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Novel Computational Protein Design Algorithms with Applications to Cystic Fibrosis and HIVRoberts, Kyle Eugene January 2014 (has links)
<p>Proteins are essential components of cells and are crucial for catalyzing reactions, signaling, recognition, motility, recycling, and structural stability. This diversity of function suggests that nature is only scratching the surface of protein functional space. Protein function is determined by structure, which in turn is determined predominantly by amino acid sequence. Protein design aims to explore protein sequence and conformational space to design novel proteins with new or improved function. The vast number of possible protein sequences makes exploring the space a challenging problem. </p><p>Computational structure-based protein design (CSPD) allows for the rational design of proteins. Because of the large search space, CSPD methods must balance search accuracy and modeling simplifications. We have developed algorithms that allow for the accurate and efficient search of protein conformational space. Specifically, we focus on algorithms that maintain provability, account for protein flexibility, and use ensemble-based rankings. We present several novel algorithms for incorporating improved flexibility into CSPD with continuous rotamers. We applied these algorithms to two biomedically important design problems. We designed peptide inhibitors of the cystic fibrosis agonist CAL that were able to restore function of the vital cystic fibrosis protein CFTR. We also designed improved HIV antibodies and nanobodies to combat HIV infections.</p> / Dissertation
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Directed Evolution of Peptide Inhibitors of HIV-1 EntryQuinlan, Brian Donald 25 February 2014 (has links)
The conflict between HIV-1 and the host immune system plays out over a time-scale of months and years, and on a grander scale in the co-evolution of lentiviruses and the immune systems of their host species. Directed evolution of HIV-1 entry inhibitors using controlled randomization together with a display system offers a means of recapitulating one side of this conflict in vitro on an accelerated time-scale. To address limitations in existing display systems, we constructed a vector (pDQ1) integrating phage-display and mammalian-expression systems. This vector displays on phage when expressed in bacteria, and as an Fc-fusion when expressed in tissue culture, thus accelerating the iterative process of randomization, display, and characterization. We demonstrated the utility of this vector in the evolution of a CD4-mimetic peptide.
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Tunnels and Grooves : Structure-Function Studies in Two Disparate EnzymesEricsson, Daniel January 2009 (has links)
This thesis describes structural and binding studies in enzymes from two different organisms: ribonucleotide reductase from Mycobacterium tuberculosis (RNR) and lipase A from Candida antarctica (CalA). RNR is viable as a target for new drugs against the causative agent of tuberculosis. The biologically active form of RNR is a heterotetramer with an α2β2 substructure. Here we show that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. An N-terminal truncation, an alanine scan and a novel statistical molecular design approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied. A full-length acetylated heptapeptide was necessary for inhibition, and Trp5 and Phe7 were also essential. Exchanging the acetyl for the N-terminal Fmoc protective-group increased the binding potency ten-fold. Based on this, several truncated and N-protected peptides were evaluated in a competitive fluorescence polarization assay. The single-amino acid Fmoc-Trp inhibits the RNR holoenzyme formation with a dissociation constant of 12µM, making it an attractive candidate for further development of non-peptidic inhibitors Lipases are enzymes with major biotechnological applications. We report the x-ray structure of CalA, the first member of a novel family of lipases. The fold includes a well-defined lid as well as a classical α/β hydrolase domain. The structure is that of the closed/inactive state of the enzyme, but loop movements near Phe431 will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation.
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Rôle de la Caspase-2 au cours des processus neurodégénératifs associés au vieillissement. Conception rationnelle d’inhibiteurs sélectifs et évaluation sur des modèles biologique / Role of Caspase-2 in neurodegenerative processes associated with aging. Rational design of selective inhibitors and their evaluation on biological modelsDavid-Bosc, Elodie 26 November 2018 (has links)
La Caspase-2 (CASP-2) est singulière de par ses multiples rôles physiologiques et par son implication dans les processus neurodégénératifs aigus et chroniques. Dans ce contexte, des études récentes ont contribué à sa validation en tant que cible thérapeutique potentielle de la maladie d’Alzheimer. Par conséquent, le développement d’inhibiteurs spécifiques constituerait des outils pharmacologiques qui permettraient de mieux appréhender ses rôles dans la physiologie et pathologie du neurone. Les inhibiteurs de Caspases actuels sont majoritairement des séquences tétra ou pentapeptidiques qui reproduisent les motifs préférentiellement reconnus par ces enzymes. Dans le cadre de ce travail de thèse, trois stratégies d’identification d’inhibiteurs ont été suivies ; (i) une approche de conception rationnelle de peptides ciblant le site actif, (ii) conception in silico de peptides de l’interface de dimérisation, (iii) criblage aléatoire et rationnel de petites molécules organiques. Parmi ces stratégies, l’inhibition du site actif s’est révélée être la plus fructueuse. Nous avons ainsi pu démontrer que des variations du résidu Alanine en P2 sur un motif VDVAD permettaient d’améliorer les paramètres de sélectivité et d’efficacité. Sur ce constat une série de peptides « LJ » avec des mécanismes d’inhibition variés a été développée. Deux composés LJ2 et LJ3, ont démontré d’excellents paramètres d’inactivation et de sélectivité envers la CASP-2. Dans des réseaux de neurones reconstruits in vitro, LJ2 et LJ3 présentent un effet synaptoprotecteur. Ces travaux de thèse ouvrent donc le champ à de nouvelles perspectives sur le plan fonctionnel ainsi que sur le plan thérapeutique. / Caspase-2 (CASP-2) is unique among Caspases since its involved in a plethora of physiological processes and in severe and chronic neurodegenerative processes. In this context, recent studies have indicated that CASP-2 is a potential therapeutic target for Alzheimer’s disease. It is therefore necessary to develop specific inhibitors which would constitute pharmacological tools to better understand the role of this protease in the physiology and pathology of the neuron. The current Caspases inhibitors are mostly tetra or pentapeptide sequences that reproduce the patterns preferentially recognized by these enzymes. During this thesis project, we used three identification strategies ; (i) a rational design approach of peptides targeting the active site, (ii) in silico design of peptides of the dimerization interface, (iii) rational an random screening of small organic molecules. Among these strategies, inhibition of the active site has been shown to be the most productive one. We have been able to demonstrate that the variation of the Alanine residue in P2 on the pattern VDVAD increased efficiency and selectivity parameters. Based on this observation, a serie of peptides “LJ” with various inhibitory mechanisms has been developed. Two compounds LJ2 and LJ3, demonstrated excellent inactivation and selectivity parameters toward CASP-2. In neuronal networks reconstructed in vitro, LJ2 and LJ3 protect against synapse loss. This thesis project opens the field to new perspectives on the functional level as well as on the therapeutic plan.
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Humanized Mice as a Model to Study Human Viral Pathogenesis and Novel Antiviral DrugsSanchez Tumbaco, Freddy Mauricio 14 February 2012 (has links) (PDF)
Animal models have greatly contributed to the understanding of different aspects of human biology, as well as a variety of human-related pathogens and diseases. In order to study them, humanized mice susceptible to pathogens that replicate in human immune cells have been developed (e.g., humanized Rag2-/-γc-/- mice). These animals are engrafted with human hematopoietic stem cells (HSCs), resulting in the de novo development and maturation of the major functional components of the human adaptive immune system and the production of a variety of human cell types. Primary and secondary lymphoid organs in the mouse are populated with human cells, and animals have long term engraftment. These features make humanized mice an excellent in vivo model to study pathogenesis of human-specific viruses in the context of a human antiviral immune response. In addition, humanized mice have been shown to be useful preclinical models for the development and validation of antiviral therapeutics. In the present study, we aimed to successfully re-establish the humanized Rag2-/-γc-/- mouse model using cord blood-derived HSCs in our laboratory. We have shown that these mice sustain long term engraftment and systemic expansion of human cells, including the major targets of Kaposi's sarcoma Herpesvirus (KSHV) and Human immunodeficiency virus type 1 (HIV-1), in peripheral blood and different lymphoid organs. Further, we have begun to evaluate the susceptibility of the humanized Rag2-/-γc-/- mouse model to infection with KSHV. We demonstrate that human lymphocytes differentiated in reconstituted Rag2-/-γc-/- mice are permissive to KSHV infection ex vivo. This finding was corroborated by detection of KSHV mRNA expression in the spleen of a humanized mouse at 6 months post infection. In a different study, we tested the in vivo antiviral efficacy of a novel HIV-1 fusion inhibitor (PIE-12-trimer) in humanized Rag2-/-γc-/- mice. We have determined the half life of PIE-12-trimer in mouse plasma. Furthermore, the administration of PIE-12-trimer to HIV-1 infected humanized Rag2-/-γc-/- mice prevents depletion of CD4+ T cells in blood, thus it may be useful to prevent AIDS in human patients.
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Développement d’une sonde de photoaffinité pour la détection sensible de formes actives de Métalloprotéases Matricielles dans des systèmes biologiques complexes / Developpement of a photoaffinity probe for the sensitive detection of Matrix Metalloprotease active forms from complex biological systemsNury, Catherine 26 November 2012 (has links)
Le développement d’une nouvelle sonde dite « activity-based probe » pour réaliser la détection de formes actives de protéases appartenant à la famille des protéases à zinc de la matrice (MMP) a été réalisé dans ce travail, en partant d’un inhibiteur phosphinique puissant des MMP dans lequel a été introduit un groupement photoactivable de type diazérine. Ce composé se révèle un inhibiteur puissant de plusieurs MMP avec des affinités nanomolaires. Ce composé incubé avec différentes MMP est par ailleurs capables de modifier de façon covalente un grand nombre de MMP au niveau de leur site actif, avec des rendements de modification variant de plus de 50% à 11%, selon la nature des MMP. En ayant choisi comme moyen de détection la radioactivité, nous démontrons qu’avec cette nouvelle sonde qu’il est possible de détecter des formes actives de MMP avec des sensibilités de l’ordre de la femtomole dans des systèmes modèles de protéomes complexes. Appliquée à l’analyse de lavages broncho alvéolaires de souris traitées par voie pulmonaire avec des nanoparticules pour induire une réponse inflammatoire, cette nouvelle sonde permet de mettre en évidence la présence de formes actives du domaine catalytique de la MMP-12, une métalloprotéase à zinc exprimée par les macrophages, mais pas dans les animaux contrôles. En revanche l’analyse de carotides humaines de patients souffrant d’athérosclérose ne nous pas conduit avec cette sonde à la détection de formes actives de MMP. Malgré ce résultat, il est à noter que la détection de forme active de MMP dans un fluide pathologique est une première dans ce domaine. Cette sonde étant validée pour sa capacité à détecter des formes actives de MMP, elle permettra dans l’avenir de tester d’autres fluides pathologiques d’origine humaine ou bien des extraits de tissu comme des tumeurs pour lesquels les MMP pourraient être des marqueurs de ces pathologies. / A new activity-based probe able to covalently modify the active site of proteases belonging to the matrix metalloprotease family (MMPs) has been developed in this thesis project. The probe was shown to behave as potent inhibitor of several MMPs, with nanomolar Ki values. This probe was also able to modify specifically only the free active site of MMPs, with particular high yields of cross-linking varying from 50 % to 11 %, depending of the MMPs tested. Using radioactivity as means of detection, this probe was able to detect active form of MMPs with a threshold of 1 femtomole. Applied to the study of bronchoalvelolar fluids (BAL) from mice exposed to nanoparticles by a lung aspiration protocol, this probe revealed the presence of the catalytic domain of MMP-12 under its active form, but not in control animals. When used to detect active form of MMPs from extracts obtained from human arteries of patient suffering from atherosclerosis, the probe was not able to detect such MMP active forms. Despite this negative result, the detection of active form of MMP in pathological fluid like BAL has never been reported before this work. Having validated this novel MMP activity-based probe, it will be possible to use it now for detecting MMPs from other pathological fluids or tissues extracts in which MMPs can be good markers of the pathology.
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