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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.

La leishmaniose est une maladie tropicale négligée à transmission vectorielle qui est endémique dans 98 pays dont les plus pauvres. Vingt espèces de Leishmania sont capables d’établir une infection intracellulaire au sein des macrophages humains, provoquant différentes manifestations cliniques. Le développement d'un vaccin contre les leishmanioses est étayé par des preuves d'immunité naturelle contre l'infection, induite par une réponse à médiation cellulaire de type Th1 dominante associée à la production d'IFN-γ, d'IL-2 et de TNF-α par des cellules T polyfonctionnelles TCD4+ et TCD8+, conduisant à l'activation classique des macrophages entrainant la destruction des parasites. Induire une protection robuste et durable et déterminer les épitopes immunodominants responsables de la protection naturelle représente un véritable défi.Les protéines sécrétées sont des facteurs de virulence jouant un rôle important dans le cycle de vie des leishmanies et sont capables d’induire une protection durable chez le chien, un bon modèle pour l’infection humaine. Notre objectif est de développer, à partir du sécrétome de Leishmania, un vaccin de seconde génération reproductible et facile à produire à bas prix dans les zones d’endémie, avec des rendements de production rendant possible son utilisation à grande échelle.Les sécrétomes des six espèces les plus pathogènes de leishmanie (plus L. tarentolae) ont été analysés et comparées par spectrométrie de masse. Les antigènes candidats ont été recherchés dans l'ensemble des données disponibles (analyses protéomiques, littérature…). 52 antigènes candidats vaccin ont ainsi été sélectionnés, dont 28 avaient déjà été décrits et 24 sont nouveaux et découverts grâce à une approche de vaccinologie réverse.Une analyse de la prédiction de liaison des épitopes in silico HLA-I et –II a été réalisée sur tous les antigènes candidats vaccin, prenant ainsi en compte le polymorphisme HLA de la population mondiale. Pour sélectionner les meilleurs épitopes parmi des milliers d’épitopes potentiels, un script R automatisé a été développé en interne, selon des critères rationnels stricts. Ainsi, 50 épitopes de classe I et 24 épitopes de classe II ont été sélectionnés et synthétisés sous forme de peptides individuels. Des essais de toxicité in vitro ont montré l’absence de toxicité cellulaire de ces peptides.Les individus guéris par chimiothérapie généralement développent des réponses immunitaires protectrices à Leishmania. Des tests de stimulation des PBMC ont donc été réalisés avec des échantillons biologiques provenant de donneurs guéris de Tunisie et la production d'IFN-γ a été évaluée par ELISpot. De plus, il était important d'inclure dans l'étape de validation expérimentale des peptides des échantillons provenant d’individus naïfs, population cible à vacciner avec un vaccin prophylactique. Les résultats montrent que des peptides spécifiques de Leishmania induisent avec succès la production d'IFN-γ par les PBMC totaux provenant de donneurs guéris et par les lymphocytes T spécifiques amplifiés à partir du répertoire naïf.Globalement, la validation expérimentale des peptides réalisée exclusivement sur des échantillons humains nous fournira une base préclinique très solide pour développer un vaccin efficace capable de protéger les populations touchées par ces maladies. Elle constituera un moyen sûr et rentable de mieux sélectionner les candidats retenus pour le vaccin et d'éliminer ceux qui présentent un risque d'échec élevé au tout début du processus de développement du vaccin.Grâce à la combinaison de l'analyse protéomique et d'outils in silico, des candidats peptidiques prometteurs ont été rapidement identifiés pour le développement d'un vaccin. Le « pipeline » de développement préclinique du vaccin proposé fournit une sélection rapide de peptides immunogènes, offrant une approche puissante pour accélérer le déploiement d'un vaccin pan-spécifique efficace contre les leishmanioses. / Leishmaniasis is a vector-borne neglected tropical disease endemic to 98 countries worldwide. Twenty Leishmania species are capable of establishing intracellular infection within human macrophages, causing different clinical presentations. Vaccine development against leishmaniases is supported by evidence of natural immunity against infection, mediated by a dominant cellular Th1 response and production of IFN-γ, IL-2 and TNF-α by polyfunctional TCD4+ and TCD8+ cells, ultimately leading to macrophage activation and parasite killing.Excreted-secreted proteins are important virulence factors present throughout Leishmania life stages and are able to induce durable protection in dogs, a good model for human infection. We aim to develop a second generation vaccine from the Leishmania secretome, with the potential for large scale dissemination in a cost-effective, reproducible approach.The secretome of six main pathogenic species (plus L. tarentolae) was analysed by Mass-Spectrometry and conserved candidate antigens were searched in the complete dataset. A total of 52 vaccine antigen candidates were selected, including 28 previously described vaccine candidates, and an additional 24 new candidates discovered through a reverse vaccinology approach.In silico HLA-I and –II epitope binding prediction analysis was performed on all selected vaccine antigens, with world coverage regarding HLA restriction. To select the best epitopes, an automated R script was developed in-house, according to strict rational criteria. From thousands of potential epitopes, the automated script, in combination with optimal IC50, homology to host and solubility properties, allowed us to select 50 class I and 24 class II epitopes, synthesized as individual peptides. In vitro toxicity assays showed these selected peptides are non-toxic to cells.The peptides’ immunogenicity was evaluated using immunoscreening assays with immune cells from human donors, allowing for the validation of in silico epitope predictions and selection, and the assessment of the peptide’s immunogenicity and prophylactic potential. Healed individuals, which had active infection and received treatment, possess Leishmania-specific memory responses and are resistant to reinfection, being considered the gold standard of protective immunity. On the other hand, the naive population is extremely important to include in the experimental validation step since it is the target population to vaccinate with a prophylactic vaccine. Importantly, a minimum specific T-cell precursor frequency is needed to induce long-lasting memory protective responses. Furthermore, there is also a positive correlation between immunodominant epitopes and a high frequency of specific T-cell precursors. Peptides able to induce Th1 and/or cytotoxic immune responses in both background are promising candidates for a vaccine formulation. Altogether,experimental validation exclusively in human samples will provide us a very strong base for a vaccine formulation and allow to accelerate translation to the field.Results show Leishmania-specific peptides successfully induce IFN-γ production by total PBMC from healed donors, and by specific T cells amplified from the naïve repertoire. Preliminary evidence exists for peptides which are immunogenic in both immune backgrounds (eight HLA-class I 9-mer peptides and five class II 15-mer peptides) which are, for now, the most promising candidates to advance for the multi-epitope peptide design.Through the combination of proteomic analysis and in silico tools, promising peptide candidates were swiftly identified and the secretome was further established as an optimal starting point for vaccine development. The proposed vaccine preclinical development pipeline delivered a rapid selection of immunogenic peptides, providing a powerful approach to fast-track the deployment of an effective pan-specific vaccine against Leishmaniases.

Overexpression of IL-12 and IL-23 has been implicated in the pathogenesis of Crohn’s disease. Targeting these cytokines with monoclonal antibodies has emerged as an effective therapy, but one with adverse reactions. In this study, we sought to develop peptide-based virus-like particle vaccines specific to p40 unit (shared by IL-12 and IL-23) or IL-12 (p35) or IL-23 (p19) and evaluate the effects of the vaccine in 2,4,6-trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulfate (DSS)-induced acute and chronic murine colitis. Three vaccines against p40 induced high-titered and long-lasting antibodies to IL-12, IL-23 and p40 without the use of adjuvants. Vaccine-induced antibodies could block IL-12- and IL-23-induced biological functions in vitro dose-dependently. One of the three p40 vaccines was selected for further evaluation in acute and chronic colitis. Administration of the vaccine before or after the commencement of TNBS or DSS delivery, significantly improved body weight loss and decreased inflammatory scores, collagen deposition, and the expression of p40, IL-12, IL-23, IL-17 and TNF in colon tissues, compared with mice receiving carrier protein (HBcAg) or saline. Moreover, in mesenteric lymph nodes, vaccinated mice exhibited a trend to lower percentages of Th1 cells in acute colitis and of Th17 cells in chronic colitis compared to carrier and saline controls. Vaccinated mice also had higher ratios of Treg/Th1 and Treg/Th17 and higher percentages of apoptosis in Th1 and Th17 cells than controls. Vaccine treatment decreased the infiltration of CD11c+ cells into the gut, but promoted the production of IL-10 from these cells. Safety evaluation indicated that vaccine immunization did not increase the susceptibility to the infection of chlamydia muridarum. Two vaccines specific to IL-12 (against p35) and one vaccine to IL-23 (against p19) were also developed. They induced specific antibodies against IL-12 and IL-23, respectively. IL-23p19 vaccine immunization, not IL-12p23 vaccine, ameliorated TNBS-induced chronic colitis. In summary, IL-12/IL-23p40 vaccine treatment ameliorated murine colitis through rebalancing Th1/Th17/Treg responses, promoting Th1 and Th17 apoptosis, and promoting IL-10 production, and did not increase the severity of chlamydia muridarum infection. This vaccine strategy may provide a novel long-term treatment for Crohn’s disease.

Overexpression of IL-12 and IL-23 has been implicated in the pathogenesis of Crohn’s disease. Targeting these cytokines with monoclonal antibodies has emerged as an effective therapy, but one with adverse reactions. In this study, we sought to develop peptide-based virus-like particle vaccines specific to p40 unit (shared by IL-12 and IL-23) or IL-12 (p35) or IL-23 (p19) and evaluate the effects of the vaccine in 2,4,6-trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulfate (DSS)-induced acute and chronic murine colitis. Three vaccines against p40 induced high-titered and long-lasting antibodies to IL-12, IL-23 and p40 without the use of adjuvants. Vaccine-induced antibodies could block IL-12- and IL-23-induced biological functions in vitro dose-dependently. One of the three p40 vaccines was selected for further evaluation in acute and chronic colitis. Administration of the vaccine before or after the commencement of TNBS or DSS delivery, significantly improved body weight loss and decreased inflammatory scores, collagen deposition, and the expression of p40, IL-12, IL-23, IL-17 and TNF in colon tissues, compared with mice receiving carrier protein (HBcAg) or saline. Moreover, in mesenteric lymph nodes, vaccinated mice exhibited a trend to lower percentages of Th1 cells in acute colitis and of Th17 cells in chronic colitis compared to carrier and saline controls. Vaccinated mice also had higher ratios of Treg/Th1 and Treg/Th17 and higher percentages of apoptosis in Th1 and Th17 cells than controls. Vaccine treatment decreased the infiltration of CD11c+ cells into the gut, but promoted the production of IL-10 from these cells. Safety evaluation indicated that vaccine immunization did not increase the susceptibility to the infection of chlamydia muridarum. Two vaccines specific to IL-12 (against p35) and one vaccine to IL-23 (against p19) were also developed. They induced specific antibodies against IL-12 and IL-23, respectively. IL-23p19 vaccine immunization, not IL-12p23 vaccine, ameliorated TNBS-induced chronic colitis. In summary, IL-12/IL-23p40 vaccine treatment ameliorated murine colitis through rebalancing Th1/Th17/Treg responses, promoting Th1 and Th17 apoptosis, and promoting IL-10 production, and did not increase the severity of chlamydia muridarum infection. This vaccine strategy may provide a novel long-term treatment for Crohn’s disease.

No description available.

No description available.

Based on the concept of covalent capture of supramolecular assemblies, the idea of mechanical encapsulation is exploited for the protection and delivery of peptidebased molecules. This thesis aims to establish a general method for the encapsulation of peptides within a rotaxane structure, as well as studying their mode of release under specific stimuli. The synthesis of such structures, relying on the elongation of short peptido[2]rotaxanes, is applied to the design of rotaxane peptide carriers whose function is to protect against biological degradation and release peptides under a biological stimulus. These molecules are composed of a rotaxane-encapsulated peptide bearing a biodegradable stopper, the enzyme-specific cleavage of which triggers peptide release, via ‘dethreading’. The synthesis and in vitro assessment of rotaxane carriers as agents for anti-cancer therapy will be described in detail. The future challenges and potential applications of the proposed systems will be addressed.

Matrix metalloproteinases (MMPs) play a significant role in degrading the extracellular matrix in cancer development and metastasis. Overexpression of matrix metalloproteinases in tumour tissues relative to normal tissues has been exploited as a target for peptide-based therapeutics, to improve therapeutic index of currently used agents. The stability of MMP-activated prodrugs in normal tissue or organs is a significant challenge for their success in the clinic. In an in vitro study, the stability of twenty six prodrugs was studied in mouse liver, kidney, lung and tumour homogenates using HPLC and LC/MS. Selected agents were studied in vivo. Each prodrug has a characteristic amino acid sequence with dominant FITC N-terminal end cap. All prodrugs were conjugated to a colchicine derivative (ICT 2552) which is a vascular disrupting agent causing tumour vasculature shutdown and consequently, tumour necrosis. ICT 3146, ICT 3019, ICT 3120 and ICT 3115 prodrugs showed significant stability in normal tissues and considerable activation in certain tumour tissues compared to the lead compound ICT 2588. Also, the selectivity of promising prodrugs to the MMP family was confirmed by using leupeptin (serine, cysteine and threonine protease inhibitor), pepstatin A (aspartate protease inhibitor), phosphoramidon (nepralysin inhibitor), ilomastat (metalloproteinase inhibitor) and BML-P115 (matrix metalloproteinase inhibitor). Moreover, members of the MMP family responsible for cleaving the selected prodrugs were identified using recombinant MMP enzymes. Furthermore, a LC/MS-MS method was developed to specifically detect and quantify MMP-16 protein expression in H460 tumour. MMP- 16 was responsible for the cleavage of ICT 3146 and ICT 3115. Therefore, MMPactivated prodrugs could be a useful therapeutic approach to avoid off-site toxicities of currently used anti-tumour agents.

Matrix metalloproteinases (MMPs) play a significant role in degrading the extra- cellular matrix in cancer development and metastasis. Overexpression of matrix metalloproteinases in tumour tissues relative to normal tissues has been exploited as a target for peptide-based therapeutics, to improve therapeutic index of currently used agents. The stability of MMP-activated prodrugs in normal tissue or organs is a significant challenge for their success in the clinic. In an in vitro study, the stability of twenty six prodrugs was studied in mouse liver, kidney, lung and tumour homogenates using HPLC and LC/MS. Selected agents were studied in vivo. Each prodrug has a characteristic amino acid sequence with dominant FITC N-terminal end cap. All prodrugs were conjugated to a colchicine derivative (ICT 2552) which is a vascular disrupting agent causing tumour vasculature shutdown and consequently, tumour necrosis. ICT 3146, ICT 3019, ICT 3120 and ICT 3115 prodrugs showed significant stability in normal tissues and considerable activation in certain tumour tissues compared to the lead compound ICT 2588. Also, the selectivity of promising prodrugs to the MMP family was confirmed by using leupeptin (serine, cysteine and threonine protease inhibitor), pepstatin A (aspartate protease inhibitor), phosphoramidon (nepralysin inhibitor), ilomastat (metalloproteinase inhibitor) and BML-P115 (matrix metalloproteinase inhibitor). Moreover, members of the MMP family responsible for cleaving the selected prodrugs were identified using recombinant MMP enzymes. Furthermore, a LC/MS-MS method was developed to specifically detect and quantify MMP-16 protein expression in H460 tumour. MMP- 16 was responsible for the cleavage of ICT 3146 and ICT 3115. Therefore, MMP-activated prodrugs could be a useful therapeutic approach to avoid off-site toxicities of currently used anti-tumour agents. / The full text will be available at the end of the extended embargo: 5th March 2027