As a promising pH-triggerable molecular switch, trans -2-aminocyclohexanol (TACH) has a variety of applications. By introducing two hydrocarbon tails, multiple TACH-based lipids (flipids) have been designed and studied that are able to perform a drastic conformational flip upon protonation, loosening the stacking of hydrocarbon tails in lipid bilayers. Liposomes constructed from such flipids (fliposomes) can be disrupted by this acid-triggered conformational flip to cause a rapid release of a cargo specifically in areas of increased acidity (such as inflammation or ischemic tissues, solid tumor, and endosome pathway). A library of flipids has been built based on structural modifications of both amino headgroups and hydrophobic tails. A series of fliposomes have been constructed and their colloidal stability, capacity and pH-dependent leakage were investigated. A good correlation between the conformational switch of flipids studied by 1 H-NMR and the fliposomes' leakage indicated that the former is a cause for the latter. The obtained results showed that all the properties of fliposomes can be manipulated by selection of the amino headgroups structure and basicity, and the length and shape of hydrophobic tails, by using mixtures of different flipids or fliposomes, and by changing the content of flipids while constructing fliposomes. As a result, we prepared the pH-triggerable fliposomes with extraordinary characteristics: high stability in storage combined with instant release of their cargo in response to a weakly acidic medium. Fliposomes encapsulating the anticancer drug methotrexate (MTX) were applied to HeLa cells and demonstrated much higher cytotoxicity than the free drug and negative controls, indicating that they could conduct more efficient cellular delivery of MTX. The MTX-loaded fliposomes inhibited tumor growth in B16F1-melanoma-bearing nude mice compared to the control group, suggesting the anticancer activity of MTX delivered by pH-triggerable fliposomes in vivo. The results of research demonstrated the potential of fliposomes to serve as a viable drug delivery system.

Trans-2-Aminocyclohexanol (TACH) is a promising model for pH-triggerable molecular switches with a variety of potential applications. In particular, such a switch, when incorporated into cationic liposomes, provides a novel design of the pH-sensitive helper lipids for gene delivery. Protonation of TACH molecules results in a strong intramolecular hydrogen bond between the amino and its neighboring hydroxyl groups, which triggers a conformational flip, and forces changes of the relative position of other substituents on the ring. In this work, a library of TACH-lipids has been designed and built based on structural modifications of both hydrophilic headgroups and hydrophobic tails, and their conformational behavior has been studied by 1 H NMR. NMR-titration has been done to quantitatively monitor the conformational switch for TACH derivatives. It was discovered that conformational behavior of TACH-lipids is independent from the length or shape of their hydrophobic tails. Therefore, a simplified model was suggested based on TACH with diethyl groups instead of hydrocarbon tails. Conformational study of these models has demonstrated that the position of equilibrium shift A [special characters omitted] BH + can be effectively changed by altering structure of NR 2 R 3 group. Furthermore, the pH-induced conformational flip occurs in a certain pH range that mostly depends on the basicity of group NR 2 R 3 , allowing a broad tuning of the pH-sensitivity of TACH-based conformational switches in a wide range of acidity. The hydrophilic OH group was also modified to influence the conformational equilibrium. External stimuli including addition of acid, change of solvent and of the solution ionic strength also showed impact on conformation equilibrium to different extents. To explore the potential to serve as pH-sensitive helper lipids in gene delivery, a variety of TACH-lipids were incorporated into lipoplexes together with the cationic lipid DOTAP to mediate DNA transfection in Bl6F1 and HeLa cancer cell lines. The lipoplex comprising TACH-lipid 3o (R 1 = C 19 H 37 ; R 2 R 3 = CF 3 CH 2 NH) exhibited one to two orders of magnitude better transfection efficiency than the one with the conventional helper lipid DOPE while only inducing slight higher cytotoxicity. Thus, the lipid can be suggested as a novel helper lipid for efficient gene transfection with low cytotoxicity.

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We have compared two gastric string tests for obtaining gastric juice for culture of Helicobacter pylori and for nested-PCR detection and PCR-based combined single- and double-stranded conformational polymorphism (SDSCP) discrimination of infecting strains. String test specimens were obtained from one seropositive volunteer for 13 consecutive weeks. The distal 10 cm of each string was suspended in 1 ml saline and quantitatively cultured. An additional nine volunteers with histories of upper-gastrointestinal complaints were given a string test for culture and nested-PCR assay. H. pylori isolates and/or gastric juice from each volunteer were extracted for DNA and analyzed by PCR-based SDSCP. Quantitative culture showed that the Entero-test was four times as sensitive as the Gastro-test but was more prone to contamination by oral flora. However, the two string tests are equally sensitive by PCR assays. Thus, the Gastro-test is more suitable for culture detection of H. pylori, since it is less prone to oral contamination and its shorter length is better tolerated. SDSCP analysis of H. pylori DNA from four PCR-positive volunteers without requiring culture showed four distinct profiles, indicating different infecting strains. SDSCP analysis of strains isolated before and after treatment of one volunteer had the same SDSCP profile, suggesting endogenous reinfection by the same strain.

Control and analysis of protein aggregation is an increasing challenge to biopharmaceutical research and development. Therefore it is important to understand the interactions, causes and analysis of particles in order to control protein aggregation to enable successful biopharmaceutical formulations. This work investigates the role of different non-ionic surfactants on protein conformational stability, as assessed by HSDSC, and on protein size stability as assessed by Dynamic Light Scattering (DLS), HIAC and MFI. BSA and IgG2 were used as model proteins. Thermal unfolding experiments indicated a very weak surfactant-immunoglobulin IgG2 interaction, compared to much stronger interactions for the BSA surfactant systems. The DLS results showed that BSA and IgG2 with different surfactants and concentration produced different levels of particle size growth. The heat treatment and aging of samples in the presence of Tween 20, Tween 80, Brij 35 and Pluronic F-68 surfactants led to an increase in the populations of larger particles for BSA samples, whereas IgG2 systems did not notably aggregate under storage conditions MFI was shown to be more sensitive than HIAC technique for measuring sub-visible particles in protein surfactant systems. Heat treatment and storage stress showed a significant effect on BSA and IgG2 protein sub-visible particle size stability. This work has demonstrated that both proteins with different Tween 20, Tween 80, Brij 35 and Pluronic F-68 concentrations, have different level of conformational and size stability. Also aging samples and heating stress bears the potential to generate particles, but this depends on surfactant type. Poor predictive correlations between the analytical methods were determined.

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Dans ce mémoire, je présente mes études sur une stratégie efficace développée pour la synthèse de cétones homoallyliques substituées à partir de l’addition en cascade de réactifs de Grignard vinyliques substitués sur des α-amino esters catalysée par des sels de cuivre. L’utilisation de ces cétones homoallyliques a permis d’obtenir des mimes peptidiques comprenant un isostère de type hydroxyéthylène du lien amide. L’étape clé de cette stratégie repose sur la synthèse de cétones homoallyliques substituées intermédiaires à partir de la réaction d’additions en cascade catalysée au cuivre, de bromure de β,β-diméthylevinyle magnésium sur des analogues d’esters de la phénylalanine et de la sérine. Les cétones homoallyliques résultantes sont réduites sélectivement en alcool, la liaison double est clivée oxydativement et l’acide carboxylique résultant est couplé à un acide aminé. Afin d’évaluer l’effet qu’ont le remplacement du lien amide central dans un coude β par un hydroxyéthylène et de la présence d’un gem diméthyle sur la chaîne carbonée sur la conformation tridimensionnelle adoptée par les tripeptides générés, des analyses à l’état solide par diffraction aux rayons X, des analyses en solution par la spectroscopie RMN et des expériences de type NOESY ont été réalisées. Ces études ont permis de définir un nouveau type de coude β. La présence de pont hydrogène intramoléculaire et l’effet de restriction de conformation induit par le gem diméthyle, généralement appelé effet Thorpe-Ingold, favorisent la formation d’un coude β. / In this thesis, I discuss my studies toward the synthesis of substituted homoallylic ketones from the copper-catalyzed cascade addition of substitued vinyl Grignard reagents to carboxylic esters. The homoallylic ketones were used to provide different peptidomimetics containing a hydroxyethylene isostere instead of an amide bond. The methyl ester of phenylalanine and serine derivatives were reacted in copper-catalyzed cascade additions of substitued vinylmagnesium bromide to provide substitued homoallylic ketone intermediates. Selective reduction of the ketone to an alcohol, oxidative cleavage of the double bond, followed by peptide coupling with amino acid lead to the desired peptidomimic. The influence of changing the central amide bond for a hydroxyethylene isostere in a β-turn and the effect of a gem dimethyl group on the backbone conformation adopted by the newly synthesized tripeptides, were studied by X-ray diffraction and solution NMR spectroscopy using NOESY experiments. From these studies, it was revealed that the iso-butyric acid hydroxyethylene isomer induced a β-turn-like conformation, and may serve as a novel scaffold for peptide mimicry.

L'enjeu des travaux effectués au cours de cette thèse est l'extraction in silico de molécules potentiellement intéressantes dans le processus d'inhibition du récepteur tyrosine kinase c-Met. La faculté de cette protéine à interagir dans les phénomènes d'embryogenèse et de réparation tissulaires rendent son inhibition cruciale dans les traitements contre les développements tumoraux où c-Met se trouve impliquée. Pour cela, la stratégie employée implique l'utilisation de méthodes in silico de conception rationnelle de médicaments. Nous avons utilisé comme support les multiples structures cristallographiques publiées sur la ProteinData Base. Un travail de modélisation par homologie fut tout d'abord nécessaire pour combler les lacunes des structures cristallographiques collectées. Afin d'échantillonner au mieux l'espace conformationnel de la kinase c-Met et de caractériser sa flexibilité, une longue campagne de simulation de Dynamique Moléculaire fut menée. Pour compléter ces simulations, nous avons également utilisé la méthode des modes normaux de vibration. De ces 2 approches, nous avons extrait un ensemble de 10 conformères considérés comme les plus représentatifs de l'espace conformationnel simulé pour la kinase c-Met et avons proposé un mode de fonctionnement de ce récepteur. Utilisant les conformations représentatives, nous avons ensuite mené une importante campagne de criblage virtuel sur plusieurs chimiothèques constituant environ 70.000 composés. L'analyse des résultats de l'arrimage moléculaire nous a conduits à la sélection de composés intéressants possédant théoriquement une bonne affinité pour la kinase c-Met. Ces molécules ont été soumises aux tests expérimentaux. / The challenge of this PhD work is the in silico identification of potentially interesting molecules concerning the inhibitory process of tyrosine kinase receptor c-Met. The faculty of this protein to interact in embryogenesis and tissue repair phenomena makes its inhibition crucial for treatments against tumor development in which c-Met is involved. For that purpose, the employed strategy involves the use of several in silico methods for rational drug design. As the basement of this work, we used the multiple crystal structures published in the ProteinData Base (PDB). A preliminary homology modeling work was needed to fill gaps in the crystal structures. To sample at best the c-Met kinase conformational space and to characterize its flexibility, a long Molecular Dynamics (MD) simulation campaign was carried out both on apo and holo forms of available crystal structures. To complete these simulations, part of this work consisted to use normal modes of vibration (NM) method. From these two approaches (DM and NM), we extracted a set of 10 conformers considered as the most representative of the kinase simulated conformational space and we suggested a mode of operation of this kinase. Using extracted conformations from the conformational sampling has enabled us to conduct an extensive campaign on several virtual screening libraries constituting a total of approximately 70,000 compounds. Analysis of the molecular docking results has led us to the selection of several theoretically interesting molecules with good potential affinity for c-Met kinase. These molecules were submitted to experimental tests performed by the biologist team associated to our work.

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