No description available.

No description available.

Synthesis of peptides anchored to DNA by intercalating chromophores can incorporate the design principle of the naturally occurring peptide based antibiotics. This work is concerned with the synthesis of DNA anchored cysteinyl peptides designed to be potentially nucleotide sequence specific with possible affinity for the AP-l transcription site. Previous work has shown that anthraquinones and anthrapyrazoles (APZs) substituted with cationic side groups are excellent DNA intercalating agents. In this work a series of APZ analogues has been synthesised which are coupled onto the amino terminus of varying peptide sequences. Three derivatives of APZs were prepared namely 2-, 2,5- and 2,7-substituted. Eight short polypeptides (see below), all varying slightly in sequence but all containing the KCR motif (with one exception where a Cys was replaced with Ser) were combined with the APZ chromophore to give a series of intercalator-peptide molecules. Peptides were synthesised using the Fmoc strategy on a solid phase peptide synthesizer (SPPS). The peptides were then isolated by reversed-phase HPLC using a water: acetonitrile gradient. Characterisation of the peptides was carried out by matrix assisted laser desorption ionisation (MALDI) mass spectrometry and two dimensional nmr (i.e. COSY and NOESy). Anthraquinone linked peptide ligands were also synthesised using similar synthetic routes, and tested for their activity. Coupling of the two components was achieved via activation of the carboxylic acid group using PyBOP or via formation of a reactive aziridinium ion. All intercalator-peptides prepared were examined for their DNA binding properties. The methods included the effect of intercalator-peptides on the thermal denaturation of DNA and the competitive displacement of ethidium by fluorimetry. It was shown that the APZ binds to DNA by intercalation. Peptides prepared were: H2N-A-K-C-R-A-C02H; H2N-A-K-C-R-A-CONH2; H2N-A-K-S-R-A-CONH2; H2N-A-K-C-R-N-A-CONH2; H2N-A-K-C-R-K-A-CONH2; H2N-A-K-C-R-N-R-A-CONH2; H2N-A-K-C-R-K-R-ACONH2; H2N-A-A-K-C-R-A-A-CONH2. The biological activities of the intercalatorpeptides were then investigated using an electrophoretic mobility shift assay (EMSA), making use of cell nuclear extracts rich in AP-l and also c-Jun homodimer recombinant proteins. It was shown that most of the intercalatorpeptides were capable of inhibiting AP-l (fos/jun) heterodimer protein from binding to the AP-l DNA consensus sequence. Importantly, the intercalatorpeptides showed superior activity over the intercalator or peptide moieties alone. The order of binding affinity was intercalator-peptide> intercalator» peptide.

This thesis describes the synthesis of a novel multimeric peptide ligand targeted to the human melanocortin 4 receptor. The synthesis of the peptide was attempted both by solid phase peptide synthesis and by solution phase peptide synthesis, leading to the conclusion that the necessary C- and N- terminal substituents were much easier to install via the solution phase route. The bifunctional peptide was purified and then multimerized in both protected and active amino acid forms using the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. The multimers were characterized using MS and UV-Vis spectroscopy. It was found that a large portion of the monomer cyclized under CuAAC conditions, though sufficient multimerization took place to form up to nonamers, as determined by mass spectrometry.

No description available.

No description available.

Immune recruiters are small molecule immunotherapeutics which redirect endogenous components of the immune system to target cells to elicit anti-cancer responses. Current immune recruiters made in the Rullo Lab are heterobispecific molecules which bind receptors on cancer cells and ligand-specific antibodies. Upon antibody binding, a proximity-induced covalent reaction with nearby nucleophilic residues installs a targeting ligand onto the protein. The resultant antibody conjugate then facilitates cancer killing through immune cell recruitment. Covalency circumvents limited binding affinity of the ligand•antibody complex, however antibody•immune receptor affinity remains an issue. This thesis presents an alternative immune recruiting strategy through direct engagement of effector immune cells; monocyte covalent immune recruiters (mCIRs). mCIRs utilize a monocyte specific peptide (cp33) to bind CD64, an activating receptor on monocytes. By incorporating a sulfonyl fluoride electrophile onto the N-terminus of cp33, selective covalent labelling of CD64 was achieved within 24 h. Furthermore, mCIRs demonstrated enhanced monocyte function relative to antibody recruiting platforms. However, these constructs have demonstrated that the order of addition to the target receptor then to CD64 is critical for bridging the two species. As a result, the effect of covalency on complex simplification and monocyte function has yet to be determined. Despite this, mCIRs represent a covalent immune recruitment strategy with the potential to address shortcomings of antibody-based therapeutics. / Thesis / Master of Science (MSc)

During infections, bacterial microorganisms initiate profound interactions with mammalian host cells. Usually defense mechanisms of the host destroy intruding bacteria in rapid manner. However, many bacterial pathogens have evolved in a way to avoid these mechanisms. By use of effector molecules, which can be small organic molecules or proteins with enzymatic activity, the host is manipulated on a molecular level. Effectors mediating post-translational modifications (PTMs) are employed by many pathogens to influence the biological activity of host proteins. In the presented thesis, two related PTMs are investigated in detail: Adenylylation, the covalent transfer of an adenosine monophosphate group from adenosine triphosphate onto proteins, and phosphocholination, the covalent transfer of a phosphocholine moiety onto proteins. Over the past years, enzymes mediating these modifications have been discovered in several pathogens, especially as a mechanism to influence the signaling of eukaryotic cells by adenylylating or phosphocholinating small GTPases. However, the development of reliable methods for the isolation and identification of adenylylated and phosphocholinated proteins remains a vehement challenge in this field of research. This thesis presents general procedures for the synthesis of peptides carrying adenylylated or phosphocholinated tyrosine, threonine and serine residues. From the resulting peptides, mono-selective polyclonal antibodies against adenylylated tyrosine and threonine have been raised. The antibodies were used as tools for proteomic research to isolate unknown substrates of adenylyl transferases from eukaryotic cells. Mass spectrometric fragmentation techniques have been investigated to ease the identification of adenylylated proteins. Furthermore, this work presents a new strategy to identify adenylylated proteins. Additionally, small effector molecules are involved in the regulation of infection mechanisms. In this work, the small molecule LAI-1 (Legionella autoinducer 1) from the pathogen Legionella pneumophila, the causative agent of the Legionnaire’s disease, was synthesised together with its amino-derivatives. LAI-1 showed are a clear pharmacological effect on the regulation of the life cycle of L. pneumophila, initiating transmissive traits like motility and virulence. Furthermore, LAI-1 was shown to have an effect on eukaryotic cells as well. Directed motility of the eukaryotic cells was significantly reduced and the cytoskeletal architecture was reorganised, probably by interfering with the small GTPase Cdc42.

Les maladies auto-immunes sont des maladies chroniques et hétérogènes caractérisées par des réactions du système immunitaire acquis contre les propres tissus sains de l'organisme. Ces maladies affectent presque 5% de la population mondiale et en particulier les jeunes adultes. La complexité de leur spectre est énorme et même si leur étiologie est encore incertaine, il a été démontré que des facteurs génétiques et environnementaux sont impliqués dans le déclenchement du mécanisme pathologique. Cependant, il est nécessaire d'utiliser des outils diagnostiques et / ou pronostiques fiables pour le diagnostic précoce avant que des dommages cellulaires irréversibles ne se produisent et pour surveiller la progression de la maladie.De nombreuses études ont mis en évidence la présence de différents auto-anticorps dans le sérum de patients atteints de maladies auto-immunes. Les auto-anticorps qui sont spécifiques d’une maladie peuvent être utilisés en tant que biomarqueurs (BM) pour son diagnostic alors que les auto-anticorps qui diffèrent en fonction de l'état de la maladie peuvent être utilisés dans le suivi des patients. En fait, dans le cas de l'auto-immunité, un BM facilement détectable et fiable peut être représenté par le titre d'un auto-anticorps spécifique.Dans ce contexte, nous nous intéressons à deux maladies différentes, la sclérose en plaques (SEP) et la gammapathie monoclonale, en utilisant l'approche chimique inverse via le criblage de librairies de peptides par des sérums de patients.En particulier, l'importance des anticorps anti-myéline, et surtout, des anticorps anti-MOG (myéline oligodendrocyte glycoprotéine) est toujours l’objet de débats, soulignant la question très controversée d'un rôle pathogène putatif d'anticorps anti-MOG dans la SEP. Dans cette thèse, nous avons étudié le rôle de MOG comme auto-Ag putatif dans la SEP en utilisant le modèle expérimental d’encéphalomyélite auto-immune (EAE). Ainsi, afin d'évaluer la présence d'un mécanisme d’« epitope spreading » des cellules B, à savoir l'apparition d'une réponse dirigée vers des épitopes distincts de l'agent pathogène induisant la réponse immunitaire, nous avons synthétisé et testé en tant que sondes antigéniques cinq peptides synthétiques qui couvrent la séquence 1-117 de MOG.La seconde étude a porté sur la sélection d'un peptide mimant l'épitope minimal reconnu par l'anticorps monoclonal commercial anti- natural killer cell-1 humain (anti-HNK-1) en utilisant la résonance plasmonique de surface (SPR). L’épitope HNK-1 est considéré comme le déterminant antigénique de la glycoprotéine associée à la myéline (MAG), un composant quantitativement mineur des gaines de myéline. On observe que les patients atteints de troubles neurologiques auto-immuns, tels que la gammapathie monoclonale à IgM et la polyneuropathie démyélinisante, développent souvent des anticorps anti-MAG ciblant spécifiquement l’épitope HNK-1. Par conséquent, l'identification et la caractérisation de ces anticorps est pertinente. Le peptide choisi suite à notre étude pourrait ensuite être utilisé chez des patients atteints de troubles neurologiques pour le développement d'un outil de diagnostic fiable ou de surveillance de l'activité de la maladie par l'identification d'anticorps anti-HNK-1 dans le sérum des patients. / Autoimmune diseases (ADs) refer to chronic and heterogeneous diseases with acquired immune system’s reactions against the body’s own healthy tissues. ADs affect more than 5% of the population worldwide and especially young adults. The complexity of their spectrum is enormous and even if their etiology is still unclear, it was demonstrated that both genetic and environmental factors are involved in triggering the pathological mechanism. Hence, a reliable diagnostic and/or prognostic tool for an early diagnosis of ADs before irreversible cellular damage occurs and for monitoring their progression is demanded.Numerous studies have revealed the presence of different autoantibodies (auto-Abs) in sera of patients suffering from ADs. Autoantibodies that are specific for a disease can be used as biomarkers (BMs) for its diagnosis while autoantibodies that differ depending on the disease state can be used in the follow up of the patients. Actually, in the case of autoimmunity, an easily detectable and reliable BM may be represented by the titer of a specific auto-Ab.In this context, we aimed to identify target(s) of the response for two different ADs, multiple sclerosis (MS) and monoclonal gammopathy, using the chemical reverse approach, which involves the screening of focused antigen (Ag) libraries with patients’ serum.In particular, the significance of anti-myelin antibodies, and especially, anti- Myelin Oligodendrocyte Glycoprotein (anti-MOG) antibodies is still matter of debate, underscoring the highly controversial issue of a putative pathogenetic role of anti-MOG antibodies in MS. In this thesis we investigated the role of MOG as putative auto-Ag in MS using the experimental autoimmune encephalomyelitis (EAE) model. Moreover, in order to assess the presence of a B-cell epitope spreading mechanism, i.e. the occurrence of a response directed toward epitopes distinct from the disease-inducing agent, we synthesized and tested as antigenic probes also five synthetic peptides covering the 1-117 sequence of MOG.The second issue focused on the selection of a peptide mimicking the minimal epitope recognized by the commercial available monoclonal antibody anti-human natural killer cell-1 (anti-HNK-1) using Surface Plasmon Resonance (SPR) technique. HNK-1 epitope, is considered as the antigenic determinant of myelin-associated glycoprotein (MAG), a quantitatively minor component of myelin sheaths. It is observed that patients affected by autoimmune neurological disorders, such as IgM monoclonal gammopathy and demyelinating polyneuropathy, often develop anti-MAG antibodies specifically targeting the HNK-1 epitope. Accordingly, identification and characterisation of these antibodies is relevant. The selected peptide could be subsequently used in earlier stage patients for the development of a novel and reliable diagnostic tool for anti-HNK-1 antibody identification in sera of patients affected by autoimmune neurological disorders monitoring disease activity.

Le but de ma thèse a été de réaliser de nouveaux composés hybrides constitués de nanotubes de carbone (ou fullerènes) et de nanotubes de peptide. L'approche choisie a consisté à greffer sur des nanotubes de carbone, ou dans un premier temps sur des fullerènes, via des bras espaceurs, des cyclopeptides « de type Ghadiri» conduisant aux nanotubes de peptide. Pour se faire, la synthèse de bras espaceurs a été nécessaire ainsi que leur fixation sur les nanotubes de carbones ou fullerènes. Les cyclopeptides sont constitués d'un nombre pair d'acides aminés (8) alternés D et L s'auto-assemblant en feuillets ß antiparallèles pour conduire à des nanotubes. Des essais de solubilisation du cyclopeptide diacide révèlent l'impossibilité d'utiliser ces composés en synthèse organique, dans la mesure ou ils ne sont pas solubles dans les solvants organiques classiques. Il a fallut ainsi revoir la synthèse en incluant soit le fullerène en cours de synthèse peptidique soit en modifiant les conditions pour permettre une solubilisation du peptide. Les composés synthétisés ont été caractérisés par différentes méthodes notamment en TEM, FT-IR, ATR, microscopie optique, diffusion de la lumière. De nouvelles applications aux peptides de Ghadiri ont été cherchées en imagerie (par encapsulation du Xénon hyperpolarisé dans les cavités). Il a été mis en évidence de nouvelles organisations cristallines des peptides possibles dans des conditions contrôlées à l'aide de contre ions tels que les éléments de la première colonne du tableau périodique (Li, Na, K, Rb, ou Cs). Ces organisations, différentes en fonction du contre ion choisi, ont un caractère fractal remarquable, une organisation cristalline régulière et on observe un réseau de liaisons hydrogène inattendu dans les conditions utilisés.