytosol at the trophozoite stage, but retained in the parasitophorous vacuole and the parasite cytosol at the schizont stage. Furthermore, our interactome analysis conducted at the trophozoite stage by IP/MS showed that PbpTKL binds many host cell proteins involved in erythrocyte cytoskeleton organization, as well as erythropoiesis and cell homeostasis. These data suggest that pTKL plays a role at the parasite/host interface, either directly or via its protein partners.Finally, in an attempt to understand the role of pTKL for the parasite development, we generated genetically modified P. berghei strains. The phenotypic study of PbpTKL KO and iKD strains did not show any difference between the defective parasites and the parental wild type ones during the intra-erythrocytic cycle, gametocyte expression and male gametocyte activation. These data suggest the dispensability of pTKL or the expression of redundant gene(s) with similar functions in these parasite stages. Whatever the explanation, it is still important to follow up this investigation in other parasite stages, from zygotes to hepatic stages.

Le nombre considérable d'interactions protéine-protéine (PPIs) existant au sein d'un organisme, ainsi que leur implication cruciale dans la vie cellulaire et dans de nombreuses pathologies, font des PPIs un immense réservoir de cibles potentielles pour la recherche de médicaments. Les PPIs sont aujourd'hui sur le devant de la scène grâce au développement de méthodologies innovantes et la validation récente de molécules chimiques modulant ces interactions dans des essais précliniques.L'étude des modulateurs d'interactions protéine-protéine (PPIM), a des implications tant dans la recherche fondamentale que thérapeutique. Les PPIMs peuvent aider à la compréhension des réseaux d'interactions. Elles permettront également de faire émerger de nouvelles familles d'agents thérapeutiques actifs dans diverses pathologies.Mon travail de thèse a principalement porté sur deux aspects de l'étude de l'inhibition des PPIs. D'une part, l'étude de l'implication des divers paramètres physicochimiques gouvernant une PPI dans sa capacité à être modulée (étude dite de la « druggabilité »), m'a amené à participer à la création d'une base de données structurale des interactions protéine-protéine : 2P2IDB (http://2p2idb.cnrs-mrs.fr/). D'autre part, j'ai contribué à l analyse de l'espace chimique des molécules présentes dans la base de données 2P2IDB. Nous avons défini la « Rule Of 4 » comme ligne de conduite pour caractériser ces molécules. Nous avons de plus utilisé le SVM afin de créer un protocole innovant (2P2IHUNTER) qui nous a permis de filtrer de grandes collections de composés afin de créer des chimiothèques dédiées aux PPIs. / The number of protein-protein interactions (PPIs) existing in an organism, and their crucial implication in cellular life and in many pathologies, demonstrates the importance of PPIs as a large reservoir of potential targets for medicinal research. Neglected for a long time by both pharmaceutical companies and academic laboratories because they were historically classified as difficult targets, PPIs are now getting into the groove due to the development of innovative methodologies and the growing number of small molecule compounds modulating these interactions.The study of PPI modulators has implications in both fundamental and therapeutics research. On the one hand, PPI modulators can be used in basic research to decipher the role of PPIs in biological networks. On the other hand, they represent a valuable source of new families of therapeutic agents in pathologic processes.In the first part of my PhD, I contributed to the development of a structural database dedicated to protein-protein interactions: 2P2IDB (http://2p2idb.cnrs-mrs.fr/). The interface descriptors of protein-protein interfaces which are typical of complexes present in 2P2IDB have been used to develop a qualitative scoring function to assess the ‘druggability' of PPI targets.In the second part of my PhD, I contributed to the analysis of the chemical space of PPI inhibitors present in the 2P2I database using chemoinformatics tools. We defined the ‘Rule-of-4' as a guideline to characterize these compounds. We have used support vector machine approaches to elaborate a protocol: 2P2IHUNTER, which allows filtering large collection of compounds to design chemical libraries dedicated to PPI targets.

Cytochromes P450 are the major xenobiotics converting enzymes. They are classified as mixed function monooxygenases (MFO). Isoform 1A1 is a extrahepatic form found mainly in the lung and other tissues. It is strongly induced by polycyclic aromatic hydrocarbons and their derivatives via the Ah receptor. As a marker reaction for this enzyme can be used hydroxylation of Sudan I, which has previously been widely used as a azo dye in industry, but since 1980s it is banned for coloring food and cosmetics for its negative influence on the organism. NADPH:cytochrome P450 reductase is the major electron donor for cytochrome P450 catalyzed monooxygenation reactions. Another electron carrier for cytochrome P450 catalyzed reactions is cytochrome b5. It was shown that cytochrome b5 can stimulate, inhibit or have no effect on P450 catalyzed reactions. This thesis aims to evaluate the influence of the ration between NADPH:cytochrome P450 reductase and cytochrome b5 on cytochrome P450 1A1 catalyzed Sudan I hydroxylation. The main goal is to characterize the influence of electron donor and electron transfer ratios on hydroxylation of Sudan I, and to determine the kinetic parameters KM and VMAX for selected protein ratios. Partial aims of the thesis were to characterize the recombinant proteins used in this study...

BACKGROUND: A long-standing challenge in the post-genomic era of Bioinformatics is the prediction of protein-protein interactions, and ultimately the prediction of protein functions. The problem is intrinsically harder, when only amino acid sequences are available, but a solution is more universally applicable. So far, the problem of uncovering protein-protein interactions has been addressed in a variety of ways, both experimentally and computationally. MOTIVATION: The central problem is: How can protein complexes with solved threedimensional structure be utilized to identify and classify protein binding sites and how can knowledge be inferred from this classification such that protein interactions can be predicted for proteins without solved structure? The underlying hypothesis is that protein binding sites are often restricted to a small number of residues, which additionally often are well-conserved in order to maintain an interaction. Therefore, the signal-to-noise ratio in binding sites is expected to be higher than in other parts of the surface. This enables binding site detection in unknown proteins, when homology based annotation transfer fails. APPROACH: The problem is addressed by first investigating how geometrical aspects of domain-domain associations can lead to a rigorous structural classification of the multitude of protein interface types. The interface types are explored with respect to two aspects: First, how do interface types with one-sided homology reveal convergently evolved motifs? Second, how can sequential descriptors for local structural features be derived from the interface type classification? Then, the use of sequential representations for binding sites in order to predict protein interactions is investigated. The underlying algorithms are based on machine learning techniques, in particular Hidden Markov Models. RESULTS: This work includes a novel approach to a comprehensive geometrical classification of domain interfaces. Alternative structural domain associations are found for 40% of all family-family interactions. Evaluation of the classification algorithm on a hand-curated set of interfaces yielded a precision of 83% and a recall of 95%. For the first time, a systematic screen of convergently evolved motifs in 102.000 protein-protein interactions with structural information is derived. With respect to this dataset, all cases related to viral mimicry of human interface bindings are identified. Finally, a library of 740 motif descriptors for binding site recognition - encoded as Hidden Markov Models - is generated and cross-validated. Tests for the significance of motifs are provided. The usefulness of descriptors for protein-ligand binding sites is demonstrated for the case of "ATP-binding", where a precision of 89% is achieved, thus outperforming comparable motifs from PROSITE. In particular, a novel descriptor for a P-loop variant has been used to identify ATP-binding sites in 60 protein sequences that have not been annotated before by existing motif databases.

Background: Foamy viruses (FVs) have developed a unique budding strategy within the retrovirus family. FV release requires co-expression and a highly specific interaction between capsid (Gag) and glycoprotein (Env), which cannot be complemented by heterologous Env proteins. The interaction domain in FV Env has been mapped in greater detail and resides mainly in the N-terminal tip of the cytoplasmic domain of the Env leader peptide subunit. In contrast, the corresponding domain within Gag is less well defined. Previous investigations suggest that it is located within the N-terminal part of the protein. Results: Here we characterized additional Gag interaction determinants of the prototype FV (PFV) isolate using a combination of particle release, GST pull-down and single cycle infectivity analysis assays. Our results demonstrate that a minimal PFV Gag protein comprising the N-terminal 129 aa was released into the supernatant, whereas proteins lacking this domain failed to do so. Fine mapping of domains within the N-terminus of PFV Gag revealed that the N-terminal 10 aa of PFV Gag were dispensable for viral replication. In contrast, larger deletions or structurally deleterious point mutations in C-terminally adjacent sequences predicted to harbor a helical region abolished particle egress and Gag – Env protein interaction. Pull-down assays, using proteins of mammalian and prokaryotic origin, support the previous hypothesis of a direct interaction of both PFV proteins without requirement for cellular cofactors and suggest a potential direct contact of Env through this N-terminal Gag domain. Furthermore, analysis of point mutants within this domain in context of PFV vector particles indicates additional particle release-independent functions for this structure in viral replication by directly affecting virion infectivity. Conclusions: Thus, our results demonstrate not only a critical function of an N-terminal PFV Gag motif for the essential capsid - glycoprotein interaction required for virus budding but also point out additional functions that affect virion infectivity.

Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5 can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...

Background: Foamy viruses (FVs) have developed a unique budding strategy within the retrovirus family. FV release requires co-expression and a highly specific interaction between capsid (Gag) and glycoprotein (Env), which cannot be complemented by heterologous Env proteins. The interaction domain in FV Env has been mapped in greater detail and resides mainly in the N-terminal tip of the cytoplasmic domain of the Env leader peptide subunit. In contrast, the corresponding domain within Gag is less well defined. Previous investigations suggest that it is located within the N-terminal part of the protein. Results: Here we characterized additional Gag interaction determinants of the prototype FV (PFV) isolate using a combination of particle release, GST pull-down and single cycle infectivity analysis assays. Our results demonstrate that a minimal PFV Gag protein comprising the N-terminal 129 aa was released into the supernatant, whereas proteins lacking this domain failed to do so. Fine mapping of domains within the N-terminus of PFV Gag revealed that the N-terminal 10 aa of PFV Gag were dispensable for viral replication. In contrast, larger deletions or structurally deleterious point mutations in C-terminally adjacent sequences predicted to harbor a helical region abolished particle egress and Gag – Env protein interaction. Pull-down assays, using proteins of mammalian and prokaryotic origin, support the previous hypothesis of a direct interaction of both PFV proteins without requirement for cellular cofactors and suggest a potential direct contact of Env through this N-terminal Gag domain. Furthermore, analysis of point mutants within this domain in context of PFV vector particles indicates additional particle release-independent functions for this structure in viral replication by directly affecting virion infectivity. Conclusions: Thus, our results demonstrate not only a critical function of an N-terminal PFV Gag motif for the essential capsid - glycoprotein interaction required for virus budding but also point out additional functions that affect virion infectivity.

5 Abstract Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) belongs to the serine/ threonine protein kinase family, which is involved in the calcium signaling pathway. The increase of intracellular calcium concentration induces the activation of calmodulin (CaM), which then activates its binding partners including CaMKII, CaMKIII, CaMKK1 and CaMKK2. CaMKK2 activates CaMKI, CaMKIV and AMP-dependent kinase, AMPK, by phosphorylation. CaMKK2 is naturally present in cells in an autoinhibited state, which is caused by the steric hindrance of the active site by the autoinhibitory domain. When calmodulin binds to the calmodulin-binding domain, the autoinhibitory domain is removed and the active site becomes accessible. Upon activation, CaMKK2 undergoes autophosphorylation, which increases its enzyme activity. Negative regulation of CaMKK2 is mediated by cAMP-dependent protein kinase A (PKA)- and GSK3-dependent phosphorylation. Sites phosphorylated by PKA have been identified for both CaMKK1 and CaMKK2. Two of them are also motifs recognized by scaffolding 14-3-3 proteins. Previous studies have shown that the 14-3-3 protein binding maintains phosphorylated CaMKK2 in an inhibited state by blocking the dephosphorylation of S495, which prevents the binding to calmodulin. However, it is unclear if it is the...