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Structural determination of triclosan derivatives as inhibitors of Plasmodium falciparum enoyl reductase (PfENR)Lucumi Moreno, Edinson 25 April 2007 (has links)
Malaria is a disease that causes more than 1 million deaths per year world wide
and more than 400 million clinical cases. Due to the acquired resistance of Plasmodium
falciparum to the drugs used to control the infection, searching for new anti-malaria
drugs is necessary in modern days. Recent studies have shown that the parasite
synthesizes fatty acids using a fatty acid synthase type II (FAS-II) instead of a type-I fatty
acid synthase (FAS-I) that is present in other eukaryotes. Plasmodium falciparum enoyl
reductase (PfENR) is responsible for the last step of fatty acid biosynthesis in the
parasite. This enzyme is located within the apicoplast, a plastid-like organelle that is
responsible for several important metabolic pathways, including fatty acid biosynthesis. It
is known that triclosan is an inhibitor of ENR in bacteria and we and others have shown
that it is also effective against ENR in apicomplexan organisms such as P. falciparum.
However triclosan cannot be used to treat malaria in humans because it has metabolic
liability (glucoronidation) which limits its inhibitory potency. We have used X-ray crystallography and a Structural Activity Relationship (SAR) strategy to design and cocrystallize
a tertiary complex of PfENR with NAD+ and triclosan derivatives to improve
their properties as drugs to treat malaria. More than five hundred triclosan derivatives
were synthesized, and their in vitro and in vivo inhibitory activity evaluated. Furthermore,
structural studies were made of their affinity to interact with residues in PfENR active
sites, as well as with the cofactor NAD+. A total of six PfENR-NAD+-triclosan
analog/complexes structures were determined. Analogs which had replacements of
chloride groups at position 5 of ring A and 4' of ring B were determined, allowing the
structural analysis of the binding of these triclosan analogs to PfENR. In addition, the
urea derivatives (modification at position 1) as well as phenylsulphonamides
(modification at position 2') have shown to be more potent inhibitors than triclosan in the
in vivo assay. The analysis of the inhibitory properties and the structure of these analogs
bound to PfENR will provide novel compounds in the search for new anti-malarial drugs.
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Étude structurale et biochimique d’un facteur d’échange atypique d’Arf / Structural and Biochemical studies of an atypical ArfGEFAizel, Kaheima 24 September 2012 (has links)
Les petites GTPases de la famille Arf, régulateurs majeurs du trafic membranaire, sont activé par plusieurs familles de facteurs d’échange nucléotidiques (ArfGEFs). Les ArfGEFs jouent un rôle essentiel dans l’intégration des signaux de régulation qui conduisent à l’activation d’Arf au niveau de compartiments cellulaires spécifiques, cependant les mécanismes par lesquels ils ciblent les Arfs activés aux membranes spécifiques et leur coordination avec l’échange de nucléotide reste peu comprise. Nous utilisons ici la cristallographie et la reconstitution des activités ArfGEF sur des membranes artificielles pour analyser ces mécanismes pour un ArfGEF humain atypique, impliqués dans l’endocytose de récepteurs et associé à l’invasion tumorale dans de nombreuses cellules cancéreuses. Les membres de cette famille ont été décrits comme des GEFs spécifique d’Arf6, et comporte un domaine de type PH après leur domaine Sec7. Dans la deuxième partie de ma thèse, nous voulions savoir comment les isoformes Arf1 et Arf6 achevaient leurs fonctions dans la cellule. Arf1 et Arf6 sont très similaires: elles possèdent plus de 60% d’identité de séquence, et des études structurales ont montré que la surface qu’ils utilisent pour interagir avec leurs régulateurs et effecteurs est essentiellement identique en séquence et en structure. Cependant, elles ont des fonctions différentes dans la cellule et des propriétés différentes in vitro, pour lesquelles aucune donnée structurale n’a donné d’explications. Nous utilisons ici la cristallographie, le SAXS et la RMN pour comprendre la différence entre ces deux isoformes. / Small GTPases of the Arf family, which are pivotal regulators of membrane traffic in eukaryotes, are activated by several families of guanine nucleotide exchange factors (ArfGEFs). ArfGEfs play a key role in processing upstream regulatory signals that lead to Arf activation onto specific subcellular compartments, yet the mechanisms by which they target activated Arfs to specific membranes and their coordination with nucleotide exchange remain poorly understood. Here we used X-ray crystallography and reconstitution of ArfGEF activities on artificial membranes to analyze these mechanisms for an atypical human ArfGEF, involved in receptor endocytosis and associated with tumour invasion in various cancer cells. Members of this family have been described as Arf6-specific GEFs, and carry a PH-like domain downstream their Sec7 domain. In a second part of the work we wanted to know how the isoforms Arf1 and Arf6 achieve exquisitely specific functions in cells. Arf1 and Arf6 are highly similar: they have over 60% sequence identity, and structural studies have shown that the surfaces they use to interact with regulators and effectors are essentially identical in sequence and structure. Yet, they have non-overlapping functions in cells. Arf1 is a major regulator of most aspects of vesicular traffic, while Arf6 is restricted to the plasma membrane where it acts at the crossroads of trafficking and cytoskeleton functions (D'Souza-Schorey and Chavrier 2006). Consistent with their cellular specificities, Arf1 and Arf6 also have distinctive biochemical properties in vitro, for which no straightforward structural explanation has been put forward. Here we used X-ray crystallography, synchrotron SAXS experiments and NMR to assess the difference between these two isoforms.
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