The Influenza virus is a major human respiratory pathogen responsible for seasonal ‘flu’ outbreaks and sporadic global pandemics. The Influenza polymerase complex is necessary for viral RNA synthesis and full virulence and requires the assembly of three conserved subunits: PA, PB1 and PB2. A recombinant chimeric protein mimetic consisting of the N-terminus (20 amino acids) of PB1 fused to Maltose Binding Protein (MBP) and Tat Nuclear Localization Signal (NLS) was designed and purified with the aim of inhibiting the assembly of the polymerase by mimicking PB1. The cell-penetrating protein mimetic was shown to efficiently enter the cell nucleus and prevent assembly of the Influenza polymerase, thus inhibiting viral replication. When MDCK cells were incubated with the mimetic and subsequently challenged with Influenza A virus, viral replication decreased up to 98% at 50 µM. Using a nuclear extraction assay, the mimetic was shown to efficiently penetrate the plasma membrane and enter the host nucleus. GST pull-down assays showed that the mimetic interacts with PA. Molecular modeling was then employed to predict the improved hypothetical free energy of binding between PB1 and PA and determined two significant substitutions for PB1 threonine at position six: glutamic acid (T6E) and arginine (T6R). These mutations increased potency of the mimetic at 25 µM (71% for T6E and 77% for T6R compared to 36% for the native construct) and 12.5 µM (27% for T6E and 70% for T6R compared to 16% for the native construct), suggesting a more stable interaction with PA consistent with molecular modeling. Using various in vitro assays, the mimetic was shown to be non-toxic to host cells. Targeting critical protein-protein interactions using a peptide fused to a cell-penetrating carrier protein presents a novel and intriguing approach in designing anti-viral therapeutics. / Thesis / Master of Science in Medical Sciences (MSMS)

Mit einer weltweiten Inzidenz von etwa 600.000 Neuerkrankten pro Jahr gehört das orale Plattenepithelkarzinom zu den sechs häufigsten malignen Tumorerkrankungen des Menschen. Zur Behandlung operabler Tumoren der Stadien III-IVb hat sich hingegen die multimodale Therapie mit primär operativem Vorgehen, gegebenenfalls mit nachfolgender adjuvanter Radiatio beziehungsweise Radiochemotherapie, etabliert. Beim Vorhandensein von Fernmetastasen hingegen ist das Therapiekonzept als palliativ einzustufen. Während sich für die adjuvante, kurativ intendierte Radiochemotherapie die Verwendung von Cisplatin oder cisplatinhaltiger Wirkstoffe etabliert hat, stellen seit einigen Jahren selektive Wirkstoffe wie Nivolumab und Cetuximab eine Alternative zur Cisplatin-Anwendung im palliativen Setting dar. Für die medikamentöse Therapie des fortgeschrittenen oralen Plattenepithelkarzinoms scheinen daher neue rationale Therapieansätze notwendig zu sein. Besonders ein hohes Maß an Toxizität sowie die individuelle Resistenzbildung vermögen den Therapieerfolg der herkömmlichen Chemotherapie zu kompromittieren. Spezifisch wirksame sogenannte "targeted agents" binden RTK und blockieren somit die nachgeschaltete Signalkaskade. In der vorliegenden Studie kam es zur Anwendung der TKI Afatinib, Volasertib und Nintedanib in alleiniger Anwendung sowie Kombinationstherapie mit Cisplatin und dem Smac-mimetic LCL-161. Die Analyse der Wirksamkeit oben genannter Stoffe erfolgte durch eine In-vitro-Evaluation an fünf Zelllinien des humanen Plattenepithelkarzinoms der Kopf- und Halsregion. Nach semiquantitativem Expressionsnachweis der Zielstrukturen erfolgte die Stimulation der Zellen mittels spezifischer Verdünnungsreihen in Mono- und Kombinationstherapie. Folgend wurden auf der Basis von Zellzahlanalysen, Kristallviolettassays und der Erstellung von Dosis-Wirkungskurven zelllinienspezifische IC50- beziehungsweise IC20-Werte ermittelt, statistisch ausgewertet und miteinander verglichen. Die Anwendung von Afatinib in Monotherapie zeigte in der vorliegenden Studie keine signifikant erhöhte Zytoreduktion im Vergleich zur alleinigen Anwendung von Cisplatin. Auch ein Zusatz von Cisplatin zur Anwendung ergab keinen erwarteten synergistischen Effekt. Die Anwendung von Nintedanib in Monotherapie zeigte in der Analyse keine signifikanten Vorteile gegenüber einer alleinigen Cisplatinanwendung. Auf der Basis der Ergebnisse der vorausgegangenen Expressionsanalysen scheint der FGFR-2 eine übergeordnete Rolle zu spielen, da hier partiell von einer verstärkten Wirksamkeit auszugehen ist. Weiterhin scheint gegenüber selektiven Angiogeneseinhibitoren kein Vorteil für Nintedanib zu bestehen. Betrachtet man die Kombinationsanwendung von Nintedanib mit Cisplatin so fallen sowohl synergistische wie auch partiell antagonistische Effekte im Vergleich zur Cisplatin-Monotherapie auf. Diese können durch die heterogene Expression der Zielstrukturen allein nicht erklärt werden, sodass hier weiterführende Untersuchungen sinnvoll wären. Bei der Anwendung von Volasertib konnte für alle Zelllinien eine sehr deutliche Zytoreduktion erzielt werden, was durch generell erhöhte Expressionsraten erklärbar scheint. Sie manifestierte sich zudem in deutlich erniedrigten mittleren inhibitorischen Konzentrationen der Monotherapie gegenüber einer alleinigen Cisplatin-Anwendung. Betrachtet man die Kombinationsanwendung von Volasertib mit Cisplatin imponierten für alle Zelllinien erstaunlicherweise sogar schlechtere Ansprechraten verglichen mit der Volasertib-Monotherapie. Hier könnte man von einer inhibierenden Wechselwirkung der Wirkstoffe ausgehen. Diese Tatsache macht weiterführende Untersuchung zur Anwendung von Volasertib in Monotherapie attraktiver als die jeweilige Kombinationsanwendung. Die Kombinationsanwendungen von LCL-161 mit Afatinib beziehungsweise Volasertib wiesen keine signifikant erhöhten zytoreduktiven Effekte auf. Hingegen waren die Untersuchungsergebnisse der Kombinationsanwendung des verwendeten Smac-Analogons und Nintedanib bemerkenswert. In vier von fünf Zelllinien sorgte ein LCL-161-Zusatz für eine signifikant erhöhte Zytoreduktion. Eine zusätzlich zur Angiogeneseinhibition durch Nintedanib induzierte Apoptoseinduktion in Kombination mit der Apoptosesensibilisierung durch LCL-161 könnte eine mögliche Erklärung dieser Beobachtung darstellen. Aufgrund der sehr heterogenen Ergebnisse dieser Studie mit partiell synergistischen aber auch antagonistischen Effekten lässt sich schlussfolgern, dass alle drei verwendeten TKI aktuell keine vielversprechende Alternative zu der herkömmlichen Chemotherapie darstellen. Diese Heterogenität verdeutlicht auch, dass die Suche nach individuellen, zielgerichteten medikamentösen Therapieansätzen essenziell bleibt. Hierbei könnten Smac-Analoge ein vielversprechendes Feld weiterführender experimenteller und klinischer Studien eröffnen. / With a worldwide incidence of about 600,000 new cases per year, oral squamous cell carcinoma is one of the six most common malignant human tumors. For the treatment of operable stage III-IVb tumors, however, multimodal therapy with primarily surgical procedures, possibly followed by adjuvant radiotherapy or radiochemotherapy, has become established. In the presence of metastases the therapy concept is classified as palliative. While the use of cisplatin or 5-FU has become established for adjuvant, curative radiochemotherapy, selective drugs such as nivolumab and cetuximab have been an alternative to the use of cisplatin in the palliative setting for several years. New rational therapeutic approaches appear to be necessary for the drug therapy of advanced oral squamous cell carcinoma. In particular, a high degree of toxicity and the individual development of resistance may compromise the therapeutic success of conventional chemotherapy. Specifically so-called "targeted agents" bind RTK and thus block the downstream signaling cascade. In the present study, TKI afatinib, volasertib and nintedanib were used alone as well as in combination therapy with cisplatin and the smac-mimetic LCL-161. The analysis of the efficacy of the above mentioned agents was performed by in vitro evaluation of five cell lines of human squamous cell carcinoma of the head and neck region. After semi-quantitative expression detection of target structures, the cells were stimulated using specific dilution series in mono- and combination therapy. Subsequently, cell line-specific IC50 or IC20 values were determined, statistically evaluated and compared on the basis of cell number analyses, crystal violet assays and the preparation of dose-response curves. The use of afatinib in monotherapy did not show a significantly increased cytoreduction in the present study compared to cisplatin alone. The addition of cisplatin to the treatment did not result in an expected synergistic effect either. In the analysis, the use of nintedanib as a monotherapy did not show any significant benefit over cisplatin alone. Based on the results of the previous expression analyses, FGFR-2 appears to play an overriding role as it is expected to partially increase efficacy. Furthermore, nintedanib does not appear to have an advantage over selective angiogenesis inhibitors. When nintedanib is used in combination with cisplatin, both synergistic and partial antagonistic effects are observed compared to cisplatin monotherapy. These cannot be explained by the heterogeneous expression of the target structures alone, so that further investigations would be useful here. When applying Volasertib, a very significant cytoreduction could be achieved for all cell lines, which seems to be explained by generally increased expression rates. It also manifested itself in significantly lowered mean inhibitory concentrations of monotherapy compared to cisplatin alone. When considering the combination of Volasertib with cisplatin, surprisingly, the response rates of all cell lines were even worse compared to Volasertib monotherapy. Here, one could assume an inhibitory interaction of the substances. This fact makes further investigation of the use of Volasertib in monotherapy more attractive than the respective combination treatment. The combination of LCL-161 with afatinib or volasertib did not show significantly increased cytoreductive effects. However, the results of the combination treatment of the Smac-mimetic and nintedanib were remarkable. In four out of five cell lines, LCL-161 supplementation resulted in significantly increased cytoreduction. An additional apoptosis induction induced by nintedanib in addition to angiogenesis inhibition in combination with apoptosis sensitization by LCL-161 could be a possible explanation for this observation. Due to the very heterogeneous results of this study with partially synergistic but also antagonistic effects, it can be concluded that all three TKI used currently do not represent a promising alternative to conventional chemotherapy. This heterogeneity also illustrates that the search for individual, targeted drug therapy approaches remains essential. Here, Smac-analogues could open up a promising field for further experimental and clinical studies.

No description available.

La macro-autophagie est une voie catabolique conservée dans l’évolution permettant la dégradation des organites endommagés ou vieux, des protéines à longue durée de vie ou agrégées et des portions du cytosol pour le recyclage métabolique afin de maintenir l'homéostasie cellulaire. L'absence d'autophagie est fréquemment observée dans de nombreuses pathologies incluant les cancers et les maladies neurodégénératives. Beclin 1, un suppresseur de tumeur,est une protéine clé dans la régulation de l’autophagie et participe à la nucléation de l’autophagosome. Beclin 1 est une protéine “BH3-only” pouvant interagir avec le site de fixation au domaine BH3 présent dans la protéine Bcl-2 et ses homologues. Cette interaction inhibe l’autophagie. Certains agents pharmacologiques tels qu’ABT737, appelés«BH3 mimetics», occupent le site de fixation du domaine BH3 de façon compétitive pour perturber l'interaction inhibitrice entre Beclin 1 et Bcl-2/Bcl-XL. Ceci permet à Beclin 1 de maintenir l’activité classe IIIphosphatidylinositol-3-kinase de Vps34 pour la formation du phagophore. L'autophagie est un processus finement régulé par de nombreux complexes protéiques. Les senseurs de la charge énergétique comme l’AMP-dependant kinase(AMPK), la cible mammalienne de la rapamycine (mTOR), la Sirtuin1 (SIRT1) ou les voies d’intégration du stress telles que celles impliquant l'inhibiteur des kinases NF-κB (IκBα) (IKK) et le suppresseur de tumeur p53, ont tous un impact majeur dans la régulation de l'autophagie. Dans de nombreux paradigmes de stimulation autophagique, ils semblent tous agir en amont de la dissociation Beclin 1-Bcl-2. Nos résultats révèlent qu’ABT737 stimule plusieurs voies pro-autophagiques pour obtenir une efficacité optimale. Ces résultats placent la SIRT1, AMPK / mTOR, HDM2et IKK en aval de la dissociation du complexe Beclin 1-Bcl-2. Cette étude démontre que les BH3-mimetics activent des voies multiples de stimulation de l’autophagie, peut-être en raison du degré élevé de connectivité qui existe entre les complexes protéiques de régulation de l’autophagie. Cela signifie qu’un effet spécifique sur l’interactome de Beclin 1 peut affecter d'autres voies dans le réseau du contrôle autophagique. Ces voies ne semblent pas suivre une hiérarchie linéaire, mais doivent être plutôt interconnectées dans un circuit complexe dans lequel la stimulation de l'autophagie par des déclencheurs physiologiques (tels que la carence en nutriments ou le stress des organites) induit un ensemble de changements intimement liés et impliqués dans une boucle de régulation positive qui constituerait un ensemble indissociable composant l’«autophagy switch». / Macro-autophagy is a conserved catabolic pathway that culminates in the degradation of old/damaged organelles,long-lived/aggregated proteins and portions of the cytosol for metabolic recycling to maintain cellular homeostasis.The absence of autophagy is frequently observed in many pathologies including cancers and neurodegenerative diseases. Beclin 1, a bona fide tumour suppressor, is the key autophagy regulatory protein that participates in autophagosome nucleation. Infect, Beclin 1 is a BH3-only protein that can interacts with the BH3 receptor domain contained within Bcl-2 and its homologues. This interaction functions as a inhibitory check on autophagy. Some pharmacological agents such as ABT737, referred to as ‘BH3 mimetics’, occupy the BH3-binding grooves to competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-XL allowing Beclin 1 to maintain the class III phosphatidylinositol-3-kinase activity of Vps34 for the phagophore formation. Autophagy is a complex process that is regulated by multiple protein complexes beyond that organized around Beclin 1. The energy sensors including AMP-dependent kinase (AMPK), mammalian target of rapamycin (mTOR), Sirtuin1 (SIRT1) as well as stress-integrating pathways such as those involving the inhibitor of NF-κB (IκB) kinases (IKK) and the tumour suppressor protein p53, all have a major impact on the regulation of autophagy. In many paradigms of autophagic stimulation, they all seem to act upstream of the dissociation of Beclin 1-Bcl-2. Our results reveal that ABT737stimulate multiple pro-autophagic pathways to be optimally efficient. These results place SIRT1, AMPK/mTOR,HDM2 and IKK downstream of the dissociation of the Beclin 1-Bcl-2 complex. This study advocates that BH3mimetics trigger multiple autophagy-stimulatory pathways maybe due to the high degree of connectivity that exists among autophagy-regulatory protein complexes meaning that a specific effect on the Beclin 1-interactome might affect other nodes in the autophagy-controlling network. These pathways cannot follow a linear hierarchy and rather must be interconnected in a complex circuitry, in which stimulation of autophagy by physiological triggers (such as starvation or organelle stress) induce an ensemble of intimately linked changes that are coupled to each other in positive feed forward loops constituting an indissociable ensemble that composes the “autophagic switch”.

This thesis presents an investigation into some of the structural colours that are produced in nature. There are many animals and plants that produce structural colour, with a particularly high structural colour diversity in insects. Of the species that exhibit structural colours, three species are the subjects for investigation of this thesis. Those comprise a group of beetles from South-East Asia, Torynorrhina flammea, a buttery, Parides sesostris and a fruit, Margaritaria nobilis, both from South American rainforests. The structures that produce the vivid colours of these species were analysed using electron microscopy. This information aided the design and creation of three inorganic, synthetic replicas of the natural structures. The fruit of Margaritaria nobilis was structurally analysed, yielding the discovery of a novel multilayer fibre. These fibres were cylindrical in design and were found to be layered together producing the epidermis of the fruit. The multilayer structure produced a vivid blue colour appearance, which is believed to offer a selective advantage because the colour deceives birds into thinking that the fruit contains nutritious flesh. This selective advantage earns M. nobilis the label of mimetic fruit. The structure found within the M. nobilis fruit epidermis inspired the synthesis of a structure which comprises single cylindrical multilayer fibres. The synthetic fibres were manufactured from elastic materials which allow the structure to be deformed under strain and, therefore, a change in colour can be observed. As the structure was stretched, this made the layers get thinner and, therefore, the colour of the fibre blue-shifted. The fibre was able to be stretched to over twice its original length which yields a shift in peak reflected wavelength of over 200 nm. Four beetles from the Torynorrhina flammea species were investigated with the aim of replicating the nanostructures responsible for their colour appearance. The initial interest in the beetles came from their strikingly vivid colour appearances. The structure responsible for the vivid colours in all four of the subspecies is a multilayer with high structural order and over 100 laminae. Both of these attributes contribute to the saturation of the colours exhibited. The multilayer was found to be intersected by an array of rods, the long axis of which is orthogonal to the surface. The rods are believed to be the cause of an interesting diffraction phenomenon exhibited by the beetles. Using imaging scatterometry, the structure was found to diffract the colour produced by the multilayers into an annulus around a specularly reflected white spot. This inspired the synthesis of a multilayer permeated with an array of holes with the aim of replicating a system that could reproduce the annular pattern of colour reflection. The initial synthesised system comprised a quarter-wave stack with a perfectly ordered hexagonal array of holes permeating the surface orthogonally. The sample displayed the scattering characteristics of a hexagonal array, and the reflection spectra of the multilayer stack. When disordered hexagonal arrays were milled into the structure with a focussed ion beam, the scattering pattern started to show more of the green colour from the multilayer and less of the ordered scattering pattern. The highly disordered, synthesised structure displayed no hexagonal scattering pattern, but instead it showed a highly scattered bluish-green colouration. One sample was created by directly mapping out the array of holes using an image of the original array from one of the beetle samples. This sample was expected the same annular diffraction pattern as the beetles, however, the sample instead exhibited the same scattering pattern as the highly disordered array. Some structurally coloured systems in nature have more than one light scattering structure, all of which contribute to the overall colour of the system. For complicated systems such as this, it is necessary to devise a technique to characterise the individual scattering structures separately. One such species that displays a complex, multicomponent system is Parides sesostris. The male of the species displays bright green patches on the dorsal side of the forewings which are made up of thousands of green wing scales. These green scales contain a 3D gyroid poly-crystal at centre with a membrane layer surrounding the underside of each scale and a scattering structure on top. Using focussed ion beam milling techniques allowed the individual characterisation of each of these structures. The gyroid poly-crystal was found to reflect not green but blue wavelengths. This led to the discovery by another group [1] that the scales contain at least one type of fluorophore. The removal of the membrane structure and some of the gyroid poly-crystal from the base of the scale resulted in the change of the overall scale structure from green to cyan. This suggests that the membrane maybe a significant source of fluorescence. Computational modelling, without fluorescence, suggests that the addition of the membrane layer to the gyroid does not shift the band-gap wavelengths; however, the overall reflection intensity does increase. The scattering structure on the top side of each scale is comprised a bi-grating which sits on top of the 3D gyroid structure. The long periodicity of the bi-grating protrudes above the surface, resulting in the very top layer of the scale to be a mono-grating. This whole structure decreases the angular-dependence of the colour by efficiently scattering the incident light into the gyroid and also scattering the reflected light from the gyroid, resulting in a double-scattering. FIB-milling was used to isolate the scattering part of the structure. Analysis of this component of the structure revealed that it was not a source of the green colour itself; however, it did show the characteristic scattering pattern of a mono-grating. The small periodicity of the bi-grating did not produce a scattering pattern since the periodicity is too small to produce optical diffraction at normal incidence. To characterise the effect of the fluorophores, the whole scale structure was photo-bleached using ultra-violet radiation for two months with the aim of destroying the fluorophores contained within the structure. The expected result occurred which was the blue-shifting of the peak reflected wavelengths. However, it could not be confirmed whether or not the photo-bleaching reduced the physical size of the light scattering structures which would, in theory, result in a blue-shift of the peak reflected wavelengths. The male P. sesostris green wing scales were also the subject for investigation for trying to make inorganic replicas of the gyroid-polycrystal. A surface sol-gel coating process was utilised to coat the green wing scales with titania. This coating process was performed using a few different methods. Half of the samples were coated with TiO2 and the other half with tin-doped TiO2. Half of each of these samples had their surfaces dendritically amplified before the coating processes and the other half were left untreated. The samples were coated with 25 surface sol-gel (SSG) cycles of each treatment at a time. After each 25 cycle treatment the samples were optically characterised. The total number of cycles applied to the samples at the end was 150. The addition of layers of titania resulted in a general red-shift that was higher for the tin-doped titania samples than for the titania samples. Another general trend found was that the samples that had their surfaces dendritically amplified, produced a lower red-shift in peak wavelength. This was contrary to the hypothesis that the amplification process was supposed to aid the SSG coating process and, therefore, increases the red-shift in peak wavelength.

Heparin, a glycosaminoglycan (GAG), is a complex biopolymer of varying chain length and consisting of uronic acid and glucosamine residues, which are sulfated at various positions. The interaction of heparin with antithrombin is the basis for anticoagulation therapy. Heparin accelerates the antithrombin mediated inhibition of factor Xa and thrombin by a conformational activation mechansism and bridging mechanism, respectively. The sequence specific pentasaccharide DEFGH in full length heparin is the most important fragment for high affinity and activation of antithrombin, without which the heparin is incapable of binding to antithrombin. Although heparin is a commonly used anticoagulant, it suffers from serious side effects including bleeding complications, heparin-induced thrombocytopenia, and intra- and inter-patient dose response variability. Desai and co-workers have shown that it is possible to replace the GAG skeleton by small, non-saccharide sulfated molecules as antithrombin activators. However, the designed molecules were found to be weak activators of antithrombin due to their binding to the extended heparin-binding site (EHBS), instead of the pentasaccharide-binding site (PBS), of antithrombin. To design better non-saccharide antithrombin activators, a virtual screening-based approach was employed. Combinatorial virtual screening of 24576 molecules based on tetrahydroisoquinoline core scaffold resulted in 92 hits that were predicted to bind preferentially in the PBS of activated antithrombin with good affinity. The work resulted in a predicted pharmacophore consisting of a 5,6-disulfated bicyclic tetrahydroisoquinoline and a 2′,5′-disulfated unicyclic phenyl ring connected by a 4- to 5-carbon linker. The work has led to several hypotheses, which are being tested in the laboratory through synthesis and biochemical evaluation. To understand the mechanism of heparin binding to thrombin in greater detail, structural biology and molecular modeling approaches were used. More specifically, the nature of the heparin binding to thrombin was studied with a special focus on understanding the specificity of recognition. Comparative analysis was performed with heparin–antithrombin interaction to assess similarities and differences between the two heparin binding systems. In antithrombin, three important amino acids are involved in heparin pentasaccharide binding, while in thrombin, at least seven basic amino acids are predicted to be involved. For biological systems, one would expect greater specificity with more interacting points. However, the heparin–thrombin system interestingly displays a lack of specificity. The molecular basis for this lack of specificity is not clear. A study of antithrombin and thrombin crystal structures with regard to surface exposure, flexibility, and geometry of basic amino acids present in the respective heparin binding site provides the basis for the specificity of recognition (or lack thereof) in the two systems. Interestingly, analysis of thrombin exosite-II showed that Arg101, Arg165 and Arg233 are spatially conserved and form a local asymmetric center. Using in-silico docking techniques, selected tetrasaccharide sequences were found to specifically recognize this triad of amino acids indicating the possibility of specific recognition of thrombin. This hypothesis led to the design of a putative lead sequence that is 50% smaller in size and contains 62.5% fewer charges in comparison to the literature reported known exosite II sequence. The design of novel putative ‘specific’ exosite II sequence challenges the idea that the thrombin–heparin interaction is completely non-specific and gives rise to novel opportunities of designing specific thrombin exosite-II ligands.

L’hypertension pulmonaire (HTP) est caractérisée par une augmentation progressive des résistances vasculaires pulmonaires, une élévation de la pression artérielle pulmonaire moyenne (≥25 mm Hg) et un déséquilibre entre la production des médiateurs endothéliaux vasoconstricteurs et vasodilatateurs conduisant ainsi à une insuffisance cardiaque droite. Les mimétiques de la prostaglandines (PG)I2 (prostacycline), tels que l'iloprost et le tréprostinil, constituent un des traitements majeurs de l’HTP. L’efficacité des traitements utilisés dans l’http étant évaluée principalement sur l’amélioration de la tolérance à l’effort et la diminution de la dyspnée, il est important d’analyser leurs effets vasodilatateurs artériels mais aussi veineux pulmonaires ainsi que les effets bronchodilatateurs. Ces travaux décrivent les effets, in vitro, de la PGI2, de la PGE2 et de leurs mimétiques sur la réactivité des préparations vasculaires (artères et veines) pulmonaires et bronchiques humaines provenant de patients contrôles ou ayant une HTP. Nous avons caractérisé les différents récepteurs aux prostanoïdes impliqués dans ces réponses. Nos travaux suggèrent que les mimétiques de la PGI2, le treprostinil et l’iloprost, utilisés en clinique, sont d’une part de puissants vasodilatateurs à la fois des artères et des veines pulmonaires et d’autre part, de puissants bronchodilatateurs. Certains mimétiques de la PGI2 (iloprost et tréprostinil) ont des effets vasodilatateurs proportionnels sur les artères et veines pulmonaires. D’autres mimétiques de la PGI2 (béraprost) entraînent une vasodilatation artérielle pulmonaire plus importante que la vasodilatation veineuse. Ceci pourrait entraîner un œdème pulmonaire ou un moindre effet bénéfique sur la correction de la dyspnée. Nous avons mis en évidence une diminution de l’expression des récepteurs IP et DP dans les préparations vasculaires des patients ayant une HTP versus les patients contrôles. Cette diminution est associée avec la réduction de la relaxation des veines pulmonaires induite par le tréprostinil. De même, nous avons montré une diminution de l’expression du récepteur EP4 dans les voies respiratoires des patients ayant une HTP de type 3 ; elle est associée à la réduction de la bronchodilatation induite par les agonistes du récepteur EP4. La correction de cette diminution pourrait être une nouvelle perspective thérapeutique chez ces patients. Nos résultats permettent une meilleure compréhension des effets bénéfiques ou délétères, pharmacologiques et thérapeutiques des mimétiques de la PGI2 déjà utilisées en clinique dans le traitement de l’HTP. A travers la voie PGE2/EP4, nos travaux ouvrent de nouvelles perspectives physiopathologiques et potentiellement thérapeutiques. / Pulmonary hypertension (PH) is characterized by a progressive increase of pulmonary vascular resistance, an increase of mean pulmonary arterial pressure (≥25 mm Hg) and an imbalance between the production of endothelial vasoconstrictor and vasodilator mediators leading to right heart failure. Prostaglandin (PG)I2 (prostacyclin) mimetics, such as iloprost and treprostinil, are major therapeutic tools for this pathology. The efficacy of the treatments used in PH is evaluated primarily on the improvement of exercise tolerance and reduction of dyspnea. For this reason it is important to analyze not only the arterial but also the venous vasodilator effects as well as bronchodilator effects. This thesis describes the in vitro effects of PGI2, PGE2 and their mimetics on the reactivity of human pulmonary vascular (ateries and veins) and bronchial preparations derived from patients with or without PH. We also characterized different prostanoid receptors involved in these responses. Our results suggest that PGI2 mimetics, treprostinil and iloprost, used clinically, are potent vasodilators for both pulmonary arteries and veins and also potent bronchodilators. Some PGI2 mimetics (treprostinil and iloprost) have proportional vasodilatory effects on the pulmonary arteries and veins. Other PGI2 mimetics, such as beraprost, cause greater pulmonary arterial vasodilation than venous vasodilation. This may cause pulmonary edema or less beneficial effects on dyspnea correction. We showed decreased expression of IP and DP1 receptors in vascular preparations of patients with PH versus control patients. This decrease is associated with reduced relaxation response of pulmonary veins induced by treprostinil. Similarly, we demonstrated a reduction in EP4 receptor expression in bronchial preparations of patients with group 3 PH. This down regulation was associated with reduced bronchodilation induced by EP4 agonists. The correction of this down-regulation may be a new therapeutic perspective in these patients. Our results enhance our understanding of the beneficial or deleterious, pharmacological and therapeutic effects of PGI2 mimetics already used clinically in the treatment of PH. Through PGE2/EP4 pathway our work opens new physiopathological and potential therapeutic perspectives.

Protein-protein interactions are key to several biological processes that facilitate signal transduction and many other processes. These interactions are involved in pathways that are critical to many human diseases. Targeting specific protein-protein interactions is a challenging goal because protein-protein interactions are predominately through hydrophobic interactions. Antagonists of the protein-protein interactions need to be perfectly fit into the binding pockets to ensure the activity. The -helical domain of the proteins behaves as the recognition motifs for numerous protein-protein, and protein-nucleic acid interactions. Research has shown that pathways of many diseases contain protein-protein interactions involving -helical domains, e.g. neurological disorders, bacterial infections, HIV and cancer, etc. It is difficult yet very important to design small molecules to target the shallow binding areas of protein-protein interactions. So far the most successful one is Hamilton’s 1,4-terphenylene scaffold, which has been used to target the interactions between p53/MDM2, Bak/Bcl-xL etc. Inspired by this, we designed and synthesized three new scaffolds of non-pepditic -helical mimetics, mimicking the i, i + 4, i + 7 positions of an -helix. There are three basic principles that were leading our design. The side chains of our designed molecules should act as mimetics of the side chains of an -helix. Second, our molecules should possess improved water solubility. Third, the molecules should be easy to synthesize to generate a focused library. Some of our molecules, including the ones whose molecular weight are as low as 294, started to show some inhibition against p53/MDM2 interactions.

Ce travail décrit la synthèse, l’étude structurale et une première évaluation des propriétés biologiques d’une nouvelle famille de mimes de l’arginine issus de motifs pyridine et pyrazine. Pour cela, nous avons mis au point la synthèse de nouveaux peptidomimétiques dans lequels la fonction amidine est remplacée par une amidoxime mime potentiel du résidu arginine. La fonctionnalisation chimique de ces motifs a permis à partir de la pyridine (ou pyrazine) 2,3-disubstituée l’obtention de nouveaux coudes non-peptidiques possédant des motifs amidoximes estérifiés par des acides aminés ou modifiés par des hydrazides d'acides aminés, des N-acylamidrazones et des résidus de 1,2,4-oxadiazole et de 1,2,4-triazole. L’analyse structurale réalisée par RMN, IR, modélisation moléculaire et par XRD a confirmé les propriétés des hétérocycles pyridine et pyrazine, en particulier dans la stabilisation des conformations et le noyau pyrazine qui semble jouer un rôle essentiel sur la stabilisation de la conformation. L’étude d'un nouveau pseudotripeptide à base de pyrazine-ProlylPhenylalanine a révélé la formation d'une liaison hydrogène entre le proton du groupe OH et l'oxygène du carbonyle de la phénylalanine C-terminale. La liaison hydrogène, ferme un pseudocycle à 7 atomes (C7). De plus, une augmentation considérable du rotamère trans (jusqu'à 98%) dans un solvant faiblement polaire (CHCl3) a été observée. Enfin, les premières amidoximes testées ont permis de révéler la libération de NO en concentration suffisante pour produire un effet pharmacologique / This work describes the synthesis, structural study and preliminary biological evaluation of new variety of pyridine and pyrazine-based arginine mimics. Initially, we have developed a convenient synthesis of new peptidomimetics with amidoxime function as a replacement of amidine one. The latter can imitate arginine residue in biological structures. Chemical functionalization of these scaffolds led to novel 2,3-disubstituted pyridine(pyrazine) turn structures bearing amidoximes esterified with amino acid or modified with amino acid hydrazides, N-acylamidrazones, 1,2,4-oxadiazole and 1,2,4-triazole residues. Additionally, all structures were analyzed by NMR, IR, molecular modelling and XRD. Conformational studies confirmed that pyridine and pyrazine heterocycles can be used to increase rigidity and the pyrazine core has stronger effect on conformation stabilization. Examination of a new ProPhe pyrazine-based pseudotripeptide revealed the hydrogen bond formation between the proton of the OH and the carbonyl oxygen of the C-terminal phenylalanine. This hydrogen bond adopts a seven-membered ?-turn conformation. Therefore, a dramatic increase of the trans rotamer up to 98% was observed in weakly polar solvent, which is CHCl3. Finally, preliminary results of the NO release assay on amidoximes demonstrated sufficient values for pharmacological effect

Le passage des médicaments a travers la membrane cellulaire représente souvent une limitation majeur dans un grand nombre de thérapies (anti-cancéreuse, anti-virale par exemple). Des peptides vecteurs connus comme les CPPs (cell penetrating peptides) ont été utilises avec succès pour introduire a l’intérieur des cellules diverses molécules (protéines, peptides, siRNA, quantum dots) et présentent un fort potentiel dans l'adressage de médicaments. Parmi les différents CPPs décrits dans la littérature la plupart sont des peptides basiques ou amphiphiles.Nous nous sommes intéressés a l'utilisation d’oligomères non charges construits a partir de motifs contraints mimes de dipeptides comme vecteurs de pénétration cellulaire. L'internalisation cellulaire et leur localisation ont été établies a l'aide de dérivés fluorescents par microscopie confocale. L' étude de pénétration cellulaire par mesure de fluorescence a montre que des oligomères de (3S)-amino-5-carbonylmethyl-2,3-dihydro-1,5-benzothiazepine-4(5H)-one] (DBT) sont aussi puissants que les oligomères d'arginine (oligoArg), vecteurs de référence. Par microscopie confocale nous avons montré que ces composés sont internalisés dans les lysosomes. L’efficacité d'internalisation de nos composés a été confirmé par une méthode de quantification par spectrométrie de masse MALDI-TOF développée dans notre groupe. Cette méthode repose sur l'utilisation conjointe d'un marqueur UV-absorbant dérivé de l'acide alfa-cyano-4-hydoxycinnamique (HCCA) et d'une matrice MALDI adaptée. Un effet important de discrimination spectrale est obtenu, permettant une amplification du signal de la molécule d' intérêt dans un mélange complexe. Ainsi les faibles concentrations internalisées peuvent être détectées. Grâce a cette technique et l'utilisation d'un étalon deutéré, nous avons calculé la concentration intracellulaire de deux CPP de référence l'octa-arginine et la pénétratine. Nous avons aussi étudier l’internalisation de petits oligomères construits a partir d'acide 2-aminomethyl-phenyl-acetique (AMPA). Par microscopie confocal nous avons constaté que ces petits oligomères sont internalisés par voie endo-lysosomale.L’efficacité de la pénétration cellulaire de ces petits oligomères aromatiques (oligoAMPA et oligoDBT) offre une nouvelle classe de vecteurs qui ont la particularité d’être non-cationiques et hydrophobes. De tels composés pourraient être utilisés pour la délivrance de médicaments dans le traitement des maladies comme le cancer, les maladies lysosomales ou la maladie d'Alzheimer. Afin de montrer que cette nouvelle classe de vecteurs est capable d'internaliser des composés biologiquement actifs, nous les avons associés a un inhibiteur puissant de la Cathepsine D (CD) la pepstatine. CD est une endopeptidase lysosomale qui dans des conditions normales est localisée dans les endosomes et les lysosomes. Pour certains cancers, la CD est surexprimée et secrétée a l’extérieur de la cellule. La CD est probablement impliquée dans la prolifération des cellules cancéreuses par l'activation de certains facteurs de croissances dans les endosomes. La pepstatine est une inhibiteur puissant de la CD. Cependant son efficacité thérapeutique potentielle est limitée par une faible capacité de pénétration des membranes cellulaires et une faible solubilité nécessitant de fortes doses pour l'inactivation de la CD in vitro et in vivo. Afin d’améliorer son efficacité et sa biodisponibilité, des conjugues de la pepstatine avec nos vecteurs de pénétration cellulaire, oligo (AMPA)4 et (DBT)4, et une partie solubilisante ont été développés. Certains de ces bioconjugués ont montre une toxicité élevée (IC50 = 2.10-6) in vitro sur différentes lignées cellulaires tumorales. Des tests in vivo sur des souris sont prévus pour le futur. / As a part of a program for foldamer design two ¦Â-turn mimetics (3S)-amino-5-(carboxylmethyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one or DBT and 2-aminomethyl-phenyl-acetic acid or AMPA were selected as frameworks from a molecular modeling study for their suitability to adopt helical structure. At first we developed a highly efficient scale up synthesis of the DBT moiety protected by 9-fluorenylmetoxycarbonyl (Fmoc) group. By standard solid phase peptide synthesis (SPPS) we synthesized DBT oligomers of different lenghts and modifications were introduced at their N-terminus. Our first task was to perform structural analysis of the oligomers by NMR and X-Ray. Numerous NOE interactions in the DBT pentamer and hexamer molecules were detected by NMR 2D NOESY experiments. These data strongly suggest the organization of these DBT oligomers. Small crystals were obtained from the same molecules in DMSO but at the time being their size is not importan t enough for X-Ray crystallography studies. In a parallel study we hypothesized that short oligomers constructed by DBT or AMPA frameworks could translocate the cellular membrane and could be used as new cell penetrating non-peptides - CPNP. Even though these compounds are not charged as most cell penetrating peptides (CPP)5 or CPNP, we considered that by virtue of their aromaticity, hydrophobicity and their well-organized structure they could have a non-specific interaction with the lipid bilayer and thus be internalized into the cell. Short oligomers were synthesized on Rink amide (RA) resin following SPPS methodology and labelled at their N-terminus with fluorescein isothiocyanate (FITC). At first the cellular uptake of the (DBT)2-4 oligomers in MDA-MB-231 breast cancer cells was analyzed by fluorescence emission measurement and compared to the potent and well-studied CPP octa-arginine (Arg)8 as a positive control and carboxyfluorescein as a negative control. The highest intracellular fluorescence intensity was found for (DBT)4 with a drastic decrease (>4-times) for (DBT)3 and (DBT)2 oligomers. Thus, the cellular uptake appeared length-dependent with an increase of the internalization with the oligomer size. Moreover, the amount of (DBT)4 that was internalized was more significant than that of (Arg)8 despite the fact that it is uncharged. By confocal microscopy we determined that (DBT)4 is mainly localized in the endosomes after 3 hours of incubation and in the lysosomes after 16 hours of incubation. Altogether, these data indicate the ability of these oligomers to target the endolysosomal pathway. Although most of the initial drug delivery studies aimed to avoid lysosomal addressing to prevent subsequent drug degradation, more recent studies demonstrated the relevant clinical utility to target this compartment for drug delivery in the treatment of lysosomal storage diseases, Alzheimer¡¯s disease, and cancer.While analyzing the internalization efficiency of our CPNP we decided to straightforward evaluate their concentration inside the cells. We studied our compounds internalization by total fluorescence emission measurement and by confocal microscopy but none of these techniques gave us the possibility to determine the exact amount of compound internalized per cell. A study reported by Burlina et al. brought a great improvement in proposing a highly reproducible quantification method based on MALDI-TOF MS to measure the concentration of the internalized peptides. However, after cell lysis, this method requires the capture of the biotin-labelled CPP by streptavidin coated magnetic beads. This step is particularly critical for the accuracy of the quantification. This is the reason why we decided to develop a new general methodology based on MALDI-TOF mass spectrometry (MS) which does not require any purification or separation steps. We studied the internalization of CPP/CPNP compou nds by using an UV light-absorbing tag alpha-cyano-4-hydroxycinnamic acid (HCCA) and preparing the samples in a neutral matrix such as alpha-cyano-4-hydroxycinnamic methyl ester (HCCE). This combination (HCCA tag and HCCE matrix) enabled us to discriminate MS signals induced by peptides of interest that were present in low concentration from those of unlabelled more abundant peptides. By addition of a precise amount of deuterated-HCCA-tagged CPP/CPNP prior the MALDI TOF MS experiment, the internalized CPP/CPNP could be quantified on the basis of the ratio between the [M+H]+ peaks of the deuterated and nondeuterated HCCA-tagged CPP.Another direction for research was to synthesize bioconjugates between our newly discovered CPNP and some biologically active compounds that are unable to cross the cell membrane. We selected pepstatine which is a powerful transition state inhibitor of the Cathepsin D (CD). Pepstatine while a very potent inhibitor of the CD is unable to cross the cellular membrane. Moreover pepstatine activity in vitro or in vivo is hampered by its poor solubility in water. CD is a soluble lysosomal aspartic endopeptidase synthesized in rough endoplasmic reticulum as preprocathepsin D (pCD).12 Upon entering the acidic endosomal and lysosomal compartments proteolytic cleavages of the pCD result in the formation of the active enzymatic form of CD. Under normal physiological conditions pCD is sorted to the lysosomes and found intracellularly but in some pathological and physiological conditions like cancer pCD/CD escape the normal targeting mechanism and is secreted from the cell. Once secreted to the outside, pCD can be endocytosed via M6PR or yet unknown receptor by both cancer cells and fibroblasts. The endocytosed pCD undergoes maturation into the enzymatically active CD. An enzymatic activity of CD outside of the cell or inside the endosomes could be responsible for the activation of several growth factors and growth factor receptors. Several groups have proven that the tumour growth is not inhibited by the powerful CD inhibitor pepstatine. These results exclude the importance of the CD enzymatic activity outside of the cell but as already mentioned pepstatine is unable to penetrate into the cell thus CD activation of growth factors inside the endosomes or the lysosomes is still a possibility. Different CPNP-Pepstatine conjugates were synthesized and tested in vitro for their ability to inhibit MDA-MB-231 breast cancer cells growth. Some of these conjugates showed high cytotoxicity, probably via a Cathepsin D inhibition in the endosomes or the lysosomes. One o f the most potent tested compounds was JMV4463. This compound was obtained by the conjugation of pepstatine with a CPNP as delivery system (AMPA4) and with solubilizing moiety composed of polyethylene glycol and D-Arginine residue. The good in vitro results obtained with the vectorized pepstatine encouraged us to perform in vivo tests. We performed scale up synthesis of JMV4463 in order to obtain enough product for anti-cancer activity on mice in the near future.