The pathway representing methionine integration and excision is an increasingly important target in drug design. Methionine aminopeptidase (MetAP), a metalloprotease responsible for cleaving the N-terminal methionine from nascent peptides, has been the object of many studies aimed to produce potential anti-bacterial, anti-fungal and anti-angiogenic agents. Though clinical trials are underway for several of these compounds, like fumagillin and CKD-731, they are still flawed based on their relatively weak inhibition and their physiological side effects. Therefore, the search for novel and potent inhibitors continues. Previous work has utilized phosphinic and phosphonic acid derivatives of methionine in co-crystallization studies with Escherichia coli MetAP (eMetAP). The aim of the research presented in this work is to study and assay various phosphorus- and sulfur-containing compounds as inhibitors and substrates in an effort to learn more about the biochemical machinery underlying MetAP catalysis. As well, we outline a predictive molecular modeling approach to MetAP inhibitor design to assist in identifying lead candidates amongst a body of possible molecular inhibitors. Ultimately, we not only hope to have identified key functional properties of molecules potentially useful as MetAP inhibitors, but also to have contributed to the knowledge base of the mechanistic features involved in this enzyme’s catalysis.

The pathway representing methionine integration and excision is an increasingly important target in drug design. Methionine aminopeptidase (MetAP), a metalloprotease responsible for cleaving the N-terminal methionine from nascent peptides, has been the object of many studies aimed to produce potential anti-bacterial, anti-fungal and anti-angiogenic agents. Though clinical trials are underway for several of these compounds, like fumagillin and CKD-731, they are still flawed based on their relatively weak inhibition and their physiological side effects. Therefore, the search for novel and potent inhibitors continues. Previous work has utilized phosphinic and phosphonic acid derivatives of methionine in co-crystallization studies with Escherichia coli MetAP (eMetAP). The aim of the research presented in this work is to study and assay various phosphorus- and sulfur-containing compounds as inhibitors and substrates in an effort to learn more about the biochemical machinery underlying MetAP catalysis. As well, we outline a predictive molecular modeling approach to MetAP inhibitor design to assist in identifying lead candidates amongst a body of possible molecular inhibitors. Ultimately, we not only hope to have identified key functional properties of molecules potentially useful as MetAP inhibitors, but also to have contributed to the knowledge base of the mechanistic features involved in this enzyme’s catalysis.

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.