Introdução: As proteinases tem um papel importante no desenvolvimento, na destruição tecidual e na produção de muco causada pela DPOC. O inibidor de proteinase de origem vegetal Enterolobium contortisiliquum Tripsin Inhibitor (EcTI) inibe tanto as proteinases da classe serina quanto da classe cisteína. Objetivos: Avaliar os efeitos do tratamento com EcTI nas alterações pulmonares induzidas pela elastase em camundongos. Métodos: Camundongos C57Bl6 receberam elastase via intratraqueal (50 uL/animal, grupo ELA) ou salina (grupo SAL). Os camundongos foram tratados com EcTI (2mg/kg) nos dias 1, 15 e 21 após a instilação de elastase (grupo ELA-EcTI) ou salina (grupo SAL-EcTI). No dia 28 do protocolo, os animais foram anestesiados, a mecânica pulmonar foi medida e o óxido nítrico exalado coletado. Posteriormente, foi realizado o lavado broncoalveolar e os pulmões foram removidos para a preparação de lâminas de histoquímica e imunohistoquímica. Por meio de morfometria analisamos o número de células positivas para neutrófilos, TNF-alfa, MMP-9, MMP-12, TIMP-1, iNOS, eNOS assim como a fração de volume de 8-iso-PGF2alfa, fibras colágenas e elásticas, nos septos alveolares e nas vias aéreas. Também foram avaliados o número de células positivas para macrófagos nos septos alveolares e MUC5ac nas vias aéreas. Resultados: O inibidor de proteinase EcTI reduziu as alterações de mecânica pulmonar (Ers, Htis e Raw), destruição do septo alveolar (Lm) e o número de células no lavado broncoalveolar (células totais, macrófagos, neutrófilos, linfócitos e eosinógilos) induzidos pela elastase. Em relação a resposta inflamatória, o EcTI reduziu o número de neutrófilos e de células TNFalfa positivas no septo alveolar e nas vias aéreas além de reduzir o número de macrófagos no septo alveolar. Considerando o remodelamento de matriz extracelular, o inibidor de proteinase atenuou a fração de volume de fibras colágenas e o número de células MMP-9 e MMP-12 positivas nos septos alveolares e nas vias aéreas. Além disso, nas vias aéreas ocorreu uma atenuação da fração de volume de fibras elásticas, e nos septos alveolares uma atenuação da quantidade de células que expressam TIMP-1. Em relação a resposta de estresse oxidativo, o EcTI reduziu a fração de volume de isoprostano e o número de células iNOS e eNOS positivas tanto nos septos alveolares quanto nas vias aéreas. O EcTI também reduziu o número de células MUC5ac positivas nas vias aéreas. Conclusões: O tratamento como inibidor EcTI modulou a mecânica pulmonar e reduziu as alterações inflamatórias, de remodelamento e de estresse oxidativo induzidas pela elastase intratraqueal. Embora sejam necessários mais estudos para elucidar os mecanismos envolvidos neste processo, o inibidor de proteinase EcTI pode ser considerado como um potencial instrumento terapêutico para o tratamento da DPOC / Background: Proteinases play a key role on emphysema development, tissue destruction and mucus production. Enterolobium contortisiliquum Tripsin Inhibitor (EcTI) is a proteinase inhibitor from plant that neutralizes serine and cysteine proteinases. Aims: To evaluated the effects of the EcTI treatment in pulmonary alterations induced by elastase in mice. Methods: C57Bl6 mice received elastase intratracheally (50 uL/animal, ELA group) or saline (SAL group). Afterwards, mice were treated with EcTI (2 mg/kg) at days 1, 15 and 21 after elastase instillation (ELA-EcTI group). Control group received saline and EcTI using the same protocol (SAL-EcTI group). At day 28, mice were anesthetized, respiratory mechanics were collected, and exhaled nitric oxide were analyzed. Afterwards, broncoalveolar lavage fluid was obtained and lungs were removed to perform histochemistry and immunohistochemistry stains. By morphometry, the number of neutrophils, TNF-alfa, MMP-9, MMP-12, TIMP-1, iNOS, eNOS positive cells as well as the volume proportion of 8-iso-PGF2alfa, collagen and elastic fibers content in alveolar septum and airways walls were performed. In airways walls, we also analyzed the number of MUC-5 positive cells and the number of macrophages. Results: The proteinase inhibitor EcTI was able to reduce the pulmonary mechanical alterations (Ers, Htis and Raw), alveolar septum disruption (Lm) and the BAL cell count (total cells, macrophages, neutrophils, lymphocytes and eosinophils) induced by elastase. Regarding the inflammatory response, EcTI also reduced the number of neutrophils and TNFalfa positive cells in both alveolar septum and airway walls, and also reduced the number of macrophages in alveolar septum. Considering the extracellular matrix remodeling, the proteinase inhibitor attenuated the volume fraction of collagen fibers, MMP-9 and MMP-12 positive cells in both alveolar septum and airway walls. Besides, in airway there were attenuation in the volume fraction of elastic fibers, and in the alveolar septa a decrease of the amount of the cells expressing TIMP-1. Regarding the oxidative stress response, EcTI reduced the volume fraction of isoprostane and the number of iNOS and eNOS positive cells in both airways walls and alveolar septa, Finally, EcTI reduced the number of MUC5ac positive cells in airway walls. Conclusions: The treatment with EcTI modulated lung mechanics and reduced inflammatory, remodeling and oxidative stress alterations induced by elastase. Although more studies need to be performed to elucidate the mechanisms involved in this process, we may considerate EcTI as a potential therapeutic tool for COPD management

Proteins that recognise the sugar surface structures on cells have an enormous potential to be used as tools in the characterisation of these structures. A group of proteins, called lectins, have been identified that can bind to carbohydrate complexes on the receptors of cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like proteins that were different from most other lectins, as they would specifically target the receptors of white blood cells and not those found on red blood cells. Therefore, the lectin isolated from G.robusta could be used as a tool to identify the specific surface structures on white blood cells. The lectin was isolated using affinity chromatography where a complex (oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to 5% of the total protein content) within the seeds of G.robusta. As a result of this low yield, the identification of the lectin by PAGE was difficult because the levels of protein were beyond the detection limit of the commercial staining reagents. The lectin was called the GR2 protein and was characterised as a monocot mannose binding lectin based on its sugar specificity for only mannose. A serine protease inhibitor was isolated from the seeds of G.robusta using two different chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration chromatography (GR1.GF). Ion exchange chromatography was used to initially separate the proteins in the crude extract and the fraction containing the GR1 protein was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the extraction process, which lead to the second purification method being established. Protease inhibitors were added to the buffers prior to being purified by gel filtration chromatography, which resulted in the GR1 protein being isolated from the crude extract without the presence of the contaminating protein. Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da and the full amino acid sequence was derived by cDNA techniques. Sequence alignment studies of the GR1 protein showed significant similarities with the Bowman-Birk inhibitor. The positioning of the cysteine residues were conserved throughout the Bowman-Birk superfamily, however these residues were not conserved within the GR1 protein. Competitive inhibition assays on the GR1 protein revealed the protein could inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-Birk superfamily of serine protease inhibitors. The three-dimensional structure of the GR1 protein was determined using two-dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and SYBYL® were used to tabulate the information taken from the 2D experiments, generate structures and minimise these structures respectively. The solution structure of the GR1 protein was found to contain a region of antiparallel β-sheet structure that corresponded to the trypsin binding site and the remainder of the protein consisted of loops and turns that were held together by disulfide bridges (the chymotrypsin-binding region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein is the first member of the Proteaceae family to be characterised as a Bowman-Birk inhibitor. This thesis outlines the isolation and biochemical characterisation of the two proteins found within Grevillea robusta and also describes the steps involved and results obtained in determining the three-dimensional structure of the GR1 protein.

This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated. The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction. The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude. Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support. The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.

Les biothérapies (anticorps monoclonaux, récepteurs solubles) ciblant les cytokines IL-6 etTNF-alpha pour le traitement des maladies inflammatoires chroniques ont constitué un succèsmajeur de l’industrie pharmaceutique. Elles présentent néanmoins des inconvénientsimportants : résistances, mode d’administration contraignant, coût élevé.Notre équipe travaille sur l’identification de petites molécules inhibant directement cescytokines, afin d’élargir l’offre thérapeutique existante. Administrées par voie orale, ellesconstitueraient une alternative particluièrement favorable aux patients.Durant ma thèse, j’ai réalisé le criblage expérimental (tests cellulaires et tests biochimiquesde liaison) des meilleurs composés identifiés par criblage virtuel d’un grande chimiothèque dediversité, ainsi que de composés dérivés de pyridazine issus d’une chimiothèque médicinale. J’aiainsi pu identifier plusieurs inhibiteurs directs du TNF-alpha et de l’IL-6. De plus, mon travail apermis d’affiner les procédures de criblage du Laboratoire.Ces travaux ouvrent de nouvelles pistes pour le développement de médicaments anti-cytokines. / Anti-cytokine biologics (monoclonal antibodies, soluble receptors) targeting TNF-alpha and IL-6in chronic inflammatory diseases have been a major success for pharmaceutical industry.However, they exhibit several drawbacks : resistance, difficult administration, high costs.Our team works on the discovery of small molecule inhibitors of cytokines suck as TNF-alphaand IL-6, in order to widen the range of therapeutic drugs. Orally active drugs would represent ahighly beneficial alternative for patients.During my PhD, I have performed an experimental screening (using cellular and biochemicalbinding testings) of the best compounds identified through virtual screening of a large chemicallibrary, and on pyridazine compounds of a medicinal chemical library. I have been able toidentify several small molecules inhibiting the interaction of TNF-! and IL-6 with their receptor.Moreover, my work will have an impact on the laboratory screening strategies.Overall, this work opens new avenues for anti-cytokine drug discovery.

During recent years, significant advances have been made in the field of molecular therapy in urologic oncology, mainly for advanced renal cell carcinoma. In this hitherto largely treatment-refractory disease, several agents have been developed targeting the von Hippel-Lindau metabolic pathway which is involved in carcinogenesis and progression of the majority of renal cell carcinomas. Although cure may not be expected, new drugs, such as the multikinase inhibitors sorafenib and sunitinib and the mammalian target of rapamycine inhibitor temsirolimus, frequently stabilize the disease course and may improve survival. Fewer data are available supporting molecular therapies in prostate, bladder, and testicular cancers. Preliminary data suggest a potential role of high-dose calcitriol and thalidomide in hormone-refractory prostate cancer, whereas targeted therapies in bladder and testicular cancers are still more or less limited to single-case experiences. The great theoretical potential and the multitude of possible targets and drug combinations, however, support further research into this exciting field of medical treatment of urologic malignancies. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Krebserkrankungen stellen in Deutschland die zweithäufigste Todesursache dar und die Anzahl der Neuerkrankungen nimmt stetig zu. Frühzeitige Diagnosen und Therapiemöglichkeiten sind daher dringend erforderlich. Cyklinabhängige Proteinkinasen (Cdk) spielen eine entscheidende Rolle bei der Regulation des Zellzyklus. Viele Tumore zeigen eine deregulierte Cdk4‑Aktivität und/oder ‑Expression. Insgesamt zeigen ca. 80% aller Tumore eine Fehlregulation der für den Zellzyklus zentralen Cdk4/CykD1/INK4/pRb/E2F Signalkaskade. Somit besitzen Cdks ein enormes therapeutisches Potential im Kampf gegen Krebs. Die spezifische Inhibierung der Cdks verhindert die Zellproliferation und damit das Tumorwachstum. In den letzten Jahren wurden verschiedenste Strukturklassen vorgestellt, die als Cdk4-Inhibitor wirken. Im Rahmen der Promotion sollen die Möglichkeiten einer funktionellen Tumordiagnose mittels cyklinabhängiger Kinasen untersucht werden. Die Entwicklung von radioaktiv markierten Inhibitoren der Cdk4/6 als Radiotracer und ihre radiopharmakologische Charakterisierung stellt dabei einen neuen Ansatz dar. Um die Rolle der Cdk4/6 im Zellzyklus von gesunden und deregulierten (z.B. Tumor-) Zellen aufzuklären, sollten mit Iod-124 und Fluor-18 markierte Inhibitoren eingesetzt werden, die hochselektiv diese Cdks blockieren. Zunächst wurden verschiedene Inhibitoren der Cdk4/6 und deren Vorstufen für die Radiomarkierung dargestellt. Die bereits aus den Vorarbeiten von VanderWel et al., 2005 und Toogood et al., 2001 bekannten Syntheserouten mussten dazu optimiert werden und für neue Verbindungen, wie die fluorethylierten Substanzen, wurden neue Reaktionswege gefunden. Die dargestellten Referenzverbindungen CKIA-E wurden anschließend mittels Durchflusszytometrie an den Zelllinien HT-29 und FaDu auf ihre inhibitorischen Wirkung untersucht. Die Untersuchungen der Verbindungen CKIA/B/E zeigte, dass ein Zellzyklusarrest unter Einwirkung der Inhibitoren erreichbar ist. Die weiteren Untersuchungen zur Radiomarkierbarkeit sowie die radiopharmakologische Evaluation sollten daher an den Verbindungen CKIA, CKIB und CKIE stattfinden. Die Darstellung der Verbindungen [124I]CKIA und [124I]CKIB erfolgte in zwei Schritten über die elektrophile Substitution durch regioselektive Destannylierung mit anschließender Entschützung der Seitenkette. Die Darstellung der fluorethylierten Verbindung erfolgte ebenfalls über eine Zweischrittsynthese beginnend mit der Synthese der prosthetischen Gruppe [18F]BFE aus der Tosylmarkierungsvorstufe. Die zur Markierung des sekundären Amins zur Auswahl stehenden prosthetischen Gruppen [18F]Fluorethyltosylat ([18F]FETos) und [18F]Bromfluorethan ([18F]BFE) wurden auf ihre Eignung untersucht, ebenso wie die Auswahl einer geeigneten Markierungsvorstufe für die Darstellung der prosthetischen Gruppe. Die optimierten Syntheserouten ermöglichten die Isolierung von ausreichenden Mengen an Produktaktivität für die radiopharmakologischen Untersuchungen. Es fanden, neben der Bestimmung der spezifischen Aktivität und der Lipophilie der Verbindungen, Zellaufnahmeuntersuchungen und Bestimmungen zur Stabilität der Verbindungen in vitro, ex vivo und in vivo statt. Die radioiodierten Verbindungen konnten des Weiteren zur Untersuchungen der Bioverteilung in normalen männlichen Wistar-Ratten eingesetzt werden. Für alle drei Verbindungen konnte eine sehr hohe in vitro-Stabilität festgestellt werden. Die Zellaufnahmeuntersuchungen zeigten vor allem für die Verbindungen [124I]CKIA und [124I]CKIB eine beträchtliche Zellaufnahme von über 1000% ID/mg Protein nach 2 h. Die Zellaufnahme der Verbindung CKIE ist geringer, sollte allerdings für eine in vivo-Anwendung ausreichend sein. Die Untersuchung der in vivo‑Stabilität der Verbindungen [124I]CKIA, [124I]CKIB und [18F]CKIE im Blut von Wistar Ratten ergab allerdings, dass alle Verbindungen schnell metabolisiert werden. Die Untersuchung der Bioverteilung der radioiodierten Verbindungen belegen eine in vivo Radiodeiodierung sowie eine hohe hepatobliliäre Auscheidungsrate. Im Hinblick auf eine Anwendung als Radiotracer konnten im Rahmen dieser Arbeit neue Erkenntnisse gewonnen werden. Die dargestellten Inhibitoren sind in der Lage am Zellmodell den Zellzyklusarrest in der G1-Phase zu induzieren. Eine Radiomarkierung der ausgewählten Strukturen liefert das Produkt mit reproduzierbarer Ausbeute in hoher radiochemischer Reinheit und ausreichender spezifischer Aktivität, allerdings ist eine Herstellung der fluorethylierten Verbindung unter GMP-Bedingungen nur schwer realisierbar. Die radiomarkierten Verbindungen zeigen eine hohe in vitro-Stabilität und werden energieabhängig in die Zelle aufgenommen. Anhand der Stabilitätsuntersuchungen in vivo wurde gezeigt, dass alle drei Verbindungen in vivo instabil sind und sehr schnell hepatobiliär eliminiert.

Le RXP470.1 est l’un des premiers inhibiteurs puissants de la MMP-12, une métalloprotéase à zinc impliquée dans de nombreuses pathologies comme l’athéroclérose et la bronchopneumopathie obstructive chronique (BPCO). Pour comprendre les bases moléculaires contrôlant l’interaction de cet inhibiteur avec sa cible, des approches pluridisciplinaires associant des relations structure-activité, avec des études de cristallographie de complexes enzymes inhibiteurs et d’études de microcalorimétrie, décrivant les contributions enthalpiques et entropiques impliquées dans la formation des complexes, ont été réalisées dans ce travail de thèse. Les affinités de trois analogues du RXP470.1 ont été tout d’abord déterminées. Puis quatre structures cristallographiques de complexes enzyme/inhibiteur décrivant le mode d’interaction duRXP470.1 et de ces trois analogues ont été obtenues avec des résolutions de 1.15 Å, 1.50 Å, 1.50Å et 1.30 Å, respectivement. Parallèlement les études de microcalorimétrie ont été menées pour étudier les facteurs énergétiques contrôlant l’interaction du RXP470.1 avec la MMP-12. Les résultats indiquent que la présence d’une chaîne latérale très longue et hydrophobe en position P1’de l’inhibiteur s’insérant dans la cavité S1’ de la MMP-12 est essentielle à la très bonne affinité de cet inhibiteur pour la MMP-12. Cette interaction met essentiellement en jeu un effet entropique très important de - 4 kcal/mol. L’interaction du RXP470.1 est aussi essentiellement dirigée par une forte augmentation d’entropie (-TDS= -10 kal/mol) et une composante enthalpique beaucoup plus faible (DH= -2.5 kcal/mol), et ce malgré l’observation dans le cristal de nombreuses interactions entre l’inhibiteur et le site actif de la MMP-12. L’étude de microcalorimétrie met aussi en lumière la prise d’un proton au cours de la formation du complexe enzyme inhibiteur impliquant deux résidus chargés négativement en solution, le résidu catalytique Glu219 et le groupe phosphoryle chélatant du zinc dans l’inhibiteur. Cette étude révèle aussi que si le groupe phosphoryle est considéré comme un chélatant faible de l’atome de zinc, il impose néanmoins des contraintes directionnelles très importantes qui ont un impact sur le positionnement des autres parties de l’inhibiteur dans le site actif de l’enzyme. Ce dernier effet pourrait expliquer pourquoi un certain nombre d’interactions entre l’inhibiteur et l’enzyme ne sont pas optimisées et pourquoi la variation d’enthalpie pour former le complexe reste relativement faible. Cette étude ouvre maintenant la voie à d’autres études en plaçant au centre des futurs travaux le rôle du groupe chélatant dans la conception des inhibiteurs de MMP, ainsi de nouveaux inhibiteurs puissants et sélectifs d’autres MMP devraient voir le jour grâce à ce travail et aux résultats obtenus. / RXP470.1 is one of the first highly potent and selective inhibitor of MMP-12, a zinc protease involved in several human diseases such as atherosclerosis and chronic obstructive pulmonary disease (COPD). To understand the molecular determinants controlling the interaction of RXP470.1 with MMP-12 active site, a multidisciplinary approach combining structure-activity data, crystallography and microcalorimetry have been performed on RXP470.1 and its three analogues. The affinities of the three RXP470.1 analogues have been determined. Then, fourcrystal structures of MMP-12 in interaction with these inhibitors have beendetermined at high resolution, 1.15 Å, 1.50 Å, 1.50 Å et 1.30 Å, respectively. These data have indicated that the presence of a long hydrophobic side chain in the P1’ position of the RXP470.1, which enters deeply inside the S1’ cavity of MMP-12, is playing a key role in the inhibitor affinity. The contribution of this side chain is mostly entropic (-TDS - 4 kcal/mol). The interaction of RXP470.1 with MMP-12 is also mostly driven by a sizeable entropy increase (-TDS= -10 kal/mol) and a more modest enthalpy contribution (DH= -2.5 kcal/mol), despite the observation in the crystal structure of several contacts between inhibitor and MMP-12 active site. Furthermore, this study reveals that the binding of RXP470.1 to MMP-12 is linked to a proton uptake involving two negatively charged residues, the catalytic Glu219 and the phosphoryl group of the inhibitor. Furthermore, despite that the phosphoryl group is considered as a weak zincbinding group, this study highlights that the interactions of this group with the active site zinc atom involved strong directionality between these two groups. This effect has strong impact on the positioning of the other parts of the inhibitor in the MMP-12 active site. This last effect could be responsible for the modest enthalpy increase associated with the binding of RXP470.1 to MMP-12, by preventing the optimization of several interactions between the inhibitor and the enzyme. The results indicate that the role of the zinc-binding group should be better consider in the future. Finally this study opens a new vision in this field and should allow the design of new selective inhibitors of other MMPs.