Atualmente, ainda é incerto se a hidrólise de L-arginil-β-naftilamida (ArgNA) e do leucotrieno (LT) A4 pela LT-A4-hidrolase (LT-A4-H) (EC 3.3.2.6) e pela aminopeptidase básica (APB) (EC 3.4.11.6) tem influência no desenvolvimento da artrite induzida por colágeno (CIA). O objetivo deste estudo foi investigar a inter-relação entre LT-A4-H, APB e LT-B4 em ratos submetidos à CIA. Cromatografia líquida de alta eficiência (HPLC), ensaio imunoenzimático (EIA), espectrofluorimetria e reação quantitativa em tempo real em cadeia da polimerase (qPCR) foram usados como metodologias. A existência dos genes para as proteínas EC 3.3.2.6 e EC 3.4.11.6 foi confirmada no tecido sinovial (TS) de ratos controles sadios. A hidrólise de ArgNA aumentou na fração solúvel (FS) dos animais submetidos à CIA que desenvolveram a doença (artríticos-CIA) em comparação com aqueles que não desenvolveram a doença (resistentes-CIA) e com os controles sadios. No líquido sinovial (SY) e no plasma sanguíneo houve menor hidrólise de ArgNA em resistentes em comparação aos artríticos e controles. Nas células mononucleares do sangue periférico (PBMCs), os níveis de hidrólise de ArgNA aumentaram na FS de controles e na fração de membrana (FM) dos resistentes em comparação aos artríticos. Em comparação com controles sadios, a hidrólise de LT-A4 aumentou no SY e na FS de PBMCs de artríticos e resistentes. A hidrólise de LT-A4 também aumentou na FM do TS de resistentes e diminuiu na FM de PBMCs em artríticos e resistentes. Em todos estes compartimentos a hidrólise de ArgNA permaneceu inalterada ou relacionou-se inversamente com a hidrólise de LT-A4, comparativamente aos controles sadios. A hidrólise de ArgNA diferiu entre os artríticos e resistentes em FM-TS, FS-TS, FM-PBMCs, SY e no plasma sanguíneo. Uma relação no mesmo sentido foi encontrada entre alterações na hidrólise de LT-A4 e nos níveis de LT-B4 apenas em SY e FM-PBMCs dos artríticos e resistentes e em FM-TS dos resistentes, comparativamente aos controles sadios. Em conclusão, a atividade APB é um novo marcador que distingue ratos artríticos e resistentes no modelo CIA. Os níveis de LT-B4 em ratos não são controlados somente pela LT-A4-H. Alterações na atividade LT-A4-H e nos níveis de LT-B4 são indistinguíveis entre artríticos e resistentes, mas tais alterações marcadamente distinguem essas duas condições da condição saudável. LT-A4-H e APB estão relacionadas de uma forma compartimento-dependente, atuando como enzimas independentes, com modulação diferencial das suas especificidades, eficiências e/ou afinidades catalíticas sobre os substratos epóxi e peptídico, ou como enzimas bifuncionais, cujas atividades são inversamente relacionadas devido à inibição concorrente de uma destas atividades / Whether L-arginyl-β-naphthylamide (ArgNA) and leukotriene (LT)-A4 hydrolyses by LT-A4 hydrolase (LT-A4-H) (EC 3.3.2.6) and basic aminopeptidase (APB) (EC 3.4.11.6) influence the development of collagen-induced arthritis (CIA) is presently uncertain. The objective of this study was to investigate the interrelationship among LT-A4-H, APB and LT-B4 in CIA rats. High-performance liquid chromatography (HPLC), enzyme immunoassay (EIA), spectrofluorometry and quantitative real time polymerase chain reaction (qPCR) were used as methodologies. The existence of genes for EC 3.3.2.6 and EC 3.4.11.6 proteins were confirmed in the synovial tissue (TS) of healthy control rats. ArgNA hydrolysis was higher in soluble fraction (FS) in rats submitted to CIA that developed the disease (CIA-arthritic) than in those that did not develop the disease (CIA-resistant) or healthy control. Synovial fluid (SY) and blood plasma had lower ArgNA hydrolysis in CIA-resistant than in CIA-arthritic or control. In the peripheral blood mononuclear cells (PBMCs) the levels of ArgNA hydrolysis increased in FS of control and in membrane-bound fraction (FM) of CIA-resistant in comparison with CIA-arthritic. Compared with healthy control, LT-A4 hydrolysis increased in SY and in FS from PBMCs of CIA-arthritic and CIA-resistant. LT-A4 hydrolysis also increased in FM from TS of CIA-resistant and decreased in PBMCs-FM of CIA-arthritic and CIA-resistant. In all these locations hydrolysis of ArgNA remained unchanged or it was inversely related with LT-A4 hydrolysis, comparatively to healthy control. ArgNA hydrolysis differed between CIA-arthritic and CIA-resistant in TS-FM, TS-FS, PBMCs-FM, SY and blood plasma. A same-sense relationship was found between changes on LT-A4 hydrolysis and LT-B4 levels only in SY and PBMCs-FM of CIA-arthritic and CIA-resistant and in TS-FM of CIA-resistant, comparatively to healthy control. In conclusion, the APB activity is a novel distinctive marker of CIA-arthritic and CIA-resistant statuses. The levels of LT-B4 in rats are not controlled only by LT-A4-H. Changes on LT-A4-H activity and LT-B4 levels are indistinguishable between CIA-resistant and CIA-arthritic, but such variations markedly distinguish these two statuses from healthy status. LT-A4-H and APB are related in a compartment-dependent manner acting as independent enzymes with differential modulation of their specificity, efficiency and/or catalytic affinity on the aminoacyl and epoxy substrates, or as bifunctional enzymes which activities are inversely related due to the concurrent inhibition of one of these

Le premier acide aminé incorporé dans une chaîne polypeptidique naissante est toujours la méthionine. On identifie donc toujours ce premier résidu à la méthionine N-terminale. Cependant, les deux tiers des protéines accumulées à l’état stationnaire ne présentent plus leur méthionine initiatrice. Cet enlèvement résulte essentiellement d’une maturation protéolytique affectant chaque protéine. Ainsi, l’Excision de la Méthionine N-terminale (NME) concerne la majorité des protéines et ce dès que les premiers résidus émergent du ribosome. Ce mécanisme est retrouvé dans tous les compartiments cellulaires où une synthèse protéique a lieu : le cytoplasme, les plastes et les mitochondries. Les enzymes responsables du clivage de la méthionine initiatrice sont les METhionine AminoPeptidases (METAPs) ; les METAPs sont conservées dans le Règne vivant. Des études fonctionnelles de délétions géniques ont montré le caractère létal du maintien de la première méthionine dans tous les organismes. Il y a plus de dix ans, les METAPs ont été identifiées comme étant la cible de composés naturels ayant des effets anticellulaires. Aujourd’hui un nombre croissant d’études rapportent que la NME est une cible prometteuse pour le traitement de nombreuses pathologies. Néanmoins, les bases moléculaires qui expliquent le caractère essentiel de la NME restent très peu comprises, en particulier dans le cytoplasme des eucaryotes supérieurs. Grâce à un système inductible permettant de moduler finement la NME cytoplasmique dans la plante modèle Arabidopsis thaliana et différentes approches incluant des analyses protéomiques et métabolomiques, j’ai pu étudier les événements moléculaires précoces associés à l’inhibition de la NME cytoplasmique. J’ai également caractérisé la contribution relative des deux types de METAP cytoplasmiques au processus. Dans ce contexte, j’ai pu démontrer chez A. thaliana que la NME cytoplasmique agit sur deux voies de signalisation fréquemment dérégulées lors de conditions pathologiques : le statut des composés thiolés et la protéolyse. La diminution de la NME cytoplasmique induit une protéolyse accrue principalement via une augmentation du nombre de protéines destinées à une dégradation rapide. Ainsi, l’activité de la NME, en modulant la sensibilité de nombreuses protéines à subir la protéolyse, est un élément fondamental de la régulation de la demi-vie protéique. Finalement, mes résultats simialires obtenus également chez les Archées, levures et les lignées de cellules humaines suggèrent l’existence d’un mécanisme ubiquitaire associé à la NME. / The first amino acid incorporated in nascent polypeptide chain is always methionine so called N-terminale methionine. However, in a given proteome, more than fifty percent of proteins have not this first methionine. Indeed, the early proteolytic event affecting a majority of proteins is N-terminal Methionine Excision (NME) as soon as few residues exit from the ribosome. Enzymes ensuring NME process are conserved along species. This mechanism takes place in all compartments where protein synthesis occurs including cytoplasm, plastids and mitochondria and the enzymes responsible of N-methionine excision are METhionine AminoPeptidases (METAP). Early functional studies of gene deletion has quickly showed that NME is an essential process. Ten years ago, METAPs have been identified as the molecular target of natural compounds with anticancer activities. Now, a growing number of studies suggest that NME is a promising target for treatment of various deseases. Nevertheless, molecular mechanisms making NME an essential process is poorly understood in particular in higher eukaryote cytoplasms.Using a dedicated inducible system in the model organism Arabidopsis thaliana and multiple approaches, including proteomics and metabolomics, I examined the earliest molecular events associated with the inhibition of this process and the contribution of both METAP to NME process. In this context, I demonstrated that cytoplasmic NME in A. thaliana orchestrates a cross-talk between two fundamental signaling pathways frequently deregulated in pathological conditions: thiol status and proteolysis. In these studies, we demonstrated that developmental defects induced by cytoplasmic NME inhibition are associated with an increase of the proteolytic activity due to an increase of the proteins available for rapid degradation. Thus, NME activity that modifies the availability of several proteins for degradation is an integral and fundamental element protein turnover regulation. Finally my preliminary results obtained in Archea, Fungi and human cells seem to suggest the existence of a ubiquitous mechanism associated with NME process.

Les parasites du genre Eimeria sont des parasites apicomplexes provoquant des maladies graves, appelées coccidioses. Ce genre compte sept espèces qui envahissent spécifiquement les cellules épithéliales des intestins de poulet. L’espèce de choix étudiée dans notre laboratoire est Eimeria tenella. La prophylaxie repose sur les vaccins qui sont onéreux et les traitements anticoccidiens, qui sont administrés dans l’alimentation dès l’éclosion du poussin. Depuis 50 ans, l’utilisation massive de ces molécules s’accompagne de l’émergence de populations parasitaires résistantes. Les modes d’action des cibles parasitaires des anticcocidiens étant peu connus, il est difficile de mettre en place une stratégie de contournement de la résistance. Dans l’optique de répondre aux besoins des éleveurs, une collaboration entre le laboratoire de chimie organique et celui de parasitologie a été mise en place en utilisant la capacité de composés chimiques à inhiber l’invasion ou le développement des parasites. Notre stratégie repose sur la syntèse, le développement ainsi que l’évaluation de l’efficacité des composés pouvant potentiellement être utilisés en tant qu’inhibiteurs de la coccidiose aviaire. Un criblage de composés nous a permis d’identifier deux composés « chefs de files », inhibant le processus d’invasion et de développement des parasites au sein de la cellule hôte. Les pharmacomodulations entreprises ont engendrés la découverte de composés plus actifs avec des valeurs d’IC50 égales à 0,8 et 3,4 µM respectivement. / Intracellular developing parasites that belong to the apicomplexan phylum represent a great threat to both animal and human being health. Apicomplexan contains a genus of parasites called Eimeria. This genus is composed of seven species which cause avian coccidiosis such as E. tenella, the most virulent agent. Control of E. tenella is presently accomplished by a prophylaxis that uses vaccines and anticoccidial drugs. However, the rapid emergence of drug resisitant parasites coupled with the expense of most vaccines has led us to a search of new approaches to control coccidiosis via the synthesis of new compounds. In order to achieve that goal, collaboration between two laboratories has been established. Our strategy relies on the laboratories scientific knowledge in two complementary domains such as oraganic chemistry and parasitology. Our research is based on the effectiveness that synthesized compounds could have to inhibit parasites first steps of infection of the host cells. The screening of compounds has led us to the discovery of two lead compound inhibiting the invasion and development process of the parasites. Undertaken pharmacomodulations of these leads have allowed us to lower these concentrations values to 0,8 and 3,4 µM

Atualmente, ainda é incerto se a hidrólise de L-arginil-β-naftilamida (ArgNA) e do leucotrieno (LT) A4 pela LT-A4-hidrolase (LT-A4-H) (EC 3.3.2.6) e pela aminopeptidase básica (APB) (EC 3.4.11.6) tem influência no desenvolvimento da artrite induzida por colágeno (CIA). O objetivo deste estudo foi investigar a inter-relação entre LT-A4-H, APB e LT-B4 em ratos submetidos à CIA. Cromatografia líquida de alta eficiência (HPLC), ensaio imunoenzimático (EIA), espectrofluorimetria e reação quantitativa em tempo real em cadeia da polimerase (qPCR) foram usados como metodologias. A existência dos genes para as proteínas EC 3.3.2.6 e EC 3.4.11.6 foi confirmada no tecido sinovial (TS) de ratos controles sadios. A hidrólise de ArgNA aumentou na fração solúvel (FS) dos animais submetidos à CIA que desenvolveram a doença (artríticos-CIA) em comparação com aqueles que não desenvolveram a doença (resistentes-CIA) e com os controles sadios. No líquido sinovial (SY) e no plasma sanguíneo houve menor hidrólise de ArgNA em resistentes em comparação aos artríticos e controles. Nas células mononucleares do sangue periférico (PBMCs), os níveis de hidrólise de ArgNA aumentaram na FS de controles e na fração de membrana (FM) dos resistentes em comparação aos artríticos. Em comparação com controles sadios, a hidrólise de LT-A4 aumentou no SY e na FS de PBMCs de artríticos e resistentes. A hidrólise de LT-A4 também aumentou na FM do TS de resistentes e diminuiu na FM de PBMCs em artríticos e resistentes. Em todos estes compartimentos a hidrólise de ArgNA permaneceu inalterada ou relacionou-se inversamente com a hidrólise de LT-A4, comparativamente aos controles sadios. A hidrólise de ArgNA diferiu entre os artríticos e resistentes em FM-TS, FS-TS, FM-PBMCs, SY e no plasma sanguíneo. Uma relação no mesmo sentido foi encontrada entre alterações na hidrólise de LT-A4 e nos níveis de LT-B4 apenas em SY e FM-PBMCs dos artríticos e resistentes e em FM-TS dos resistentes, comparativamente aos controles sadios. Em conclusão, a atividade APB é um novo marcador que distingue ratos artríticos e resistentes no modelo CIA. Os níveis de LT-B4 em ratos não são controlados somente pela LT-A4-H. Alterações na atividade LT-A4-H e nos níveis de LT-B4 são indistinguíveis entre artríticos e resistentes, mas tais alterações marcadamente distinguem essas duas condições da condição saudável. LT-A4-H e APB estão relacionadas de uma forma compartimento-dependente, atuando como enzimas independentes, com modulação diferencial das suas especificidades, eficiências e/ou afinidades catalíticas sobre os substratos epóxi e peptídico, ou como enzimas bifuncionais, cujas atividades são inversamente relacionadas devido à inibição concorrente de uma destas atividades / Whether L-arginyl-β-naphthylamide (ArgNA) and leukotriene (LT)-A4 hydrolyses by LT-A4 hydrolase (LT-A4-H) (EC 3.3.2.6) and basic aminopeptidase (APB) (EC 3.4.11.6) influence the development of collagen-induced arthritis (CIA) is presently uncertain. The objective of this study was to investigate the interrelationship among LT-A4-H, APB and LT-B4 in CIA rats. High-performance liquid chromatography (HPLC), enzyme immunoassay (EIA), spectrofluorometry and quantitative real time polymerase chain reaction (qPCR) were used as methodologies. The existence of genes for EC 3.3.2.6 and EC 3.4.11.6 proteins were confirmed in the synovial tissue (TS) of healthy control rats. ArgNA hydrolysis was higher in soluble fraction (FS) in rats submitted to CIA that developed the disease (CIA-arthritic) than in those that did not develop the disease (CIA-resistant) or healthy control. Synovial fluid (SY) and blood plasma had lower ArgNA hydrolysis in CIA-resistant than in CIA-arthritic or control. In the peripheral blood mononuclear cells (PBMCs) the levels of ArgNA hydrolysis increased in FS of control and in membrane-bound fraction (FM) of CIA-resistant in comparison with CIA-arthritic. Compared with healthy control, LT-A4 hydrolysis increased in SY and in FS from PBMCs of CIA-arthritic and CIA-resistant. LT-A4 hydrolysis also increased in FM from TS of CIA-resistant and decreased in PBMCs-FM of CIA-arthritic and CIA-resistant. In all these locations hydrolysis of ArgNA remained unchanged or it was inversely related with LT-A4 hydrolysis, comparatively to healthy control. ArgNA hydrolysis differed between CIA-arthritic and CIA-resistant in TS-FM, TS-FS, PBMCs-FM, SY and blood plasma. A same-sense relationship was found between changes on LT-A4 hydrolysis and LT-B4 levels only in SY and PBMCs-FM of CIA-arthritic and CIA-resistant and in TS-FM of CIA-resistant, comparatively to healthy control. In conclusion, the APB activity is a novel distinctive marker of CIA-arthritic and CIA-resistant statuses. The levels of LT-B4 in rats are not controlled only by LT-A4-H. Changes on LT-A4-H activity and LT-B4 levels are indistinguishable between CIA-resistant and CIA-arthritic, but such variations markedly distinguish these two statuses from healthy status. LT-A4-H and APB are related in a compartment-dependent manner acting as independent enzymes with differential modulation of their specificity, efficiency and/or catalytic affinity on the aminoacyl and epoxy substrates, or as bifunctional enzymes which activities are inversely related due to the concurrent inhibition of one of these

The parasite Plasmodium falciparum is the causative agent of the most severe form of human malaria. During its intraerythocytic life cycle, P. falciparum transports red blood cell contents to its acidic organelle, known as the food vacuole, where a series of proteases degrade a majority of the host hemoglobin. Two metalloaminopeptidases, PfAPP and PfA-M1, have been previously localized to the food vacuole (in addition to distinct secondary locations for each), implicating them in the final stages of hemoglobin catabolism. Prior genetic work has determined these enzymes are necessary for efficient parasite proliferation, highlighting them as potential anti-malarial drug targets. This study presents the biochemical basis for the catalytic roles of these two enzymes in the hemoglobin degradation pathway. PfAPP, an aminopeptidase P homolog, is specific for hydrolyzing the N-termini of peptides containing penultimate prolines. PfA-M1 is a member of the expansive M1 family of proteases and exhibits a broad specificity towards substrates. The two enzymes are ubiquitous, found in organisms across all kingdoms of life. Their presence in an acidic environment is unique for aminopeptidase P proteins and rare for M1 homologs. Our immunolocalization results have confirmed the dual distribution of these two enzymes in the parasite. Vacuolar targeting was found to be associated with the Plasmodium specific N-terminal extension found in the PfA-M1 sequence by yellow fluorescent protein fusion studies. Kinetic analysis of recombinant forms of PfAPP and PfA-M1 revealed both enzymes are stable and catalytically efficient in the substrate rich, acidic environment of the parasite food vacuole. In addition, mutagenic exploration of the PfA-M1 active site has determined a residue important in dictating substrate specificity among homologs of the same family. These results provide insight into the parasite's functional recruitment of these enzymes to deal with the final stages of hemoglobin catabolism and necessary considerations for inhibitor design. / Ph. D.

Melflufen (melphalan flufenamide, chemical name L-melphalanyl-p-L-fluorophenylalanine ethyl ester hydrochloride, previously called J1) is a derivative of the classical alkylating agent melphalan. Melflufen is potentiated by hydrolytic cleavage by aminopeptidase N (APN), leading to high intracellular concentrations of alkylating moieties and subsequent cell death. Increased APN expression is associated with the malignant phenotype of several human cancers, including acute myeloid leukemia, lymphoma and ovarian cancer, and plays a functional role in tumor angiogenesis. Therefore investigations of melflufen activity in these malignancies as well as detailed studies of inhibition of angiogenesis are interesting. The aim of this project was to investigate the cytotoxic and antiangiogenic effect, in vitro and in vivo, of melflufen, compared to melphalan and other cytotoxic drugs used in the clinic. We showed that melflufen was more effective than its parental drug melphalan in lymphoma, AML and ovarian cancer in cell lines as well as in primary patient samples. An improved in vitro therapeutic index was demonstrated by an increased cytotoxic activity in the patient samples compared to normal peripheral blood mononuclear cells (PBMCs). Furthermore, melflufen in combination with cytarabine was synergistic in an AML cell line in a sequence-dependent manor. Melflufen was shown effective in several animal models using lymphoma, AML and ovarian cell xenografts (single drug or in combination), including an intraperitoneal ovarian xenograft. Finally, we demonstrated that melflufen had antiangiogenic properties in several different models.

Les aminopeptidases sont des cibles thérapeutiques importantes pour plusieurs maladies, car elles sont impliquées dans divers processus physiologiques et pathologiques comme la progression tumorale, l'angiogenèse, et certaines infections (virales, bactériennes, et parasitaires). Il en existe deux classes : les aminopeptidases avec un ion métallique (Aminopeptidase N [APN ou CD13] et leukotrien A4 hydrolase [LTA4H]) et les aminopeptidases avec deux ions métalliques (Aminopeptidase de l'Aeromonas proteolytica [APaero], Leucine Aminopeptidase cytosolique [LAPc] et Méthionine aminopeptidase 1 ou 2 [MetAP]). Deux types de composés dérivés des amino-benzosubérones ont été envisagés pour inhiber sélectivement chacune de ces classes d'aminopeptidases. L'étude des relations structures-activités (RSA) nous a permis de découvrir une molécule très puissante et sélective de l'APN (Ki 60 pM). L'APN est une enzyme monométallique considérée aujourd'hui comme une nouvelle cible pour la lutte contre le cancer car son inhibition bloque le processus de l'angiogenèse et donc la progression tumorale. L'étude d'une nouvelle classe de molécules trisubstituées dérivées des amino­benzosubérones a abouti à la découverte d'une seconde molécule active et sélective des enzymes bimétalliques notamment l'APaero (Ki 10 nM). / The aminopeptidases are important therapeutic targets for several diseases, because they are implied in various physiological and pathological processes like the tumoral progression, the angiogenesis, and certain infections (viral, bacterial, and parasitic). There are two classes: aminopeptidases with one metal ion (Aminopeptidase N [APN or CD13] and leukotriene A4 hydrolase [LTA4H]) and aminopeptidases with two metal ions (Aminopeptidase of Aeromonas proteolytica [APaero], cytosolic leucine aminopeptidase [LAPc] and Methionine aminopeptidase 1 or 2 [MetAP]). Two types of compounds of amino-benzosuberone derivatives were envisaged to inhibit selectively each one of these classes of aminopeptidases. The study of the structure-activity relationship (SAR) enabled us to discover a very powerful and selective molecule of the APN (Ki 60 pM). The APN is a monometallic enzyme considered today as a new target for the fight against cancer because its inhibition blocks the angiogenesis process and thus the tumoral progression. The study of a new class of trisubstituted molecules derived from the amino-benzosuberone led us to discover another molecule which is active and selective of the bimetallic enzymes in particular APaero (Ki 10 nM).

Nach einem Myokardinfarkt setzen Wundheilungsprozesse ein, um die Durchblutung wieder herzustellen und nekrotisches Muskelgewebe durch Narbengewebe zu ersetzen. Die Einsprossung neuer Kapillaren vom bestehenden Gefäßnetz aus wird als Angiogenese bezeichnet. Das dabei vermehrt exprimierte proteolytische Enzym Aminopeptidase N (APN) spielt eine entscheidende Rolle bei der Einsprossung von Endothelzellen. Beim kardialen Remodeling werden abgestorbene Myozyten mithilfe der Einwanderung von Fibroblasten durch Binde- oder Stützgewebe ersetzt, dabei übernimmt das Fibroblasten-Aktivierungs-Protein alpha (FAP) Aufgaben bei der Proliferation und Fortbewegung von Fibroblasten. Durch ihre erhöhte Expression bei den Wundheilungs- und Remodelingprozessen nach einem Herzinfarkt stellen die Metalloprotease APN und die Serinprotease FAP molekulare Targets für die Diagnostik und Therapie dar. Als Diagnosemethode besonders geeignet ist die Positronen-Emissions-Tomographie (PET), die es ermöglicht, biochemische Prozesse in Echtzeit im zu untersuchenden Organismus zu visualisieren und zu quantifizieren. Eine als Radiopharmakon oder Tracer bezeichnete biochemische Sonde kann im Falle eines Enzyms dessen radioaktiv markiertes Substrat oder ein Inhibitor sein. Ziel dieser Arbeit war es, spezifische APN- und FAP-affine Tracer für die nicht-invasive Untersuchung der APN- und FAP-Expression mittels PET zu entwickeln und dadurch die Rolle von APN und FAP bei Remodelingprozessen nach Myokardinfarkt besser verstehen bzw. klären zu können. Um die Protease APN mittels PET zu untersuchen, wurden die für APN affine Verbindung NOTA-NGR (Komplexbildner + cyclisches Peptid inkl. Asparagin-Glycin-Arginin) mit dem Positronen-emittierenden Nuklid Gallium-68 (68Ga) markiert. Das Potential von 68Ga-NOTA-NGR als PET-Tracer wurde in vivo am Infarktmodell mittels Kleintier-PET untersucht und mit 68Ga-NOTA-RGD, einem zur Visualisierung des neo-angiogenetischen alphavbeta3-Integrins etablierten Tracer, verglichen. Untersuchungen ergaben, dass 68Ga-NOTA-NGR einen vielversprechenden neuen PET-Tracer für die Visualisierung und Quantifizierung der APN-Expression im Rahmen der Angiogenese nach einem Myokardinfarkt darstellt. 68Ga-NOTA-NGR zeigte eine erhöhte Aufnahme im Bereich des Myokardinfarkts im Sinne einer vermehrten Angiogenese. Die Aufnahme des Tracers in infarzierten Arealen war quantitativ höher als in der Untersuchung mit 68Ga-NOTA-RGD. In Autoradiographie-Experimenten wurde 68Ga-NOTA-NGR ex vivo untersucht. Die Akkumulation von 68Ga-NOTA-NGR im ischämischen Bereich war deutlich höher als im gesunden Myokard. Der Nachweis der unterschiedlichen Bereiche des Herzens erfolgte mit HE-Färbung. Die Expression von APN wurde immunohistochemisch mittels spezifischer Antikörper bestätigt. Zum Vergleich wurden ebenso einige andere an der Angiogenese beteiligte Faktoren untersucht. APN stellte sich auch hier als geeignetes Target zum Nachweis der Angiogenese heraus. Um die Protease FAP mittels PET zu untersuchen, wurden eine Reihe peptidomimetischer Inhibitoren, die die Erkennungssequenz Glycin-Prolin mit einer Carbonitril-Gruppe als elektrophiler Einheit zur kovalent-reversiblen Hemmung des Enzyms enthalten, entwickelt. Ausgehend vom N-Acetylglycin-pyrrolidin-(2S)-carbonitril als Leitstruktur wurden Inhibitoren und Vorstufen zur Radiomarkierung inkl. verschieden substituierter Benzoesäuren dargestellt. Zusätzlich wurden noch bereits bekannte Inhibitoren synthetisiert, die zum Vergleich in den Enzymassays dienten. Drei Verbindungen zeigten gute inhibitorische Wirkung an FAP und außerdem Selektivität gegenüber DPP IV. Keine der entwickelten Verbindungen zeigte einen KI-Wert im nanomolaren Bereich, erforderlich für einen potentiellen Tracer zur in-vivo-Visualisierung einer Enzymexpression mittels PET. Um die Inhibitoren mit der besten Hemmung an FAP zum PET-Tracer weiterzuentwickeln, mussten sie mit einem Positronenemitter markiert werden. Die Markierung erfolgte über Isotopenaustausch, bei dem nicht-radioaktives Iod am aromatischen Ring des Precursors durch das radioaktive Iod-124 (124I) substituiert wurde. Es konnten dadurch die radioiodierten Verbindungen 1-(2-[124I]Iodhippursäure)-pyrrolidin-(2S)-carbonitril und 1-(4-[124I]Iod-hippursäure)-pyrrolidin-(2S)-carbonitril synthetisiert werden. Trotz der relativ niedrigen Affinität für FAP wurde das neue 1-(2-[124I]Iodhippursäure)-pyrrolidin-(2S)-carbonitril in Ratten am Infarktmodell mittels Kleintier-PET getestet. Die Lage der ischämischen Zone wurde im Anschluss durch HE-Färbung bestimmt. In vivo zeigte sich eine nur sehr geringe Aufnahme des Radiopharmakons in der ischämischen Zone des Myokards. Damit ist 1-(2-[124I]Iod-hippursäure)-pyrrolidin-(2S)-carbonitril kein für den gewünschten Zweck geeigneter PET-Tracer. Nichtsdestotrotz war der Ansatz vielversprechend und es wurde zum ersten Mal ein PET-Tracer dieser Art zur Untersuchung des FAP im Myokardinfarkt hergestellt. / After myocardial infarction, processes of wound healing are initiated in order to regain perfusion and to replace necrotic muscle tissue with soft tissue. The sprouting of new capillaries from the vasculature is called angiogenesis. During Angiogenesis, Aminopeptidase N (APN) plays an important role in the sprouting of endothelial cells. Cardiac remodeling is the process of replacement of necrotic myocytes with soft tissue through invasion of fibroblasts. For this cause, also a lot of proteases are activated. During the process of cardiac remodeling, fibroblast activation protein alpha (FAP) is involved in proliferation and migration of cardiac fibroblasts. Due to their increased expression during remodeling processes after myocardial infarction, the metalloprotease APN and the serine protease FAP have been identified as potential molecular targets for diagnosis and therapy. Diagnosis of the heart by nuclear imaging techniques is a well established method in clinical cardiology. Most of all positron emission tomopgraphy (PET) provides information on biochemical processes in vivo using specific radiotracers in real time. This imaging probe is labeled with a positron emitting radionuclide and is called radiopharmaceutical or tracer. In case of an enzyme, the tracer might for example be a labeled substrate or inhibitor of the enzyme. To visualize the protease APN with PET, NOTA-NGR (chelating agent + peptide sequence incl. asparagine-glycine-arginine), a compound that shows high affinity for APN, was labeled with the positron emitting nuclide Gallium-68 (68Ga). 68Ga-NOTA-NGR was developed including an improved synthesis, isolation and formulation of the tracer. Its potential as a PET-tracer was assessed in vivo using micro-PET and compared to the established tracer 68Ga-NOTA-RGD, used to visualize the integrin alphavbeta3 in angiogenesis. Studies in rats with ischemia/reperfusion showed high uptake of the new radiopharmaceutical 68Ga-NOTA-NGR in myocardial infarction area being used in diagnostic PET imaging of APN. The new tracer shows even a slightly higher uptake in angiogenetic areas compared with results obtained with 68Ga-NOTA-RGD. 68Ga-NOTA-NGR was also examined ex vivo using autoradiography, confirming the significant higher accumulation of the tracer in the ischemic area compared with the healthy myocardium. The different areas of the tissue were displayed by HE staining. For the purpose of immunohistochemistry, the expression of the enzyme APN was verified using antibody staining. Additionally several other factors that are involved in angiogenesis were stained. Through antibody staining APN was shown to be a suitable target for the evidence of angiogenesis. With 68Ga-NOTA-NGR, the development of a new PET-tracer for diagnosis of the expression of APN during angiogenesis after myocardial infarction was successful. In order to develop an imaging probe suitable for investigation of the protease FAP using PET, several peptidomimetic inhibitors containing the dipeptide motif glycine-proline and the electrophilic moiety carbonitrile were designed. With N-Acetylglycine-pyrrolidine-(2S)-carbonitrile being the basic structure, modifications were introduced through a benzoylic residue at the N-terminus. In addition, some well-known inhibitors were synthesized for comparison to the new ones in enzymatic assay. To evaluate their inhibitory effect, the new inhibitors were tested in enzymatic assays using FAP and dipeptidyl peptidase IV, a prolyl peptidase from the same family in order to compare the results with regard to selectivity. None of the new compounds showed a KI-value in the nanomolar range, required for visualization of an enzyme expression using PET. In order to investigate a PET-Tracer, the best inhibitors against FAP had to be labeled with a positron emitter. The radioactive analogues of the inhibitors were obtained using isotopic exchange of the natural iodine-nuclide by iodine-124 (124I), resulting in 1-(2-[124I]Iodohippuric acid)-pyrrolidine-(2S)-carbonitrile und 1-(4-[124I]Iodohippuric acid)-pyrrolidine-(2S)-carbonitrile. 1-(2-[124I]Iodohippuric acid)-pyrrolidine-(2S)-carbonitrile was tested in vivo using microPET in rats with myocardial infarction. Very low uptake of the radiopharmaceutical was observed in the ischemic area of the rat´s heart. Locations of ischemic and surviving parts of the myocardium were confirmed using HE staining. To our knowledge, 1-(2-[124I]Iodohippuric acid)-pyrrolidine-(2S)-carbonitrile is the first FAP-affine tracer developed for PET investigation. However, its potential as tracer for the FAP-expression within the myocardial infarction in vivo using PET could not be proven in the present study. Therefore, developments based on the structure of 1-(2-[124I]Iodohippuric acid)-pyrrolidine-(2S)-carbonitrile are going on, with view to identify a PET-tracer suitable for in-vivo-investigation of FAP in healing processes and remodeling after myocardial infarction using PET.

Copper is a widely distributed transition metal in the earth's crust and has been adopted in a variety of biological systems. In many ways the biochemical usefulness of copper stems from its positive redox potential. This positive redox potential allows copper to assist in the movement of electrons. Copper ions can be found in natural systems as either CuI, CuII or CuIII in part due to this redox potential. While CuII -centered biochemistry has been studied for years, mechanistic details in certain CuII -centered redox reactions remain unresolved. This study presents two methodologies for studying natural systems with known CuII -centered redox capabilities in order to better elucidate the mechanistic intricacies of Copper ion chemistry. The first method explored involves the promiscuous enzyme Streptomyces griseus aminopeptidase (SgAP) which although known primarily as a peptidase has been shown to oxidize catechol under near physiological conditions in vitro when its native ZnII ions are replaced by CuII ions. Protein engineering techniques were utilized toward expression a functional recombinant enzyme in wild type and mutant forms. The goal was to utilize Site directed mutagenesis of residues in the active site to determine which residues are involved in both the hydrolysis and the oxidative activities of SgAP. The second methodology explored was the use of the N-terminus of Histatin-5, a naturally occurring peptide that is known to form complexes with CuII, as a model system to study CuII -centered oxidation chemistry. Metal-Peptide complexes are much more simplified model systems which use the same building blocks as proteins, but reduce the structure to the minimal functional unit necessary for activity. This in turn, simplifies the study of their catalytic chemistry as influences outside of the active region are greatly reduced. Furthermore, chemical synthesis of short peptides is easily performed and inexpensive in comparison to protein engineering, thus enabling further exploration, if deemed necessary, to be a feasible and economically viable possibility.

Antimalarial drug discovery remains a challenging endeavour as malaria parasites continue to develop resistance to drugs, including those which are currently the last line of defence against the disease. Plasmodium falciparum is the most virulent of the malaria parasites and it delivers its deadliest impact during the erythrocytic stages of the parasite’s life cycle; a stage characterised by elevated catabolism of haemoglobin and anabolism of parasite proteins. The present study investigates the use of nanotechnology in the form of metallic silver nanoparticles (AgNPs) against P. falciparum leucine aminopeptidase (PfLAP), a validated biomedical target involved in haemoglobin metabolism. AgNPs were also tested against the human homolog cytosolic Homo sapiens leucine aminopeptidase (HsLAP) to ascertain their selective abilities. PfLAP and HsLAP were successfully expressed in Escherichia coli BL21(DE3) cells. PfLAP showed optimal thermal stability at 25 °C and optimal pH stability at pH 8.0 with a Km of 42.7 mM towards leucine-p-nitroanilide (LpNA) and a Vmax of 59.9 μmol.ml⁻¹.min⁻¹. HsLAP was optimally stable at 37 °C and at pH 7.0 with a Km of 16.7 mM and a Vmax of 17.2 μmol.ml⁻¹.min⁻¹. Both enzymes exhibited optimal activity in the presence of 2 mM Mn²⁺. On interaction with polyvinylpyrrolidone (PVP) stabilised AgNPs, both enzymes were inhibited to differing extents with PfLAP losing three fold of its catalytic efficiency relative to HsLAP. These results show the ability of AgNPs to selectively inhibit PfLAP whilst having much lesser effects on its human homolog. With the use of available targeting techniques, the present study shows the potential use of nanotechnology based approaches as “silver bullets” that can target PfLAP without adversely affecting the host. However further research needs to be conducted to better understand the mechanisms of AgNP action, drug targeting and the health and safety issues associated with nanotechnology use.