Molecular and Functional Characterization of Histone Derived Antimicrobial Peptides from Marine Organisms

Antimicrobial peptides (AMPs) are gene encoded, small sized, generally cationic, amphiphathic peptides characterized by antimicrobial activity against bacteria, fungi, viruses and other pathogens. They are a major component of the innate immune defense system of almost all living organisms, ranging from bacteria to humans and represent the first line of defense against the invading microbial pathogens (Boman, 1995; Zasloff, 2002). Antimicrobial peptides represent a heterogeneous group displaying multiple modes of action that are determined by the sequence and concentration of peptides. Their remarkable specificity for prokaryotes with low toxicity for eukaryotic cells has favored their investigation and exploitation as new antibiotics

Novel inhibitors of the tRNA-dependent amidotransferase of "Helicobacter pylori": Peptides generated by phage display and dipeptide-like compounds

Cette thèse présente la découverte de nouveaux inhibiteurs de l’amidotransférase ARNt-dépendante (AdT), et résume les connaissances récentes sur la biosynthèse du Gln-ARNtGln et de l’Asn-ARNtAsn par la voie indirecte chez la bactérie Helicobacter pylori. Dans le cytoplasme des eucaryotes, vingt acides aminés sont liés à leur ARNt correspondant par vingt aminoacyl-ARNt synthétases (aaRSs). Ces enzymes sont très spécifiques, et leur fonction est importante pour le décodage correct du code génétique. Cependant, la plupart des bactéries, dont H. pylori, sont dépourvues d’asparaginyl-ARNt synthétase et/ou de glutaminyl-ARNt synthétase. Pour former le Gln-ARNtGln, H. pylori utilise une GluRS noncanonique nommée GluRS2 qui glutamyle spécifiquement l’ARNtGln ; ensuite, une AdT trimérique, la GatCAB corrige le Glu-ARNtGln mésapparié en le transamidant pour former le Gln-ARNtGln, qui lira correctement les codons glutamine pendant la biosynthèse des protéines sur les ribosomes. La formation de l’Asn-ARNtAsn est similaire à celle du Gln-ARNtGln, et utilise la même GatCAB et une AspRS non-discriminatrice. Depuis des années 2000, la GatCAB est considérée comme une cible prometteuse pour le développement de nouveaux antibiotiques, puisqu’elle est absente du cytoplasme de l’être humain, et qu’elle est encodée dans le génome de plusieurs bactéries pathogènes. Dans le chapitre 3, nous présentons la découverte par la technique du « phage display » de peptides cycliques riches en tryptophane et en proline, et qui inhibent l’activité de la GatCAB de H. pylori. Les peptides P10 (CMPVWKPDC) et P9 (CSAHNWPNC) inhibent cette enzyme de façon compétitive par rapport au substrat Glu-ARNtGln. Leur constante d’inhibition (Ki) est 126 μM pour P10, et 392 μM pour P9. Des modèles moléculaires ont montré qu’ils lient le site actif de la réaction de transmidation catalysée par la GatCAB, grâce à la formation d’une interaction π-π entre le résidu Trp de ces peptides et le résidu Tyr81 de la sous-unité GatB, comme fait le A76 3’-terminal de l’ARNt. Dans une autre étude concernant des petits composés contenant un groupe sulfone, et qui mimiquent l’intermédiaire de la réaction de transamidation, nous avons identifié des composés qui inhibent la GatCAB de H. pylori de façon compétitive par rapport au substrat Glu-ARNtGln. Cinq fois plus petits que les peptides cycliques mentionnés plus haut, ces composés inhibent l’activité de la GatCAB avec des Ki de 139 μM pour le composé 7, et de 214 μM pour le composé 4. Ces inhibiteurs de GatCAB pourraient être utiles pour des études mécanistiques, et pourraient être des molécules de base pour le développement de nouvelles classes d’antibiotiques contre des infections causées par H. pylori.

A Novel Siderophore-based Chelator to Develop Targeted 68Ga and 89Zr Labelled RGD Peptides for Positron Emission Tomography (PET)

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Antiviral host defence peptides.

The on going global mortality and morbidity associated with viral pathogens highlights the need for the continued development of effective, novel antiviral molecules. The antiviral activity of cationic host defence peptides is of significant interest as novel therapeutics for treating viral infection and predominantly due to their broad spectrum antiviral activity. These peptides also display powerful immunomodulatory activity and are key mediators of inflammation. Therefore, they offer a significant opportunity to inform the development of novel therapeutics for treating viral infections by either directly targeting the pathogen or by enhancing the innate immune response. In this chapter, we review the antiviral activity of cathelicidins and defensins, and examine the potential for these peptides to be used as novel antiviral agents.

Lipid-Based Liquid Crystals As Carriers for Antimicrobial Peptides: Phase Behavior and Antimicrobial Effect

International audience

Amélioration des propriétés pharmacocinétiques de peptides par différentes alkylations N-terminales

Modélisations moléculaires réalisés avec le logiciel HyperChem 8.

Studies of the gastro-oesophageal junction in normal and overweight healthy volunteers

Introduction: Oesophageal adenocarcinoma has increased dramatically in incidence over the past three decades with a particularly high burden of disease at the gastro-oesophageal junction. Many cases occur in individuals without known gastro-oesophageal reflux disease and in the absence of Barrett’s oesophagus suggesting that mechanisms other than traditional reflux may be important. Distal squamous mucosa may be prone to acid damage even in the absence of traditional reflux by the mechanism of distal opening of the lower oesophageal sphincter. This is splaying of the distal segment of lower oesophageal sphincter allowing acid ingress without traditional reflux. It has been suggested that the cardiac mucosa at the gastro-oesophageal junction, separating oesophageal squamous mucosa and acid secreting columnar mucosa of the stomach may be an abnormal mucosa arising as a consequence of acid damage. By this theory the cardiac mucosa is metaplastic and akin to ultra-short Barrett’s oesophagus. Obesity is a known risk factor for adenocarcinoma at the gastro-oesophageal junction and its rise has paralleled that of oesophageal cancer. Some of this excess risk undoubtedly operates through stress on the gastro-oesophageal junction and a predisposition to reflux. However we sought to explore the impact of obesity on the gastro-oesophageal junction in healthy volunteers without reflux and in particular to determine the characteristics of the cardiac mucosa and mechanisms of reflux in this group. Methods: 61 healthy volunteers with normal and increased waist circumference were recruited. 15 were found to have a hiatus hernia during the study protocol and were analysed separately. Volunteers had comprehensive pathological, physiological and anatomical assessments of the gastro-oesophageal junction including endoscopy with biopsies, MRI scanning before and after a standardised meal, prolonged recording of pH and manometry before and after a meal and screening by fluoroscopy to identify the squamo-columnar junction. In the course of the early manometric assessments a potential error associated with the manometry system recordings was identified. We therefore also sought to document and address this on the benchtop and in vivo. Key Findings: 1. In documenting the behaviour of the manoscan we described an immediate effect of temperature change on the pressure recorded by the sensors; ‘thermal effect’ and an ongoing drift of the recorded pressure with time; ‘baseline drift’. Thermal effect was well compensated within the standard operation of the system but baseline drift not addressed. Applying a linear correction to recorded data substantially reduced the error associated with baseline drift. 2. In asymptomatic healthy volunteers there was lengthening of the cardiac mucosa in association with central obesity and age. Furthermore, the cardiac mucosa in healthy volunteers demonstrated an almost identical immunophenotype to non-IM Barrett’s mucosa, which is considered to arise by metaplasia of oesophageal squamous mucosa. These findings support the hypothesis that the cardia is metaplastic in origin. 3. We have demonstrated a plausible mechanism of damage to distal squamous mucosa in association with obesity. In those with a large waist circumference we observed increased ingress of acid within but not across the lower oesophageal sphincter; ‘intrasphincteric reflux’ 4. The 15 healthy volunteers with a hiatus hernia were compared to 15 controls matched for age, gender and waist circumference. Those with a hiatus hernia had a longer cardiac mucosa and although they did not have excess traditional reflux they had excess distal acid exposure by short segment acid reflux and intrasphincteric acid reflux. Conclusions: These findings are likely to be relevant to adenocarcinoma of the gastro-oesophageal junction

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

Factors associated with gastro-duodenal disease in patients undergoing upper GI endoscopy at the Korle-Bu Teaching Hospital, Accra, Ghana.

Background: There is a high prevalence of gastro-duodenal disease in sub-Saharan Africa. Peptic ulcer disease in dyspeptic patients, 24.5%, was comparable to prevalence of gastro-duodenal disease among symptomatic individuals in developed countries (12 – 25%). Limited data exists regarding its associated risk factors despite accumulating evidence indicating that gastroduodenal disease is common in Ghana. Objectives: This study investigates risk factors associated with gastro-duodenal disease at the Korle-Bu Teaching Hospital, Accra, Ghana. Methods: This study utilized a cross-sectional design to consecutively recruit patients referred with upper gastro-intestinal symptoms for endoscopy. The study questionnaire was administered to study participants. Helicobacter pylori infection was confirmed by rapid-urease examination at endoscopy. Results: Of 242 patients sampled; 64 had duodenal ulcer, 66 gastric ulcer, 27gastric cancer and 64 non-ulcer dyspepsia. Nineteen (19) had duodenal and gastric ulcer while 2 had gastric ulcer and cancer. A third (32.6%) of patients had history of NSAIDuse. H. pylori was associated with gastric ulcer (p=0.033) and duodenal ulcer (p=0.001). There was an increased prevalence of duodenal ulcer in H. pylori-infected patients taking NSAIDs, P=0.003. Conclusion: H. pylori was a major risk factor for peptic ulcer disease. However, NSAID-related gastro-duodenal injury has been shown to be common in H. pylori infected patients. It highlights the need for awareness of the adverse gastro-intestinal effects in a H. pylori endemic area.

Síntese, estudos fotofísicos e imageamento celular de peptóides-cumarínicos fluorescentes baseados em Reações de Ugi

Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2016.

Enhancement of non-viral transfection efficiency with nuclear localization signal peptides

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Development of a novel platform for high-throughput gene design and artificial gene synthesis to produce large libraries of recombinant venom peptides for drug discovery

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e Biomédicas

Síntese de peptóides lineares e cíclicos via reação multicomponente de Ugi e cicloadição Azido/Alcino catalisada por cobre, realizada sob regime de fluxo contínuo

Tese (doutorado)–Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2015.