Dissertations / Theses on the topic 'Peptides'

Part 1: Development of a racemisation free process of attaching the first amino acid to a solid resin support for peptide synthesis. To overcome the high degree of racemisation of the amino acid in this process, we have explored a new kind of method of attaching this first amino acid to a resin. This approach involves activating the alcohol of the polyamide resin, instead of activating the amino acid. To test this theory we used benzyl alcohol as a model for the resin, and activated the alcohol with diisopropylcarbodiimide to form an O-isourea derivative. A detailed kinetic study was carried out to establish the rate of reaction with different benzyl alcohols, which proved that solution phase formation of the benzyl esters was successful. We were unable to attach the first amino acid to the resin support, which we believe is due to the reagents being unable to interact with the alcohol sites which are inside the bead of the resin. Part 2: Development of a novel peptidomimetic constraint. Current interest in the development of β-turn stabilising constraints for biologically active peptide motifs led us to synthesise a novel peptidomimetic molecule which in its cis form would provide a scaffold to constrain the conformation of a linear peptide. In stereochemically pure form the BocNH and CO₂Et would be pointing in the right direction to mimic a β-bend, and could be a prototype for peptidomimetics based on e.g. cell adhesion and anti-aggregatory peptides. The synthesis proved demanding as this dioxopiperazine combines an α-diamino with a β-keto ester system, and although all linear precursors were satisfactorily synthesised, the final step which involved a cyclisation reaction did not work. To remove the complication of a β-keto ester system we then attempted to synthesise (36). (Fig. 3754) Again the pre-cyclisation stages were achieved satisfactorily to yield Z-NHCH(NHBoc)CONHCH(CH₂OH)CO₂CH₃

Doctor of Philosophy
Department of Biochemistry
John M. Tomich
Peptide-based delivery systems show great potential as safer drug delivery vehicles. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. We have designed and synthesized a set of 15 and 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. They undergo supramolecular self-assembly and form solvent-filled, bilayer delineated spheres with 50-150 nm diameters (confirmed by TEM and DLS). Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these structures are further stabilized by β-sheet hydrogen bonding and are stable at very low concentrations and even in the presence of SDS, urea and trypsin as confirmed by circular dichroism spectroscopy. Given sufficient time, they fuse together to form larger assemblies and trap compounds of different sizes within the enclosed space. They are prepared using a protocol that is similar to preparing lipid vesicles. We have shown that different concentrations of the fluorescent dye, 5(6)-Carboxyfluorescein can be encapsulated in these assemblies and delivered into human lens epithelial cells and MCF-7 cells grown on coverslips. Besides fluorescent dyes, we have delivered the plasmid (EGFP-N3, 4.7kb) into N/N 1003A lens epithelial cells and observed expression of EGFP (in the presence and absence of a selection media). In the case of large molecules like DNA, these assemblies act as nanoparticles and offer some protection to DNA against certain nucleases. Linear peptides that lacked a branching point and other branched peptides with their sequences randomized did not show any of the lipid-like properties exhibited by the branched peptides. The peptides can be chemically decorated with target specific sequences for use as DDS for targeted delivery.

Une conception générale de synthèse de phosphonopeptides renfermant un motif phosphore du cote c-terminal ou du cote n-terminal est proposée à partir d'un même substrat acide 2-dialkylphosphonoalcanoique. L'objectif est de préparer des inhibiteurs de peptidases présentant une activité thérapeutique. L'approche la plus efficace pour introduire le motif phosphonate sous forme enantiomeriquement pure dans les phosphonopeptides n-terminaux consiste à utiliser la chaine peptidique chirale pour induire une asymétrie sur le carbone directement lié au phosphore. Le couplage peptidique a également été mis au point entre l'acide phosphonoacetique et l'acide 6-aminopenicillanique dans le but de préparer de nouveaux antibiotiques. L'étude du comportement des phosphonopeptides n-terminaux en tant que réactifs de Honer a été abordée pour préparer des peptides insaturés, substituts possibles de peptides à usage thérapeutique. Enfin, la réactivité de thiols sur les chlorures d'acides 2-dialkylphosphonoalcanoiques constitue une voie de synthèse de dialcoxyphosphorylalcane thioates de s-alkyle, qui conduisent eux-mêmes par réaction de Horner à des thioesters éthyléniques, cette réaction des généralisable aux dérivés chrysanthemiques

L’utilisation de peptides pour des applications biomédicales reste limitée, en raison de leur courte demi-vie biologique. Le greffage de polymères aux peptides a conduit à l’amélioration des propriétés du peptide. Les conjugués peptide-polymère peuvent être synthétisés par méthode convergente ou divergente. Alors que la première approche conduit à de faibles rendements, la deuxième est limitée à la polymérisation de monomères vinyliques. Nous proposons une méthode de synthèse divergente des conjugués, basée sur la fonctionnalisation des liaisons peptidiques, via le greffage de polyéthers. En effet, la polymérisation anionique par ouverture de cycle (AROP) des epoxides, à partir d’amorceurs amide, à été démontrée. Cette méthode polyvalente donne accès à des chaines de type PEG sans activation préalable. Les fonctions NH des amides de peptides ont été déprotonnées par une base phosphazène pour générer un amorceur de l’AROP. Des dipeptides cycliques (DKP) ont d’abord été utilisés pour démontrer la fonctionnalisation des fonctions NH. Des conditions de polymérisation ont été identifiées pour synthétiser de manière contrôlée des chaînes de polymères fonctionnalisées par une DKP. Le même système d’amorçage a été appliqué à un dipeptide, et des conjugués ont pu être synthétisés. Des essais ont été menés sur un tripeptide protégé et non protégé, mais se sont révélés peu concluants
Peptides as drugs are facing drawbacks such as short in-vivo half life and low resistance to enzymes, which limits a larger scale use. To overcome these shortcomings, conjugation of polymers to peptides leads to improvements of pharmacokinetic properties of the peptide. Peptide-polymers conjugates are synthesized either by convergent or divergent synthesis. While the first strategy faces low yields, the second one is limited to vinyl-based polymers. We aim to functionalize peptides on amide bonds by divergent synthesis of polyether from the peptide. Anionic Ring-Opening Polymerization (AROP) of epoxides has already proven to be feasible, from amide-based initiators. The approach is polyvalent and gives access to PEG-like polymers without activation of peptides prior synthesis. In this project, NH amide functions from small peptides were deprotonated by phosphazene base to generate an AROP initiator. First, cyclic dipeptides, were used to demonstrate the possible functionalization on NH functions. Polymerization conditions were identified for a controlled AROP, to afford polymers end-capped by a DKP. Initiator’s complexity was increased to protected linear dipeptide. Similar initiating system was used as previously, and conjugates could actually be synthesized. Initiating capacities of tri-peptides and protected tri-peptides were also investigated but were found to be inefficient

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002.
Includes bibliographical references (leaves 419-420). Also available in electronic version. Access restricted to campus users.

Les peptides à visée thérapeutique suscitent une attention croissante auprès des industries pharmaceutiques en raison de leur forte activité biologique. Parmi ces peptides, les peptides antimicrobiens (AMP) peuvent éliminer une grande gamme de microorganismes tels que les bactéries et les champignons pathogènes en perturbant leurs membranes tout en ne provoquant qu’une résistance bactérienne limitée contrairement aux antibiotiques couramment utilisés. D’autre part, les peptides vecteurs (CPP) se distinguent par leur capacité à traverser les membranes cellulaires eucaryotes sans les endommager. Cette particularité peut être exploitée pour l’internalisation intracellulaire de molécules bioactives. Malgré leurs différences, les AMP et les CPP sont des peptides membranotropes qui partagent de nombreuses similitudes telles que la taille, la présence d’acides aminés chargés positivement et les structures secondaires qu’ils peuvent adopter, telles que la structure en hélice α. Le projet décrit ici propose de déterminer quels sont les paramètres qui peuvent conférer une activité antimicrobienne ou pénétrante à une séquence peptidique. À cet effet, des séquences inspirées d’un AMP naturel et modifiées par substitution d’acide(s) aminé(s) afin de promouvoir la capacité à pénétrer les membranes eucaryotes ont été établies et synthétisées pour en étudier l’activité antimicrobienne et la pénétration dans les cellules. Les propriétés physico-chimiques de ces peptides (taille, charge globale, hydrophobie …) ont alors modifiés afin d’étudier l’importance de ces paramètres pour chacune des activités
Recently, therapeutic peptides has particularly retained attention of pharmaceutical industries because of their diversified biological activity and their high specificity. Among them, there are noticeably antimicrobial peptides (AMPs), which can straightly kill microorganisms such as pathogenic bacteria and fungi by disrupting prokaryote cell membranes and induce minimal bacterial resistance unlike commonly used antibiotics. Contrastingly, cell-penetrating peptides (CPPs) are distinguished by their ability to cross eukaryote cell membranes without causing any damages, a property that can be used for intracellular drug delivery. Despite their differences, AMPs and CPPs are both membranotropic peptides which are similar in many aspects considering that they might share similar sizes, polycationic charges and secondary structures such as α-helical structure. This project proposes to determine the parameters that might confer AMP versus CPP properties to a peptide sequence. To achieve this purpose, short sequences inspired from a natural AMP were slightly modified by amino acid substitution to promote cell penetration have been designed and synthesized in order to study their antimicrobial activities and uptake potency in mammalian cells. New peptides varying in size, charge and hydrophobicity were obtained. The study demonstrated that antimicrobial and cell-penetration activities can respectively be induced by a small increase in hydrophobicity and global charge from a non-active peptide

Although peptides and proteins are considered as lead compounds for the discovery and development of new therapeutic agents, poor metabolic and physical properties have limited their optimisation as drug candidates (Adessi & Soto, 2002). Research by medicinal chemists however, generated the discovery of structural similarities between some peptides and diketopiperazines and the common occurrence of such compounds in natural products. This discovery initiated the synthesis of diketopiperazines from amino acids in an attempt to bypass the previously mentioned limitations of using peptides as drug candidates (Dinsmore & Beshore, 2002). Diketopiperazines (DKPs) are the simplest form of cyclic dipeptides, and a class of unexplored bioactive peptides that have great potential for the future. The compounds are relatively simple to synthesise and are prevalent in nature (Prasad, 1995). The DKP backbone is rigid and therefore poses conformational constraint on the compounds. This rigidity allows for simple conformational analysis of the compounds and also gives insight into the conformational requirements for interaction with the targets involved in their biological activity. The reduced conformational freedom also increases the receptor specificity and thus the compounds are proposed to have less unfavourable effects (Anteunis, 1978). The aim of the study was to synthesise compounds that would exhibit metabolic stability, receptor specificity and enhanced lipophilicity which would increase the bioavailability of the compounds. This was to be achieved by the introduction of fluorine and chlorine elements into the DKPs. The structure of the DKPs would be altered which in turn would improve the physicochemical properties and the biological activity of the compounds (Naumann, 1999). Cyclo(D-Phe-2Cl-Pro) and cyclo(Phe-4F-Pro) were synthesised using the method of Milne et al. (1992) and by boiling the linear counterparts under reflux in sec-butanol-toluene. The structures of the synthesised DKPs were elucidated using mass spectrometry, nuclear magnetic resonance spectroscopy, infrared spectroscopy and molecular modeling. Qualitative analysis and evaluation of the physicochemical properties of the DKPs were performed using high-performance liquid chromatography, scanning electron microscopy, thermogravimetric analysis, differential scanning calorimetry and x-ray powder diffraction. The study aimed to determine the biological activity of cyclo(D-Phe-2Cl-Pro) and cyclo(Phe-4F-Pro) with respect to their anticancer, antimicrobial, haematological and antidiabetic effects. The anticancer results obtained indicated that the percentage inhibition produced by both DKPs were lower than those proposed by Graz et al. (2000) for proline-containing DKPs where, a greater than 50% inhibition was observed for cyclo(Phe-Pro). Antimicrobial studies revealed that both DKPs demonstrated marginal effects on Gram-positive and Gram-negative organisms but showed significant effects against C. albicans. The haematological studies revealed that cyclo(D-Phe-2Cl-Pro) at a screening concentration of 12.5 mM, significantly decreased the levels of D-dimer (P < 0.0001). The antidiabetics studies showed limited activity of the DKPs in inhibiting the activity of α-glucosidase and α-amylase enzymes.

A rich gallery of novel systems based on the sequence KLVFF (namely KLVFF, FFKLVFF, AAKLVFF, AAKLVAA, βAβAKLVFF, KLVFF-PEG, FFKLVFF-PEG, AAKLVFF-PEG, AAKLVAA-PEG, FF-PEG, FFFF-PEG) was synthesized and characterized. For the first time structure-properties relationships of this class of materials were systematically explored, with emphasis on self-association properties in both bulk and solution. Such a comparative investigation, essentially absent from the literature, provides insights into the underlying mechanism of amyloid fibrogenesis, given that KL VFF has been identified as critical for amyloid fibril formation of the amyloid-β peptide. In this respect, this virtually unique, bottom-up approach contributes to the development of therapeutics for Alzheimer's disease. Despite the fact that all the peptides were found to organize into predominantly β-sheet conformations (in a variety of solvents and in bulk) they exhibit a versatile range of morphological features, such as fibrils, twisted assemblies of protofilaments, hollow tubes and single tapes. Interestingly, FFKLVFF was found to exhibit strong fibrillization properties, while helically twisted ribbons were obtained from βAβAKL VFF. The self-organization of KLVFF towards fibrillar symmetries gives rise, under certain conditions, to time-dependant hydrogels with potential for application in tissue engineering. Notably, only FFKLVFF-PEG and FFFF-PEG retain the β-sheet conformation of the peptide precursor, and they form fibrils bearing a peptidic core surrounded by a corona of PEG chains. At high volume fractions packing of the FFKLVFF-PEG fibrils leads to the evolution of nematic and hexagonal columnar phases. Owing to their inherent biocompatible character, those hybrids carry great promise to substitute a number of conventional polymers in applications such as tissue engineering, cell growth and drug delivery. In the solid state, the interplay between two competing factors, polymer crystallization and peptide fibrillization, was studied for several conjugates. Two distinct cases were identified; solidification of FFKL VFF-PEG controlled by peptide- peptide interactions, while KLVFF -PEG and AAKL V AA-PEG crystals reflected the characteristics of PEG spherulites.

Antibiotics have been used widely for the treatment of bacterial infections for over half a century. However, the emergence of resistance to antibiotics has aroused public health concern, leading to the development of antimicrobial peptides (AMPs) as potential alternative therapeutic agents against bacterial infections. AMPs are naturally found in many species and have important roles in our innate immune defense systems. AMPs are usually cationic amphipathic peptides with membrane destabilizing property. They have a relatively broad spectrum of antimicrobial activity and pathogens are less likely to develop resistance against AMPs. The major challenge of using AMPs as therapeutic agents is their toxicity towards mammalian cells. The biological stability of AMPs to protease in human body is another concern. To address the latter problem, instead of the naturally occur L-enantiomers, Denantiomeric AMPs were introduced to enhance their stability. This study aimed to test the hypothesis that the D-enantiomeric AMPs are more resistant than the Lenantiomeric AMPs against proteolytic degradation. Three pairs of synthetic D-/LAMPs (D-LAO160-P13/LAO160-P12; D-LAO160-H/LAO160-H; and D-LAK-120-HP13/LAK-120-HP13) were employed to test for their stability when treated with trypsin, serum and gastric fluid, and the samples were analyzed by high performance liquid chromatography (HPLC). Generally, all the D-enantiomeric AMPs were found to be resistant towards proteolysis. Besides, to compare the cytotoxicity of D-/LAMPs, MTT and LDH assays of the D/L-LAK120-HP13 pair were carried out on two different cell lines, A549 cells (human lung adenocarcinoma epithelial cells) and RAW264.7 cells (mouse macrophage cells). Significant difference in cytotoxicity of D-LAK120-HP13 and LAK120-HP13 on RAW264.7 cells were obtained from MTT assay, but not in LDH assays or on A549 cells. Further analysis has to be done to validate the findings obtained from this research.
published_or_final_version
Pharmacology and Pharmacy
Master
Master of Medical Sciences

Thesis (Ph.D.)--Tufts University, 2000.
Submitted to the Dept. of Chemistry. Adviser: Clemens Richert. Includes bibliographical references (leaves 103-107). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis advisor: Jianmin Gao
As the threat of microbial resistance to antibiotics grows, we must turn in new directions to find new drugs effective against resistant infections. Antimicrobial peptides (AMPs) and host-defense peptides (HDPs) are a class of natural products that have been well-studied towards this goal, though very few have found success clinically. However, as there is much known about the behavior of these peptides, work has been done to manipulate their sequences and structures in the search for more drug-like properties. Additionally, novel sequences and structures mimicking those seen in nature have been discovered and characterized. Herein, we demonstrate our ability to finely tune the antimicrobial activity of various peptides, such that they can be provided with more clinically desirable characteristics. Our results show that gramicidin A (gA) can be made to be less toxic via incorporation of unnatural cationic amino acids. This is achieved by synthesizing lysine analogues with diverse hydrophobic groups alkylated to the side-chain amine. Through exploring different groups, we achieved peptide structures with improved selectivity for bacterial over mammalian membranes. Additionally, we were able to achieve novel broad-spectrum gram-negative activity for gA peptides. In efforts to combat bacterial resistance to cationic antimicrobial peptides (CAMPs), we have directed our reported amine-targeting iminoboronate chemistry towards neutralizing Lys-PG in bacterial membranes. Originally incorporating 2-APBA into gA, we found this to hinder the peptide’s activity. However, we were successful in increasing the potency of gA3R, a cationic mutant of gA, towards S. aureus by using a co-treatment of this peptide with a Lys-PG binding structure. Currently, we are exploring this strategy further. Finally, we describe our work towards establishing a novel cyclic peptide library incorporating a 2-APBA warhead for iminoboronate formation with a given target. In this, we have achieved intermolecular reduction of iminoboronates, strengthening the stringency of library screening. Although we were unsuccessful in finding a potent hit for binding of the lipid II stem peptide, screening against human transferrin yielded selective hits. Currently we are investigating these hits to understand their activity and therapeutic potential
Thesis (PhD) — Boston College, 2017
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

Les récepteurs à sept domaines transmembranaires couplés aux protéines G (RCPG) représentent une grande famille de récepteurs ubiquitaires impliqués dans de nombreux processus métaboliques. Leur grande diversité d'action en fait des cibles pharmacologiques privilégiées pour le développement de médicaments avec des applications dans tous les champs d'activité clinique en particulier au niveau cardio-vasculaire. Les récepteurs 2-adrénergique (2AR) et M2 muscariniques (M2Ach-R) appartiennent à la famille des RCPG et sont impliqués dans la régulation du système cardiovasculaire. De nombreuses études ont montré l'importance de la seconde boucle extracellulaire pour l'activité de ces récepteurs dans certaines pathologies autoimmunes. La première partie du travail concerne le 2AR. Les scFvs issus d'AcM dirigés contre un peptide dérivé de la seconde boucle extracellulaire (SBE) du 2AR ont été produits. La capacité de cette protéine recombinante de reconnaître son peptide antigénique de façon spécifique a été mise en évidence, et les constantes cinétiques d'interaction ont pu être déterminées. Leur capacité à reconnaître le récepteur a été démontrée par immuno-cyto-fluorescence sur cellules A431 exprimant de façon constitutive le 2AR. Sur ces mêmes cellules, ce fragment d'anticorps est capable de façon dose dépendante de diminuer l'augmentation intracellulaire d'AMPc induite par 10 nM de salbutamol (agoniste 2), il est également capable seul de diminuer le niveau basal d'AMPc ce qui en fait un agoniste inverse. Il inhibe de façon non compétitive l'augmentation des battements spontanés de cardiomyocytes de rats nouveau-nés induits par différentes concentrations de clenbutérol (agoniste ). Des souris traitées par injections i. V. De scFv montrent une diminution du rythme cardiaque. Les peptides dérivés des CDRs du scFv 6H8 ont été synthétisés et cyclisés. Le CDR H1, totalement insoluble, n'a pu être utilisé. Les constantes cinétiques d'interaction avec le peptides dérivé du 2AR ont pu être déterminées par SPR pour le CDR L2, L3 et H3. Les CDR H2, L1 et L2 se sont révélés être des modulateurs allostériques négatifs du 2AR en inhibant de façon non compétitive l'augmentation des battements induite par une dose croissante de clenbutérol alors que les CDR H3 et L3 se sont révélés être des modulateurs allostériques positifs en augmentant l'effet du clenbutérol sur ces cellules. La deuxième partie du travail concerne la modulation de l'activité du récepteur M2 muscarinique et cela par deux approches différentes. Dans un premier temps, un lot de souris a été immunisé avec un peptide dérivé de la SBE du M2Ach-R (souris M2). Les sérums issus des souris M2 sont capables de reconnaître le M2Ach-R en immuno-empreinte. Ils sont également capables d'augmenter l'activité du carbachol sur le M2Ach-R in vitro sur des cellules CHO transfectées avec ce dernier. Pour mettre en évidence une modulation du récepteur induite par les anticorps anti-M2Ach-R, les souris ont été traitées par injection i. V. Avec plusieurs ligands muscariniques et adrénergique. Leurs ECGs ont été enregistrés et l'évolution de leurs rythme cardiaque suite à l'injection ainsi que les spectres de variabilité de leur rythme ont été calculés. Suite à l'injection de carbachol, les souris M2 diminuent plus fortement leur rythme que les souris contrôles. De plus, les souris M2 sont moins sensibles à l'injection de gallamine, atropine et isoprotérenol. L'analyse de l'évolution et de la variabilité de leur rythmes cardiaques suite aux différents traitements montre une augmentation du tonus parasympathique chez les souris M2 par rapport aux souris contrôles. Les anticorps anti-M2Ach-R agissent dans ce cas comme des ligands allostériques positifs. Une deuxième approche a également permis de moduler l'activité du M2Ach-R. De la même manière que pour le scFv anti-récepteur 2 adrénergique, un scFv anti-M2Ach-R a été produit et caractérisé à l'aide de la technologie SPR. Des expériences menées in vitro sur cellules CHO transfectées avec le M2Ach-R et sur cardiomyocytes de rats nouveau-nés ont permis de mettre en évidence une activité pharmacologique sur ce récepteur. Il est capable d'inhiber de manière non-compétitive l'action d'un agoniste tel que le carbachol. De plus, seul, il est également capable de diminuer l'activité basale du récepteur ce qui fait également de ce fragment d'anticorps un agoniste inverse. Il est également capable in vivo de modifier la réponse au carbachol et à l'isoprotérénol sur l'activité cardiaque de souris via le M2Ach-R par injection i. V. . Ce travail montre que des fragments d'anticorps recombinants monovalents dirigés contre la SBE de RCPG représentent des agonistes inverses allostériques alors que des anticorps (bivalents) induits contre ce même site, sont des ligands allostériques positifs. De plus, les peptides dérivés des CDRs du scFv anti 2AR, ont gardé une activité pharmacologique sur leur récepteur cible. Outre l'aspect développement de nouvelles molécules, ces fragments d'anticorps ainsi que les peptides mimant les CDRs représentent des outils puissants pour la compréhension des mécanismes d'activation des RCPG
G protein coupled receptor are involved in various metabolic functions. This receptor family represents the main target of cardiovascular and neurological used drugs. The 2 adrenergic receptor (2AR) and the M2 muscarinic receptor (M2Ach-R) belong to this family and are implicated in the regulation of the cardiovascular system. Many studies show the importance of the second extracellular loop of GPCR on their activity in particular in some autoimmune diseases. The first part of this work concern the 2AR. Using a functional monoclonal antibody against the human 2AR, a scFv fragment with high affinity for the target epitope was constructed and produced. The fragment recognized the ß2-adrenergic receptors on A431 cells, blocked cAMP accumulation induced by the ß2-agonist salbutamol, and decreased basal cAMP accumulation in the same cells. Their in vitro activity was tested on neonatal rat cardiomyocytes. The antibody fragments blocked the chronotropic activity induced by the ß2-agonist clenbuterol. They also decreased the in vivo heart beating frequency of mice pretreated with bisoprolol (a ß1-adrenergic receptor antagonist) for 4 minutes after injection. Cyclic peptides derived from the CDR of the scFv 6H8 were produced. The CDR H1 was not used because of its total insolubility in water. Kinetic contants of interaction with their target peptides were determined by SPR technology for the CDR peptides L2, L3 and H3. The CDR H2, L1 et L2 behaved as negative allosteric modulator of the 2AR, they inhibited in a non competitive manner the increase of spontaneous beating rate of neonatal rat cardiomyocytes induced by an increasing dose of clenbuterol. The CDR H3 and L3 behaved as positive allosteric modulators of the 2AR by increasing the clenbuterol effect on these cells. The second part of this work concern the modulation of the M2Ach-R activity using two different strategies. First, we investigated the in vivo consequences on heart rate of such antibodies in mice immunized with a peptide derived from the second extracellular loop of the M2AChR. Nine mice, immunized with a peptide corresponding to the N-terminus of the second extracellular loop of the M2AChR were compared to 9 mice immunized with an irrelevant peptide. Sera of mice immunized with the M2ACh-R derived peptide recognized the M2ACh-R on immunoblots and enhanced the agonist activity of carbachol towards the M2AChR transfected in CHO cells. In vivo, no difference could be shown in heart rate nor in heart rate variability between the two groups of mice. In contrast, the decrease in heart rate induced by carbachol was more pronounced in the M2AChR immunized mice compared to the control mice. The increase in heart rate induced by atropine, gallamine and isoproterenol were alike significantly attenuated in the M2ACh-R immunized mice compared to the control mice. Analysis of heart rate variability further argued for an increased parasympathetic response to the different drugs in the M2ACh-R immunized mice. Antibodies raised against the M2AChR can behave as positive M2AchR allosteric modulators in vivo. They might be protective in boosting the activity of the parasympathetic drive to the heart, in particular in patients with a high sympathetic tone. The second strategy was, to construct and produce a single chain variable fragment, from a partial agonist monoclonal antibody directed against the M2ACh-R. It showed high affinity for its target epitope. The fragment is able to recognize its receptor on Chinese hamster ovary cells transfected with the M2ACh-R, to block the effect of carbachol on this receptor and to exert an inverse agonist activity on the basal activity of the receptor. The antibody fragment is also able to increase the basal rhythm of cultured neonatal rat cardiomyocytes, and to inhibit in a non-competitive manner the negative chronotropic effect of carbachol. This antibody fragment is able to exert its inverse agonist activity in vivo on mice heart activity. This works shows that recombinant monovalent antibody fragments directed against te second extracellular loop of GPCR have inverse agonist activity. Bivalent antibodies directed against the same target, show an allosteric positive activity on the receptor. Peptides derived from CDR of the scFv 6H8, keep a pharmacological function, they act as allosteric modulators of the 2AR. These scFv and cyclic peptides may represent leads for a new class of allosteric modulators of GPCR. They also represent tools for understanding of the activation mechanisms of GPCR

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xvii, 143 p.; also includes graphics. Includes bibliographical references (p. 106-111). Available online via OhioLINK's ETD Center

Plant natriuretic peptides (PNP) are novel peptides which, like in vertebrates, have been shown to have a function associated with water and salt homeostasis. Two PNP-encoding genes have been identified and isolated from Arabidopsis thaliana, namely; AtPNP-A and AtPNP-B. In this study, the focus was on PNP-B, which has not been extensively studied. Bioinformatic analysis was done on the AtPNP-B gene. This included the bioinformatic study of its primary structure, secondary structure, tertiary structure, transcription factor binding sites (TFBS) and its relation to other known proteins. The AtPNP-B gene was shown to be a 510 bp long, including a predicted 138 bp intron. AtPNP-B was also shown to have some sequence similarity with AtPNP-A and CjBAp12. The TFBS for AtPNP-B and OsJPNP-B were compared and they comprised of TFBS that are related to water homeostasis and pathogenesis. This suggested two possible functions; water stress and homeostasis and a pathogenesis related function for PNP-B. Following bioinformatic analysis, the heterologous expression of the AtPNP-B was attempted to investigate whether the AtPNP-B gene encoded a functional protein and to determine the functional role of PNP-B. However, expression was unsuccessful. An evolutionary study was then carried out which revealed that there were some plants without the intron such as, rice, leafy spurge, oilseed rape, onion, poplar, sugar cane, sunflower and tobacco. These plants would therefore be used for expression and functional studies in the future. The evolutionary studies also revealed that PNP-B had a relationship with expansins and the endoglucanase family 45. Other PNP-B related molecules were also obtained from other plant genomes and therefore used in the construction of a phylogenetic tree. The phylogenetic tree revealed that AtPNP-B clustered in the same group as CjBAp12 while AtPNP-A had its own cluster group. There were also other PNP-B like molecules that clustered in the same group as expansins (α- and β-). Thus, we postulate that, like PNP-A, PNP-B also has a possible function in water and salt homeostasis. However, due to the clustering iii of AtPNP-B into the same group as CjBAp12, a possible role of PNP-B in pathogenesis-related response is also postulated.

Microgels are lightly cross-linked hydrogel particles in the sub-micrometer to micrometer size range with a capacity to drastically change their volume in response to changes in the external environment. Microgels have an ability to bind and store substances such as biomacromolecular drugs, notably proteins and peptides, and release them upon stimuli, making them potential candidates as drug delivery vehicles and functional biomaterials. This thesis aims at clarifying important factors affecting peptide-microgel interactions. These interactions were studied by micromanipulator-assisted light and fluorescence microscopy focusing on microgel deswelling in response to peptide binding, as well as re-swelling in response to peptide release or enzymatic degradation. To evaluate peptide uptake in microgels, solution depletion measurements were used whereas the peptide secondary structure was investigated by circular dichroism. In addition, the peptide and enzyme distribution within microgels was analyzed with confocal microscopy. Results presented in this thesis demonstrate that peptide incorporation into microgels, as well as peptide-induced microgel deswelling, increases with peptide length and charge density. In addition, results demonstrate that the peptide charge (length) rather than peptide charge density determines microgels deswelling. End-to-end cyclization is shown to not noticeably influence peptide-microgel interactions, suggesting that peptide cyclization can be used in combination with oppositely charged microgel carriers to improve the proteolytic and chemical stability of the peptide compared to the corresponding linear variant. Peptide secondary structure is found to drastically affect peptide incorporation into, and release from, oppositely charged microgels. Furthermore, it is shown that microgel charge density, peptide molecular weight, and enzyme concentration all greatly influence microgel bound peptide degradation. Of importance for applications, protective effects of microgels against proteolytic peptide degradation are observed only at sufficiently high microgel charge densities. Enzyme-mediated microgel degradation is shown to increase with increasing enzyme concentration, while an increased peptide loading in microgels causes a concentration-dependent decrease in microgel degradation. Taken together, results obtained in this work have provided some insight into factors of importance for rational use of microgels as delivery systems for protein or peptide drugs, but also in a host of other biomedical applications using weakly cross-linked polymer systems.

L'ilot génomique pks de Escherichia coli et d'autres Enterobacteriaceae code des synthases depolycétides et de peptides non ribosomaux qui permettent l'assemblage d'un composé hybride polycétidepeptidenon ribosomal putative. Ce composé nommé colibactine induit des cassures double-brin de l'ADN descellules eucaryotes.La machinerie enzymatique codée par l'ilot pks comporte une protéine essentielle ClbP, atypique dansce type d'ilot. Nous avons montré que ClbP possède une partie N-terminale catalytique et périplasmique, et unepartie C-terminale associée à la membrane cytoplasmique. La structure cristalline de ClbP et des expériences demutagenèse ont révélé un site actif à sérine et des caractéristiques structurales originales, qui sont compatiblesavec une activité peptidase, confirmée par des analyses biochimiques. Dix homologues de ClbP ont été identifiésin silico dans des ilots génomiques de synthases de peptides non ribosomaux d'espèces bactériennes proches etéloignées. Tous les homologues testés ont présenté une promiscuité fonctionnelle avec ClbP. ClbP est donc leprototype d'une nouvelle sous-famille de peptidases, qui sont probablement impliquées dans la maturation decomposés peptidiques non ribosomaux.Par ailleurs, nous avons réalisé deux études épidémiologiques sur la prévalence de l'ilot pks dansl'espèce E. coli dans deux contextes physiopathologiques, l'urosepsis et les cancers coliques et rectaux. L'ilotpks était significativement associé aux souches issues d'urosepsis comparé à des souches commensales, et auxsouches issues de biopsies de tumeurs coliques comparé à des souches commensales ou issues de biopsies detumeurs rectales, de diverticuloses et de lésions iléales de maladie de Crohn.

La mucoviscidose est une maladie génétique létale induite par des mutations du canal ionique CFTR, provoquant une perte de sa fonctionnalité au niveau des tissus épithéliaux de divers organes. Le poumon est particulièrement touché et devient sujet à des infections bactériennes chroniques. Dans le but de traiter la maladie, nous avons développé des « stabilisateurs » de la protéine CFTR : il s’agit de peptides inhibant l’interaction de la protéine CFTR avec le médiateur-clé de sa demi-vie à la membrane apicale des cellules épithéliales, la protéine CAL. En particulier, le peptide iCAL36 a démontré une hausse de fonctionnalité de la protéine CFTR mutée. Le but de cette thèse a été de renforcer cet effet biologique en améliorant ses caractéristiques pharmacologiques : pénétration cellulaire (vectorisation), stabilité métabolique et affinité pour la protéine CAL.Le premier axe d’optimisation a été l’internalisation du peptide iCAL36 par 7 différents peptides vecteurs (CPP). Les conjugués correspondants ont été évalués suivant leur cytotoxicité, leur efficacité d’internalisation et leur capacité à maintenir cette efficacité en présence de sérum. Le mécanisme d’entrée des deux meilleurs conjugués a ensuite été étudié. Divers biais couramment rencontrés lors de l’analyse de l’efficacité d’internalisation de peptides vecteurs par des méthodes de fluorescence ont également été identifiés et expliqués. La séquence du peptide iCAL36 a ensuite été modulée par inclusion d’acides aminés non-naturels. Le criblage des interactions peptide/protéine a été réalisé par une procédure optimisée dans le cadre de cette thèse (méthode PIPEPLUS) et a permis d’identifier 32 analogues prometteurs de la séquence d’iCAL36 incluant différentes substitutions. En particulier, une des séquences identifiées (iCAL-Q27) a démontré une affinité 70 fois supérieure à celle du peptide iCAL36 pour la protéine CAL, indiquant une inhibition plus complète de l’interaction CAL/CFTR.Ces résultats majeurs permettent dans leur ensemble de développer des « stabilisateurs » peptidiques de seconde génération pouvant avoir un effet biologique accru dans le contexte de la mucoviscidose
Cystic fibrosis is a lethal disease induced by genetic mutations of the CFTR chloride channel, leading to a loss of its function in the epithelial tissues of various organs. The lung is particularly affected and becomes a target for chronical bacterial infections. To cure the disease, we developed so-called CFTR “stabilizers”, which are peptides inhibiting the interaction between the CFTR protein and the key mediator of its half-life at the apical membrane of epithelial cells, the CAL protein. In particular, the iCAL36 peptide showed an increase of the functionality of the mutated CFTR protein. The aim of this thesis was to increase this biological effect by improving its pharmacological parameters: cellular internalization (vectorization), metabolic stability and affinity for the CAL protein.The first axis of optimization was the internalization of the iCAL36 peptide by 7 different cell-penetrating peptides (CPP). The corresponding conjugates were evaluated upon their cytotoxicity, their uptake efficiency and their capacity to maintain this efficiency in the presence of proteases. The mechanism of entry of the two best candidates was then studied. Various bias frequently encountered during the analysis of CPP uptake efficiency by fluorescence methods were also identified and explained. Afterwards, the iCAL36 sequence was modulated by inclusion of non-natural amino acids. The screening of the peptide/protein interactions was performed by a method optimized during this thesis (PIPEPLUS process) and allowed the identification of 32 promising analogues of the iCAL36 sequence including several substitutions. In particular, one of these sequences (iCAL-Q27) showed an affinity 70 times stronger for the CAL protein compared to iCAL36, hinting a more complete inhibition of the CAL/CFTR interaction.Overall, these major results grant the access to second-generation “stabilizers” potentially showing an improved biological effect in the context of cystic fibrosis

Modern medicine is challenged continuously by the increasing prevalence of multi-drug resistant bacteria. Therefore, the development of alternatives to traditional antibiotics is an urgent necessity. Cationic antimicrobial peptides (CAMPs) are components of the innate immune defense system. Histones, generally known as proteins that package and regulate the transcription of DNA, share all of the essential antimicrobial traits of CAMPs, and could be promising alternatives to antibiotics. In this study, I investigated the antimicrobial properties of nucleated-erythrocyte-specific linker histone H5 and its derived peptides. Histone H5 was extracted and purified from chicken erythrocytes using an acid extraction followed by ion exchange chromatography using a step salt gradient; the purity (>95%) was verified by densitometry and proteomics analysis. Purified histone H5 demonstrated potent antimicrobial activity against various Gram-positive and Gram-negative planktonic bacteria, including resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), as well as anti-biofilm activity against Listeria monocytogenes and Pseudomonas aeruginosa. Furthermore, scanning electron microscopy (SEM) revealed significant damage to L. monocytogenes and P. aeruginosa bacterial cell surfaces after histone H5 treatment. The potential for histone toxicity towards mammalian cells was investigated with a hemolytic assay which determined that even at the highest concentration tested (1 mg/mL), histone H5 was non-hemolytic. An in silico analysis determined the predicted antimicrobial domain of histone H5 of which six histone H5-derived peptides with potential antimicrobial activity were identified. These six histone H5-derived peptides were synthesized and tested against bacterial pathogens to determine their antimicrobial properties. Although the H5-derived peptides were identified within the predicted antimicrobial domain of histone H5, they did not possess more potent antimicrobial activity than the full length protein. Overall, this study demonstrates that histone H5 and histone H5-derived peptides could be promising candidates in the development of novel anti-infective therapeutics.

Une reaction modele, l'hydrolyse de l'hemoglobine par la pepsine, a permis de mettre au point plusieurs procedes: production d'hydrolysats, methodes analytiques (determination du degre d'hydrolyse, chromatographie d'exclusion (colonne superose 12), chromatographie en phase inverse, analyse sensorielle), et methodes de fractionnement permettant d'isoler des composes amers. Afin de les generaliser, les techniques ont ete egalement appliquees a d'autres hydrolysats d'hemoglobine obtenus avec des enzymes de type et de specificite divers (proctase, alcalase, neutrase, papaine), ainsi qu'a d'autres types d'hydrolysats: hydrolysats de gelatine et de caseine produits par les cinq proteases precedemment citees, hydrolysats industriels. Il decoule de cette etude qu'il n'est pas possible de prevoir l'apparition d'un gout amer en considerant les parametres d'hydrolyse. En effet, la formation d'amertume semble dependre principalement du substrat de depart, mais aucune relation ne peut etre etablie avec le type ou la specificite de l'enzyme ou avec le degre d'hydrolyse. Cependant, il apparait que les fractions ameres sont extraites preferentiellement et selectivement par le butanol 2 et correspondent toujours a une gamme de poids moleculaires de 500 a 5000 da, determinee lors d'ultrafiltrations en cascade. De plus, les peptides principaux responsables de l'amertume des hydrolysats d'hemoglobine obtenus avec la pepsine, la proctase et l'alcalase ont ete isoles et identifies (ex: vv hemorphine 7). Il s'avere qu'ils sont specifiquement adsorbes sur la matrice de la colonne superose 12, faisant ainsi de cette technique une methode de detection potentielle de l'amertume des hydrolysats. Elle a d'ailleurs ete validee avec les hydrolysats non amers (hydrolysats d'hemoglobine produits par la papaine et la neutrase, hydrolysats de gelatine et hydrolysats industriels). Par contre, elle n'est pas forcement generalisable puisqu'elle n'est pas respectee dans le cas des hydrolysats amers de caseine. Elle reste toutefois interessante et doit etre envisagee lors d'etudes d'hydrolysats

The goal of this project was to clone and express the antimicrobial peptide protegrin 1 (PG-1). Initially a yeast system was chosen but was discarded due to technical difficulties. Invitrogen's bacterial T7 expression system was chosen next to express the peptide. PG-1 expression was verified by anti-his immunoblot and then the peptide was purified by IMAC. Its activity was verified using a Bacillus subtillis radial diffusion assay.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2004.
Includes bibliographical references (leaves 148-151). Also available in electronic version. Access restricted to campus users.

La synthèse de β-aminoacides, analogues structurels des -aminoacides, constitue un enjeu essentiel dans le développement d'oligopeptides. Un long travail a été mené sur le comportement des β-peptides (enchaînement de β-aminoacides) ainsi que les peptides mixtes (mélange d'β- et β- aminoacides). Il en résulte que la préférence conformationnelle des β-aminoacides va induire l'apparition d'une structure tridimensionnelle ordonnée de l'oligopeptide. Ainsi, plusieurs types d'hélices, des feuillets et des coudes ont été observés sur des β-oligopeptides. En plus de cette fascinante particularité, les β-peptides et les peptides mixtes ont montré une grande stabilité vis-à-vis des enzymes digestives. Ces caractéristiques sont importantes dans le développement de nouveaux médicaments, mimes de structures protéiques. Au laboratoire, nous travaillons sur le β-aminoacide cyclobutanique comme bloc de construction de structures moléculaires élaborées dont les β-peptides. La partie bibliographique, scindée en deux parties, décrit la synthèse deβ-aminoacides carbocycliques puis les différents types de structures secondaires obtenus à partir de peptides composés de β-aminoacides. Des travaux ont été menés sur les β-aminoacides cycliques à 6, 5 et à 4 chaînons. Cependant, aucuns travaux n'ont été décris sur le trans-β-aminoacide cyclobutanique " trans-ACBC ". Nous avons entrepris d'accéder au cis- et trans-ACBC optiquement enrichis et nous avons mis au point deux voies de synthèse à l'échelle multigramme. L'étape clé, commune aux deux synthèses et la photocycloaddition [2+2] entre l'éthylène et un partenaire ènone pour créer le cyclobutane. A l'aide de méthodes de couplages peptidiques, nous avons préparé le dimère, le tétramère, l'hexamère et l'octamère du 1R,2R- trans-ACBC. Grâce à plusieurs techniques de caractérisation couramment utilisées dans l'étude conformationnelle de polypeptides, nous avons pu déterminer avec certitude l'existence d'une hélice- 12 dès l'hexamère. En parallèle, nous avons mené une étude complète de modélisation moléculaire qui nous a conduits à un résultat identique. Ce travail sur la synthèse des ACBC optiquement pures et sur le comportement des β-peptides cyclobutaniques complète le tableau des β-peptides carbocycliques. Nous envisageons de préparer des β-aminoacides cyclobutaniques fonctionnalisés, afin de construire des β-peptides structurés, destinés à une application en chimie médicinale.

Peptides have attracted increasing attention as therapeutics in recent years, at least partially as a consequence of the widespread acceptance of protein therapeutics; but also as possible solutions to problems such as short half-life and delivery of molecules, and as therapeutics in their own right. The current work presents three projects that involve applications of peptides in a therapeutic environment. The first project studies the use of ER retaining peptides and CPPs (Cell penetrating peptides) in enhancing the effective concentration of DNJ (1-deoxynojirimycin), an α-glucosidase inhibitor, in cells. DNJ constructs with ER retaining peptides (6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL and 6-[N-(1-deoxynojirimycino)]-hexanoyl-KKAA) and CPPs (6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT and 6-[N-(1-deoxynojirimycino)]-hexanoyl-MAP) were synthesised and analysed for their inhibitory activity against α-glucosidase I and II in vitro. The constructs were then analysed in a cell-based assay to determine their inhibitory activity on α¬-glucosidase-mediated hydrolysis of N-linked oligosaccharides. FITC-labelled ER retaining peptides were also synthesised to determine the internalisation and trafficking of the constructs by FACS and IF-microscopy. While none of the DNJ-constructs showed higher cellular inhibition than NB-DNJ (N-butyl DNJ; Miglustat), the CPP construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT showed comparable activity and the ER retaining construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL showed a small but significant increase in activity following long-term administration. The second project focuses on beauveriolides, a cyclic depsipeptide family shown to have activity as ACAT inhibitors and thus a possible treatment for Alzheimer’s disease by the decrease in the production of Amyloid β (Aβ). A published total synthetic method was improved by the use of a cross-metathesis to reduce the total synthesis by 5 steps and increase its flexibility to allow the production of analogues. The synthesised beauveriolide III was used in attempts to develop an IF-FACS-based assay to measure the intracellular concentrations of Aβ. However, the location of γ-secretase in the used cell-line meant that levels of intracellular Aβ were not sufficient to track any decrease caused by ACAT inhibition. The third project involves the design of a cyclic peptide that could block the binding site for the influenza virus in the host cell. The cyclic peptide (cGSGRGYGRGWGVGA) was developed from a comparative study of four different sialic acid-binding proteins and synthesised by solution cyclisation of the linear peptide synthesised by traditional solid phase peptide synthesis (SPPS). An in silico study showed that the cyclic peptide allowed overlap with the binding site of Hemagglutinin. A 1H NMR titration determined the dissociation constant of the cyclic peptide to sialic acid. The KD corresponded to a low binding affinity, however the observed binding seemed to be specific and caused by a single bound conformation.

For decades the opioid receptors have been an attractive therapeutic target for the treatment of pain. Since the first discovery of enkephalin, approximately a dozen endogenous opioid peptides have been known to produce opioid activity and analgesia, but their therapeutics have been limited mainly due to low blood brain barrier penetration and poor resistance to proteolytic degradation. One versatile approach to overcome these drawbacks is the cyclization of linear peptides to cyclic peptides with constrained topographical structure. Compared to their linear parents, cyclic analogs exhibit better metabolic stability, lower offtarget toxicity, and improved bioavailability. Extensive structure-activity relationship studies have uncovered promising compounds for the treatment of pain as well as further elucidate structural elements required for selective opioid receptor activity. The benefits that come with employing cyclization can be further enhanced through the generation of polycyclic derivatives. Opioid ligands generally have a short peptide chain and thus the realm of polycyclic peptides has yet to be explored. In this review, a brief history of designing ligands for the opioid receptors, including classic linear and cyclic ligands, is discussed along with recent approaches and successes of cyclic peptide ligands for the receptors. Various scaffolds and approaches to improve bioavailability are elaborated and concluded with a discourse towards polycyclic peptides.

La modification de la liaison amide dans les peptides a plusieurs conséquences potentielles: une biodégradabilité réduite, une possible modulation structurale due aux perturbations du réseau de liaisons hydrogène, et éventuellement, une bio-activité modulée pour l'analogue pseudopeptidique résultant, sans nécessiter de changer les chaines latérales. Nous avons étudie les perturbations structurales induites par la substitution d'une liaison amide par un groupe hydrazide dans diverses séquences mono-, di- et tripeptidiques protégées à leurs deux extrémités par une fonction amide, et contenant l'analogue hydrazine de la proline ou de l'alanine. L'analyse conformationnelle a été conduite à l'état solide par diffraction des rayons X, en solution par spectroscopie infrarouge et résonance magnétique nucléaire du proton, et par modélisation moléculaire. Après des considérations générales sur les structures des peptides et pseudopeptides, l'aspect chimique et technique de ce travail est présenté dans le second chapitre. Le troisième chapitre rassemble les données spectroscopiques et les conclusions sur les structures présentes qui sont ensuite discutées en référence aux séquences peptidiques originelles. La conclusion principale est que le groupe hydrazide induit localement un repliement du à la nucléophilie de l'oxygène du carbonyle et de l'azote alpha qui participent tous les deux a une liaison hydrogène bifide avec le site N-H amide immédiatement situe en aval. La conformation repliée qui en résulte est très stable, et capable de donner naissance à une forme globalement repliée de la molécule, de façon très semblable au repliement beta dans les peptides

Neurokinin A, (NKA), belongs to the tachykinin family, a group of small amphipathic peptides that bind to specific membrane-embedded G-protein-coupled receptors. The cell membrane acts as a solvent to accumulate peptide and an inducer of peptide secondary structure. The 3-dimensional shape that the peptide assumes when associated to the cell membrane will be an important parameter with regards receptor selectivity and affinity. Receptor affinity appears to depend on the different secondary structures of each tachykinin, which share the same hydrophobic C-terminal sequence, FXGLM. Binding of tachykinins to phospholipid bilayers may take place both on the aqueous membrane surface and in the hydrophobic region. Therefore, neutron diffraction measurements were carried out on highly aligned phospholipid multi-bilayers in order to define the location of the N- and C-terminus of NKA. This study reports that the bilayer location of NKA is remarkably similar to that of substance P, thereby inferring that finer levels of structure must control receptor specificity. Circular dichroism studies were carried out in order to define the conformation of NKA in structure-inducing solvents and in a range of disperse systems. NKA was found to be in a random coil conformation in many of the solvents but exhibited considerable b-sheet conformation in octan-1-o1. These results are related to the neutron diffraction data. A model of the NKA-bilayer interaction is presented using the results from these experiments and from other biophysical techniques. X-ray diffraction measurements shown that the peptide reduces the d-repeat of the membrane prior to the onset of an inverted cubic phase. This suggests that membrane thinning may play a role in peptide-induced model membrane fusion and strengthens the link between the fusion pathway and inverted cubic phase formation. The results of this study are interpreted in relation to models of the membrane fusion mechanism. Also included in this thesis is a study that aims to improve lamellar neutron diffraction data collection and analysis.

Les bêta-peptides possèdent deux propriétés remarquables : la stabilité face aux enzymes lytiques alors que les peptides naturels sont rapidement hydrolysés ; la propension à adopter des structures secondaires de type hélice, feuillet et coude. Cette propriété est très intéréssante car il est possible d'obtenir une structure secondaire particulière selon l'enchaînement spécifique de bêta-aminoacides constituant le bêta-peptide. Au laboratoire, nous nous sommes intéressés à la première synthèse de bêta-aminoacides cyclobutaniques de configuration "trans" et optiquement purs. Pour cela, nous avons mis au point deux voies de synthèse, diastéréosélective pour l'une et par dédoublement racémique pour l'autre, pour ensuite préparer des bêta-peptides cyclobutaniques. Nous avons mené une étude conformationnelle poussée sur les différents bêta-peptides basées sur des expériences de résonance magnétique nucléaire à une et deux dimensions le dichroïsme circulaire, la modélisation moléculaire et les rayons X d'un monocristal. Ces analyses nous ont permis d'affirmer l'existence d'une structure secondaire en hélice-12 des bêta-peptides cyclobutaniques à partir de six résidus bêta-trans-aminoacides cyclobutaniques

Les peptides de la famille du LAH4 sont des peptides cationiques capables de se replier en hélice α amphiphile. Ces peptides sont riches en histidines ce qui permet de moduler les interactions de ces peptides de manière pH dépendante et dans une gamme physiologique. Leurs capacités à interagir et perturber les membranes ont été utilisées pour divers applications biologique, et notamment pour l'amélioration de systèmes de transport de gènes.Le travail de cette thèse a été divisé en trois parties dans le but de caractériser de manière biophysique les différentes interactions ayant lieu lors de la livraison du système de transport de gènes à l’intérieur d’une cellule. L’interaction peptide-peptide : avec l’étude de l’agrégation en fibrilles de la VF1 ; l’interaction peptide-membrane : avec l’effet du LAH4L1 en présence de membranes ; et l’interaction peptide-ADN : avec le suivit de l’interaction entre le LAH4L1 et de l'ADN
The LAH4 family consists of cationic amphiphilic peptides with propensity to fold in α-helical secondary structures. They contain histidines allowing the modulation of their interactions in a pH dependent manner in the physiological range. In membranes, at neutral or acidic pH the peptide assumes a transmembrane or an in planar configuration, respectively.In the field of gene delivery systems, peptides like LAH4 are used. They are able to firstly interact with different cargoes in order to form stable complexes, then interact with the cell membrane, and finally, promote to escape from the endosome.This PhD has been divided into three parts in order to characterize, with biophysical methods, the interactions occurring during the delivery of these gene systems: peptide-peptide interactions with a focus on the study of VF1 fibre formation; peptide-membrane interactions: with the investigation of the effect of LAH4L1 in different membranes; and peptide-DNA interactions, where the interactions of LAH4L1 with a small DNA fragment were measured

Head-to-tail cyclic peptides with a wide range of ring sizes have been discovered in various organisms including bacteria, fungi, plants and animals. Many of them exhibit remarkable biological activities with high potency. Daptomycin, a cyclic lipodepsipeptide isolated from soil bacteria Stretomyces roseoporus, is the first natural product antibiotic launched in a generation. Daptomycin has potent bactericidal activity against otherwise antibiotic-resistant Gram-positive pathogens including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and vancomycin-resistant S. aureus. Daptomycin contains a 31-membered ring made up of 10 amino acids and a linear 3-amino acid side chain modified with an n-decanoyl lipid at the N-terminus. The complex structure of daptomycin, the presence of two non-proteinogenic amino acids (kynurenine and 3-methyl glutamic acid) and the macrolactamization of a 31-membered ring render daptomycin a challenging target for total synthesis. We recently developed a chemoselective serine/threonine ligation (STL) allowing peptide ligation at Ser/Thr site using side chain unprotected segments. We have successfully applied STL intramolecularly in the key cyclization step for the synthesis of daptomycin molecule, which allows us to finally achieve the first total synthesis of daptomycin. With this technique in hand, the chemical synthesis of daptomycin analogues, which are difficult to obtain otherwise becomes possible and is now ongoing. This would allow for the search for optimized analogues. We further investigated if STL could be applied for the synthesis of cyclic tetrapeptides, which are extremely difficult to be synthesized in the absence of turninducing components due to their rigid structural framework. To our delight, a series of cyclic tetrapeptides without any turn-inducing component, like Gly, Pro or Damino acids have been successfully synthesized by intramolecular STL. The synthesis of cyclic tetrapeptides with drug-like scaffold would be useful for the therapeutic development. We also applied intramolecular STL to successfully synthesize some natural cyclic peptides with different ring sizes, including anticancer stylopeptide 1, phakellistatin 4 and anti-inflammatory cyclosquamosin D.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2004.
"A dissertation in pharmaceutical sciences and chemistry." Advisor: Thomas P. Johnston. Typescript. Vita. Description based on contents viewed Feb. 28, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 135-148). Online version of the print edition.

Milk-derived peptides are milk components able to influence specific physiologicalfunctions, finally acting on body health condition. At present, the bioactivitiesdescribed for milk-derived peptides include opiate, antithrombotic, antihypertensive, immunomodulating, antioxidative, antimicrobial, anticancer, mineral-carrying andgrowth-promoting activities. In this thesis, special attention has been given to bioactive peptides with ACE-inhibitory and immunomodulatory activities. In the Experiment 1 Enterococcus faecalis TH563 (E. faecalis TH563) and Lactobacillus delbrueckii subsp. bulgaricus LA2 (L. delb. bulgaricus LA2), two bacterial strains isolated from traditional North Eastern Italy dairy products, have been evaluated for their ability to produce fermented milk rich in ACE-inhibitory and immunomodulatory activities. The preliminary results obtained from this experiment demonstrated that E. faecalis TH563 produced a fermented milk with high ACEinhibitory activity while L. delb. bulgaricus LA2 showed an immunomodulatory activity on bovine lymphocytes. To better understand the mechanisms underlying the immunomodulatory activity of fermented milks, in the Experiment 2 the immunomodulatory effects of the milkderived bioactive tri-peptide YGG have been examined. YGG could be generated during milk fermentation from a–lactalbumin hydrolysis operated by bacterial enzymes, so it could be present in milk fermented by L. delb. bulgaricus LA2. YGG has been administered to purified peripheral blood lymphocytes in different culture conditions (presence/absence of activators of lymphocyte proliferation, different concentration of newborn calf serum) and its effects on lymphocyte proliferation and cytokine RNA expression (IL2 and INFg) have been analyzed. YGG modulated lymphocytes proliferation, in a manner dependent from culture conditions but its effects did not seem mediated by the modulation of IL2 or INFg RNA expression. An important limiting factor of the large-scale diffusion of food carrying potential bioactivities is the bioavailability of the peptides responsible of such bioactivities. The main factors influencing the bioavailability of peptides are the resistance to digestion enzymes of and the absorption by the intestinal epithelium. In the Experiments 3 and 4 the sensitivity to gastrointestinal enzymes and the mechanisms of absorption of the peptide b-CN (193-209) have been evaluated. b-CN (193-209) is a long hydrophobic peptide derived from b-casein that has been already isolated and identified from fermented milks and yogurt and displayed immunomodulatory properties. The pattern of digestion and the mechanisms of absorption have been evaluated in well-known in vitro models for the intestinal epithelium, as the brush border membrane vesicles (BBMV) and the Caco-2 cell line. The results of these studies demonstrated that the b-CN (193-209) peptide is absorbed intact by the Caco-2 monolayer, probably via a vesicles-mediated mechanism. In conclusion, the main contribution of this PhD thesis was to provide new knowledge about milk-derived products with bioactivities. In particular, original contributions are in relation to the mechanisms by which milk-derived bioactive peptides are generated, express their bioactivities, and their fate in the gastrointestinal tract. As a result, new questions have arisen on this area that could constitute the objective of further research programs in the future.
I peptidi bioattivi derivati dal latte costituiscono una parte importante del latte, in grado di influenzare lo stato di salute. Attualmente nel latte e nei suoi derivati sono stati identificati e caratterizzati peptidi ad azione oppioide, anti-trombotica, antiipertensiva, immunomodulatoria, antiossidante, antimicrobica, anticancro, stimolanti l’assorbimento di minerali e la crescita. In questa tesi particolare attenzione è stata rivolta ai peptidi bioattivi ad attività ACE-inibitoria e immunomodulatoria. Nell'Esperimento 1 Enterococcus faecalis TH563 (E. faecalis TH563) e Lactobacillus delbrueckii subsp. bulgaricus LA2 (L. delb. bulgaricus LA2), due ceppi batterici isolati da formaggi tradizionali del Nord Italia, sono stati caratterizzati per la loro capacità di produrre latti fermentati arricchiti in attività ACE-inibitoria e immunomodulatoria. I risultati preliminari hanno dimostrato che il ceppo E. faecalis TH563 è in grado di produrre un latte fermentato con elevata attività ACE-inibitoria mentre il ceppo L. delb. bulgaricus LA2 produce un latte fermentato con attività immunomodulatoria su linfociti bovini. Per meglio comprendere i meccanismi che regolano l’attività immunomodulatoria manifestata dal latte fermentato, nell’Esperimento 2 sono stati riportati i risultati di un esperimento atto a valutare gli effetti immunomodulatori del peptide bioattivo YGG. Tale tripeptide può essere generato durante il processo di fermentazione del latte dalla proteina alfa–lattoalbumina mediante l’azione proteolitica degli enzimi batterici, e quindi anche durante la fermentazione operata dai ceppi E. faecalis TH563 e L. delb. bulgaricus LA2. YGG è stato somministrato a linfociti isolati da sangue bovino e ne è stata studiata la capacità di modulare la proliferazione dei linfociti e l’espressione (RNA) di due citochine (IL2 e INFg) in diverse condizioni di coltura (presenza/assenza di attivatori della proliferazione, diverse concentrazioni di siero bovino). Lo studio ha dimostrato che il peptide YGG è in grado di modulare la proliferazione delle cellule e che tale modulazione è influenzata dalle condizioni di coltura ma non sembra essere mediata dalle citochine oggetto di studio. Un fattore importante che limita l’impiego su larga scala di alimenti con proprietà bioattive è la biodisponibilità dei peptidi portatori di tali bioattività. I fattori che maggiormente influenzano la biodisponibilità dei peptidi sono la resistenza alla digestione operata dagli enzimi gastrointestinali e la possibilità che tali peptidi possano essere assorbiti dall’epitelio intestinale. A questo scopo, negli Esperimenti 3 e 4 sono stati esaminati il profilo di digestione e i meccanismi di assorbimento del peptide b-CN (193-209). b-CN (193-209) è un peptide bioattivo lungo e idrofobico,derivato dalla b-caseina ed è già stato isolato e identificato in diversi prodotti derivati dal latte come yogurt e latte fermentati. Tale peptide possiede inoltre diverse attività immunomodulatorie. Il profilo di digestione di tale peptide e i meccanismi di assorbimento intestinale sono stati studiati in modelli in vitro adatti a rappresentare la mucosa intestinale, come le vescicole della membrana a orletto a spazzola (BBMV) e la linea cellulare Caco-2. Tali esperimenti hanno dimostrato che il peptide viene assorbito intatto dalle cellule Caco-2, probabilmente attraverso un trasporto mediato da vescicole. In conclusione, il contributo principale di questa tesi di dottorato è stato il fornire nuova conoscenza sui prodotti derivati dal latte ad azione bioattiva. Più specificatamente, questa tesi ha permesso di ottenere nuove informazioni sui meccanismi di produzione dei peptidi bioattivi derivati dal latte, sul loro meccanismo d’azione e sulla loro stabilità nel sistema gastrointestinale. Infine, i risultati ottenuti hanno contribuito a generare nuove idee che potranno costituire nuovi spunti per futuri progetti di ricerca.

Peptides are small proteins that are crucial in many biological pathways such as antimicrobial defense, hormone signaling, and virulence. They often exhibit tight specificity for their targets and therefore have great therapeutic potential. Many peptide-based therapeutics are currently available, and the demand for this type of drug is expected to continue to increase. In order to satisfy the growing demand for peptide-based therapeutics, new engineering approaches to generate novel peptides should be developed. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of peptides that have the potential to be effective scaffolds for in vivo peptide engineering projects. These natural RiPP peptides are enzymatically endowed with post-translational modifications (PTMs) that result in increased stability and greater target specificity. Many RiPPs, such as microcin J25 and micrococcin, can tolerate considerable amino acid sequence randomization while still being capable of receiving unique post-translational modifications. This thesis describes how we successfully engineered E. coli to produce the lasso peptide microcin J25 using a two-plasmid inducible expression system. In addition, we characterized the protein-protein interactions between PTM enzymes in the synthesis of micrococcin. The first step in micrococcin synthesis is the alteration of cysteines to thiazoles on the precursor peptide TclE. This step is accomplished by three proteins: TclI, TclJ, and TclN. We found that a 4-membered protein complex is formed consisting of TclI, TclJ, TclN, and TclE. Furthermore, the TclI protein functions as a central adaptor joining two other enzymes in the Tcl pathway with the substrate peptide.

Dermatophilosis commonly known as "lumpy wool" in sheep in Australia, is a skin disease of animals and man caused by Dermatophilus congolensis. In sheep dermatophilosis causes significant wool production losses both directly and indirectly due to down grading of wool and hides, low meat production and high mortality. particularly in young sheep. Control of the disease by vaccination is desirable but has only been partially successful and antigens are difficult to produce in sufficient quantity from D. congolensis. Dr T.M. Ellis and colleagues (Agriculture WA) have developed a vaccine that can produce a significant reduction in ovine dermatophilosis against several strains of the bacterium. In pen and field studies, the haemolysin based vaccine did not prevent initial infection but approximately 70% were protected against the development of lumpy wool. Their previous research showed that a vaccine prepared from a serine protease produced by D. congolensis also gave some protection against the disease. This antigen was expensive to prepare by conventional culture. An alternative approach is to use recombinant protein as an antigen. However, this is not a simple task if the protective antigens have not been identified and even if they have been, it can be difficult to prepare a recombinant protein in high yield. Random peptide libraries provide an alternative approach to the identification of peptides which might be useful in a vaccine. While these libraries have been used to identify epitopes and prepare diagnostic tests, their potential to produce antigens which could generate a protective immune response has still to be explored. The main aim of this thesis was to use random peptide libraries to identify new antigens which could be used in the future to immunise sheep and other animals against dermatophilosis. Because of the commercial availability of large random peptide libraries displayed on phage and flagellin there is an opportunity to produce low cost and immunologically potent peptide vaccines. To explore the effectiveness and reliability of random peptide libraries (Ph.D.™ and FliTrxTM libraries) for the selection of peptides, polyclonal antibodies against a recombinant serine protease from D. congolensis were used to pan the libraries. This recombinant serine protease was produced using a pQE expression vector and purified by immobilised metal affinity chromatography. Clones selected from the libraries were sequenced and the peptides aligned with the original amino sequence of serine protease. Many of the peptides aligned with varying homology to the serine protease sequence which demonstrated that the antibodies were selecting specific sequences from the libraries rather than just random peptides. To obtain peptides, which might be associated with a protective immune response to D. congolensis sheep which had been immunised with a crude enzyme preparation form D. congolensis by Dr T.M. Ellis were used as a source of antibodies. Four peptides from Ph.D.TM and three peptides from FliTrx™ libraries were chosen for vaccination of sheep. Sheep were given two doses of vaccine one month apart. Twentyone days after the second vaccination each sheep was challenged with a zoospore suspension of the MB and W14 strains of D. congolensis and the presence of lesions and their severity were measured at 7, 14 and 21 days after challenge. The immune response to D. congolensis antigens was also studied. There was a striking production of antibodies to antigens from D. congolensis induced by the peptides selected from the random peptide libraries. Vaccination with recombinant serine protease and with the peptides selected from the Ph.D.™ library increased the rate of resolution of the lesions caused by one strain of D. congolensis. The results in this thesis provide the first demonstration in large animals that phage displayed peptides can induce a specific immune response against an infectious agent.