Gotowa bibliografia na temat „Α/γ Peptide”

As with all classical monomeric autotransporters, IgA protease of Neisseria meningitidis is a modular protein consisting of an N-terminal signal sequence, a passenger domain and a C-terminal translocator domain (TD) that assists in the secretion of the passenger domain across the outer membrane. The passenger of IgA protease consists of three separate domains: the protease domain, the γ-peptide and the α-peptide that contains nuclear localization signals (NLSs). The protease domain is released into the extracellular milieu either via autocatalytic processing or via cleavage by another autotransporter, NalP, expression of which is phase-variable. NalP-mediated cleavage results in the release of a passenger that includes the α- and γ-peptides. Here, we studied the fate of the α-peptide when NalP was not expressed and observed strain-dependent differences. In meningococcal strains where the α-peptide contained a single NLS, the α-peptide remained covalently attached to the TD and was detected at the cell surface. In other strains, the α-peptide contained four NLSs and was separated from the TD by an IgA protease autoproteolytic cleavage site. In many of those cases, the α-peptide was found non-covalently associated with the cells as a separate polypeptide. The cell surface association of the α-peptides may be relevant physiologically. We report a novel function for the α-peptide, i.e. the binding of heparin – an immune-modulatory molecule that in the host is found in the extracellular matrix and connected to cell surfaces.

ABSTRACT Spermidine and cadaverine were found to be constituents of the cell wall peptidoglycan of Anaerovibrio lipolytica, a strictly anaerobic bacterium. The peptidoglycan was degraded with theN-acetylmuramyl-l-alanine amidase and endopeptidase into two peptide fragments, peptide I and peptide II, at a molar ratio of 4:1. Peptides I and II were identified asl-alanine–d-glutamic acid(αcadaverine)γ meso-diaminopimelic acid (DAP)–d-alanine andl-alanine–d-glutamic acid(αspermidine)γ meso-DAP–d-alanine, respectively. The N1-amino group of spermidine was linked to the α-carboxyl group of the d-glutamic acid residue of peptide II.

Autoimmune disorder is a chronic immune imbalance which is developed through a series of pathways. The defect in B cells, T cells, and lack of self-tolerance has been greatly associated with the onset of many types of autoimmune complications including rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and chronic inflammatory demyelinating polyneuropathy. The SLE is an autoimmune disease with a common type of lupus that causes tissue and organ damage due to the wide spread of inflammation. In the current study, twenty anti-inflammatory peptides derived from plant and animal sources were docked as ligands or peptides counter to proinflammatory cytokines. Interferon gamma (IFN-γ), interleukin 3 (IL-3), and tumor necrosis factor alpha (TNF-α) were targeted in this study as these are involved in the pathogenesis of SLE in many clinical studies. Two docking approaches (i.e., protein-ligand docking and peptide-protein docking) were employed in this study using Molecular Operating Environment (MOE) software and HADDOCK web server, respectively. Amongst docked twenty peptides, the peptide DEDTQAMMPFR with S -score of -11.3018 and HADDOCK score of − 10.3 ± 2.5 kcal/mol showed the best binding interactions and energy validation with active amino acids of IFN-γ protein in both docking approaches. Depending upon these results, this peptide could be used as a potential drug candidate to target IFN-γ, IL-3, and TNF-α proteins to control inflammatory events. Other peptides (i.e., QEPQESQQ and FRDEHKK) also revealed good binding affinity with IFN-γ with S -scores of -10.98 and -10.55, respectively. Similarly, the peptides KHDRGDEF, FRDEHKK, and QEPQESQQ showed best binding interactions with IL-3 with S -scores of -8.81, -8.64, and -8.17, respectively.

Abstract An achiral tripeptide, namely, Boc-γ-Abu-m-ABA-Aib-OMe (γ-Abu: γ−amino butyric acid; m-ABA: meta-aminobenzoic acid) was synthesized by solution phase procedure. The α, γ-hybrid peptide was designed in such a way that two dissimilar γ−amino acids, one flexible and another rigid, were positioned sidewise along with α-amino isobutyric acid (Aib) as C-terminal residue. The single crystal X-ray diffraction analysis revealed that two kinks were generated around centrally placed m-ABA. Interestingly, the peptide self-assembled via three intermolecular N–H···O and one intermolecular C–H···O hydrogen bonding interactions to supramlecular helical architecture.

Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the α- and γ-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the γ-subunit at sites flanking a 43 residue inhibitory tract (γE144-K186). To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the γ-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the γ-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the γ-subunit further identified a key 11-mer tract, R158-F168 (RFLNLIPLLVF), which inhibited wild-type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native γ inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the γ inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel.

Seven novel, amphiphilic AApeptides were prepared. Two cationic, lipo-α-AApeptides, NA-75 and NA-77 were found to possess potent antimicrobial activity against Gram-positive bacteria, with almost no hemolytic activity. In addition to NA-75 and 77, four amphiphilic, γ-AApeptides, NA-133, 135, 137, and 139, and one anionic lipo-α-AApeptide, NA-81, were prepared for molecular self-assembly studies, with several interesting nanostructures observed by TEM. Mineralization of calcium carbonate from gaseous CO2 and Ca2+ in the presence of the 7 AApeptide amphiphiles was also observed by optical microscopy. Several AApeptides were found to be able to influence CaCO3 crystal morphology. Another α-AApeptide, NA-63, was synthesized by a novel, alternative method, which has several potential advantages over the previous synthesis methods.

Le travail présenté concerne la conception et la synthèse de peptides modifiés et se divise en deux parties. La première est l'introduction d'un résidu aminé vinylogue à stéréochimie cis et trans dans un undécapeptide, la substance P pour comprendre ses interactions avec le récepteur NK-1. Une seconde étude a été orientée vers l'obtention de g-aminopeptides par hydrogénation des acides aminés vinylogues précédents. Cette modification structurale a donné un nouvel analogue de la SP. La seconde partie est de la méthodologie de synthèse en utilisant les peptides vinylogues. Un premier chapitre, la dihydroxylation de ces motifs avec une induction asymétrique en utilisant des réactifs chiraux, permet l'obtention de g-aminoacides dihydroxylés. Un second chapitre, une application par la synthèse totale d'un produit naturel issu d'une plante nyctinastique. Et le dernier chapitre est une autre application, la synthèse de lactames dihydroxylés conduisant à la synthèse de nouveaux azasucres
This work concerns conception and synthesis of modified peptides and is divided in two parts. Firstly, it's the insertion of vinylogous amino acids with cis and trans conformation in neuropeptide of eleven amino acids which is substance P in order to understand its interaction with NK-1 receptor. A second study has been oriented on the preparation of g-amino peptides by hydrogenation of previous vinylogous amino acids. This structural modification has given a new SP's analog which has been tested. The second part is the methodology of synthesis using vinylogous peptides and is divided in three chapters. First one presents dihydroxylation's results of these residues with an asymmetric induction using chiral catalyst to obtain dihydroxylated g-amino acids. Second one is an application of these studies with a total synthesis of natural product extracted from nyctinastic plant. And the last one deals with preparation of dihydroxylated lactams leading to the synthesis of new azasugars

L’utilisation d’oligomères d’α-amino-γ-lactame (Agl-αAA) comme vecteurs de pénétration intracellulaire est décrite dans ce manuscrit. Nous avons montré que ces oligomères structurés en ruban sont capables de traverser la membrane plasmique pour atteindre le cytosol et y délivrer un cargo biologiquement actif. A la différence des séquences peptidiques, ces oligomères ont une très bonne résistance enzymatique. Une nouvelle famille de foldamères d’α-amino-γ-lactame (Agl-βAA) obtenus à partir de séquences /β peptidiques est également décrite. La structure secondaire de ces composés a été étudiée par RMN, IR-TF, CD et DRX. Nous avons montré que ces oligomères sont capables d’adopter une structure stable en hélice 12. De façon remarquable, ces oligomères sont solubles en milieux aqueux malgré une absence totale de chaînes latérales hydrophiles
The use of α-amino-γ-lactam oligomers (Agl-αAA) as cell penetrating vectors are described in this work. These ribbon structured oligomers are able to cross the cell membrane to reach the cytosol and deliver a biologically active cargo. Unlike peptide sequences, these oligomers display a strong enzymatic resistance. A new family of α-amino-γ-lactam oligomers (Agl-βAA) obtained from conversion of /β peptide sequences are also described. Secondary structure of these molecules have been studied by NMR, FTIR, CD and XRD. These oligomers are able to adopt a stable 12-helix structure. Unexpectedly, these oligomers are soluble in aqueous mediums without any hydrophilic side chains

Structural studies of peptides are of great importance in developing novel and effective biomaterials ranging from drugs and vaccines to nano materials with industrial applications. In addition, they provide model systems to study and mimic the protein conformations. The ability to generate folded intramolecularly hydrogen bonded structures in short peptides is essential for peptide design strategies, which rely on the use of folding nuclei in the construction of secondary structure modules like helices and β-hairpins. In these approaches, conformational choices at selected positions are biased, using local stereochemical constraints, that limit the range of accessible backbone torsion angles. X-ray crystallographic studies of designed peptides provide definitive proof of the success of a design strategy, and provide essential structural information that can be utilized in the future design of biologically and structurally important polypeptides. Recent trends in peptide research focus on the incorporation of β-, γ- and higher homologs of the α-amino acid residues in designed peptides as they confer more proteolytic stability to the polypeptides. X-ray crystallographic studies of such modified peptides containing non-protein residues are essential, since information on the geometric and stereochemical properties of modified amino acids can only be gathered from the systematic structural studies of synthetic peptides incorporating them. This thesis reports a systematic study of the structures and conformations of amino acid derivatives and designed peptides containing stereochemically constrained α-, β- and γ-amino acid residues and the structural studies of polymorphic peptide helices. The structures described in thesis contain the Cα,α-dialkyalted α-residues α-aminoisobutyric acid (Aib) and 1-aminocyclohexane-1-carboxylic acid (Ac6c), the β-amino acid residue 1-aminocyclohexane acetic acid (β3,3Ac6c) and the γ-amino acid residue 1-aminomethylcyclohexaneacetic acid (gabapentin, Gpn). The crystal structure determination of peptides incorporating conformationally constrained α-, β- and γ- amino acid residues permitted the characterization of new types of hydrogen bonded turns and polymorphs. The studies enabled the precise determination of conformational and geometric parameters of two ω-amino acid residues, gabapentin and β 3,3Ac6c and provided detailed information about the conformational excursions possible for peptide molecules. This thesis is divided into 10 chapters. Chapter 1 gives a general introduction to the stereochemistry of the polypeptide chain, description of backbone torsion angles of α- and ω- amino acid residues and the major secondary structures of α-peptides, β-peptides, γ-peptides and hybrid peptides. A brief introduction to polymorphism and weak interactions, in particular aromatic interactions, is also provided, followed by a discussion on X-ray diffraction and solution to the phase problem. Chapter 2 describes the crystal structures of gabapentin zwitterion and its eight derivatives (Ananda, Aravinda, Vasudev et al., 2003). The crystal structure of the gabapentin zwitterions determined in this study is identical to that previously reported (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643). Eight of the nine achiral compounds crystallized in centrosymmetric space groups P21/c, C2/c or Pbca, while one derivative (Tos-Gpn-OH) crystallized in non-centrosymmetric space group Pna21 with four independent molecules in the asymmetric unit.The structural studies presented in this chapter reveal that the geminal substituents on the Cβ atom limits the values of dihedral angles θ1 and θ2 to ±60°, resulting in folded backbone conformations in all the examples. Intramolecular hydrogen bonds with 7-atoms in the hydrogen bond turn (C7) are observed in three derivatives, gabapentin hydrochloride (GPNCL), Boc-Gpn-OH (BGPNH) and Piv-Gpn-OH (PIVGPN), while a 9-atom hydrogen bonded turn (C9) is observed in Ac-Gpn-OH (ACGPH). Unique structural features, such as an unusual anti conformation of the COOH group (in ACGPH) and positional disorder of the cyclohexane ring (in BGPNN), indicating the co-existence of both the interconvertible chair conformations, are revealed by the crystal structure analyses. Chapter 3 describes the structural characterization of novel hydrogen bonded conformations of homo oligomers of Gpn. The crystal structures of three peptides, Boc-Gpn-Gpn-NHMe (GPN2), Boc-Gpn-Gpn-Leu-OMe (GPN2L) and Boc-Gpn-Gpn-Gpn-Gpn-NHMe (GPN4) provide the first crystallographic characterization of two new families of polypeptide structures, the C9 helices and C9 ribbons (Vasudev et al., 2005, 2007), in which the molecular conformations are stabilized by contiguous C9 turns formed by the hydrogen bonding between the CO group of residue (i) and the NH group of residue (i+2). The C9 hydrogen bond is characterized by a specific combination of the four torsion angles for the Gpn backbone, with the torsion angles θ1 and θ2 adopting g+/g+ or g /g- conformations. The structural analysis also permits precise determination of hydrogen bond geometry for the C9 structures, which is highly linear in contrast to the analogous γ-turn hydrogen bonds in α-peptides. A comparison of the backbone conformations in the three peptides reveals two classes of C9 hydrogen bonded secondary structures, namely C9 helices and C9 ribbons. The packing arrangement in these γ-peptides follows the same patterns as the helix packing in crystals of α-peptides. Chapter 4 describes ten crystal structures of short hybrid peptides containing the Gpn residue (Vasudev et al., 2007). In addition to the C7 and C9 hydrogen bonded turns which are defined by the backbone conformations at the Gpn residue, hybrid turns defined by a combination of backbone conformations at the α and γ-residues or at the β and γ-residues have been determined. Peptides Boc-Ac6c-Gpn-OH (ACGPH), Piv-Pro-Gpn-Val-OMe (PPGPV) and Boc-Val-Pro-Gpn-OH (VPGPH) reveal molecular conformation stabilized by intramolecular C9 hydrogen bonds, while Boc-Ac6c-Gpn-OMe (ACGPO) and Boc-Gpn-Aib-OH (GPUH) are stabilized by a C7 hydrogen bonded turn at the Gpn residue. An αγ hybrid turn with 12 atoms in the intramolecular hydrogen bonded rings (C12 turns) has been observed in the tripeptide Boc-Ac6c-Gpn-Ac6c-OMe (ACGP3), while βγ hybrid turns with 13 atoms in the hydrogen bonded ring (C13 turns) have been characterized in the tripeptides Boc-βLeu-Gpn-Val-OMe (BLGPV) and Boc- βPhe-Gpn-Phe-OMe (BFGPF). The two βγ C13 turns belong to two different categories and are characterized by different sets of backbone torsion angles for the β and γ residues. A γα C10 hydrogen bond, which is formed in the N→C direction (NHi ••• COi+2), as opposed to the regular hydrogen bonded helices of α-peptides, has also been observed in BFGPF. The Chapter provides a comparison of the backbone torsion angles of the Gpn residue in various hydrogen bonded turns and a brief comparison of the observed hydrogen bonded turns with those of the α-peptides. Chapter 5 describes the crystal structures of three αγ hybrid peptides which show C12/C10 mixed hydrogen bond patterns (Vasudev et al., 2007, 2008a; Chatterjee, Vasudev et al.,2008a). The insertion of gabapentin in the predominantly α-amino acid sequences in Boc-Ala-Aib-Gpn-Aib-Ala-OMe (AUGP5) and Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe results in the observation of helices stabilized by αα C10 (310-turn) and αγ C12 turns. The tetrapeptide Boc-Leu-Gpn-Leu-Aib-OMe reveals a novel conformation, stabilized by C12 (αγ) and C10 (γα) hydrogen bonds of opposite hydrogen bond directionalities. The conformations observed in crystals have been extended to generate C12 helix and C12/C10 helix with alternating hydrogen bond polarities in ( αγ)n sequences. The structure determination of three crystals, providing five molecular conformations, presented in this chapter provides the first crystallographic characterization of two types of helices predicted for the regular αγ hybrid peptides from theoretical calculations. The crystal structure of Boc-Ala-Aib-Gpn-Aib-Ala-OMe also provides an example for the co-existence of left-handed and right-handed helix in the asymmetric unit. Chapter 6 describes the structural studies of αγ hybrid peptides containing Aib and Gpn residues, and is divided into two parts. The first part presents the crystal structure analysis of peptides of sequence length 2 to 4, with alternating Aib and Gpn residues, and illustrates the conformational variability in αγ hybrid sequences as evidenced by the observation of conformational polymorphs (Chatterjee, Vasudev et al., 2008b; Vasudev et al., 2007; Ananda, Vasudev et al., 2005). The peptide Boc-Gpn-Aib-NHMe (GUN), Boc-Aib-Gpn-Aib-OMe (UGU), Boc-Gpn-Aib-Gpn-Aib-OMe (GU4O), Boc-Aib-Gpn-Aib-Gpn-OMe (UG4O) and Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), all of which are potential candidates for exhibiting αγ C12 hydrogen bonds, reveal molecular conformations stabilized by diverse hydrogen bonded turns such as C7, C9, C12 and C17 in crystals. The conformational heterogeneity in this class of hybrid peptides is further evidenced by the observation of three polymorphs in the monoclinic space group P21/c for the tetrapeptide Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), providing four independent peptide molecules adopting two distinct backbone conformations. In one polymorph, C12 helices terminated with an unusual three residue ( γαγ) C17 turn is observed, while the unfolding of helical conformation by solvent insertion into the backbone is observed in the other two polymorphs. The studies indicate the possible utility of Gpn residue in stabilizing locally folded conformations in the folding pathway, thus permitting their crystallographic characterization in multiple crystal forms. A discussion of the structural and conformational features of Gpn residues determined from all the crystal structures is presented in the Chapter, along with a φ-ψ plot for the Gpn residue. Part 2 of Chapter 6 describes the crystal structures of two octapeptides, Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (GU8) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (LFVUG8), featuring C12 turns at the Aib-Gpn segments (Chatterjee, Vasudev et al., 2009). GU8 folds into a C12 helix flanked by C9 hydrogen bonds at both the termini, while LFVUG8 adopts β-hairpin conformation with a chain-reversing C12 turn at the central Aib-Gpn segment. A remarkable feature of the Aib-Gpn turn in the β-hairpin structure is the anti conformation about the Cβ-Cα (θ2) bond, which is the only example of a Gpn residue not adopting gauche conformation for both θ1 and θ2. The crystal structures of the two peptides, mimicking the two major secondary structural elements of α-peptides in hybrid polypeptides, permits a comparative study of the mode of molecular packing in crystals of α-peptides and hybrid peptides. The chapter also discusses theoretical calculations on αγ hybrid sequences, which reveal new types of C12 hydrogen bonded turns. Chapter 7 describes the crystal structures of conformationally biased tert-butyl derivatives of Gpn. The crystallographic characterization of the E (trans) and Z (cis) isomers of the residue,three protected derivatives and a tripeptide provides examples of C7 and C9 hydrogen bonded conformations, suggesting that the C7 and C9 hydrogen bonds can be formed by Gpn residues with both the chair conformations of the cyclohexane ring. Chapter 8 describes the systematic structural studies of the derivatives and peptides of the stereochemically constrained β- amino acid residue, β3,3Ac6c (Vasudev et al., 2008c). The backbone torsion angles φ and θ adopt gauche conformation in majority of the examples, owing to the presence of a cyclohexane ring on the Cβ atom. In contrast to Gpn, β3,3Ac6c does not show strong preference for adopting intramolecularly hydrogen bonded conformations. Of the 16 crystal structures determined, intramolecular hydrogen bonds involving the β-residue are observed only in 4 cases. The amino acid zwitterion (BAC6C), the hydrochloride (BACHCL) and the dipeptide Boc-β3,3Ac6c-β3,3Ac6c-NHMe (BAC62N) form N-H•••O hydrogen bonds with 6-atoms in the hydrogen bond ring (C6 turns). An αβ hybrid C11 hydrogen bonded turn is characterized in the dipeptide Piv-Pro-β3,3Ac6c-NHMe, which is distinctly different from the C11 hydrogen bonds observed in αβ hybrid peptide helices. Several unique structural features such as a dynamic disorder of the hydrogen atom of the carboxylic acid group (in BBAC) and cis geometry of the urethane bond (in BBAC, BAC62N and BPBAC) have been observed in this study. A comparison of the backbone conformations of β3,3Ac6c with other β- amino acid residues is also provided. Chapter 9 describes the crystallographic characterization of a new polymorph of gabapentin monohydrate and crystal structures of the zwitterions of E and Z isomers of tert-butylgabapentin and its hydrochloride and hydrobromide (Vasudev et al., 2009). A comparison of the crystal structures of the monoclinic form (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643) of gabapentin monohydrate and the newly characterized orthorhombic form reveals identical molecular conformations and intermolecular hydrogen bond patterns in both the polymorphs. The two polymorphs show differences in the orientation of molecules constituting a layer of hydrophobic interactions between the cyclohexyl side chains. A comparison of the packing arrangements of the zwitterionic amino acid molecules in the crystal structures of gabapentin monohydrate, the tert-butyl derivatives and other co-crystals of gabapentin that had been characterized so far, is provided which would facilitate prediction of new polymorphs of the widely used drug molecule, Gpn. Chapter 10 describes the crystallization of α-peptide helices in multiple crystal forms (Vasudev et al., 2008b). Crystal structures of two peptides, Boc-Leu-Aib-Phe-Phe-Leu-Aib-Ala-Ala-Leu-Aib-OMe (LFF), Boc-Leu-Aib-Phe-Ala-Leu-Ala-Leu-Aib-OMe (D1) in two crystal forms and the crystal structure of a related sequence, Boc-Leu-Aib-Phe-Ala-Phe-Aib-Leu-Ala-Leu-Aib-OMe (D10) permit an analysis of the molecular conformation and packing patterns of peptide helices in crystals. The two polymorphs of LFF, crystallized in the space groups P21 and P22121, reveal very similar molecular conformation (α/310-helix) in both the polymorphic crystals; the two forms differ significantly in the pattern of solvation. The crystal structure determination of a monoclinic (P21) and an orthorhombic polymorph (P21212) of D1 provides five different peptide conformations, four of which are α-helical and one is a mixed 310/α-helix. The crystal structure determination of the three peptides provide an opportunity to compare the nature and role of aromatic interactions in stabilizing molecular conformation and packing and its significance in the observation of polymorphism. An analysis of the Cambridge Structural Database and a model for nucleation of crystals in hydrophobic peptide helices are also discussed.

Les accouchements prématurés restent un défi pour la médecine moderne. Malgré les efforts de préventions déployés, les taux de naissances prématurés sont en constante augmentation dans les pays industrialisés. Le récepteur interleukine-1 (IL-1R) a été étudié dans le but de développer des agents thérapeutiques pouvant prolonger la gestation et mener à des prognostiques néonataux. Dans cette optique, il y a été démontré que le peptide 101.10 possède a démontré une capacité à moduler le récepteur IL-1R, retarder les accouchements prématurés et réduire l’incidence de la rétinopathie prématurée par un mécanisme allostérique impliquant une signalisation biaisée. Dans le but d’étudier la conformation active du peptide 101.10 et d’en améliorer l’activité, nous avons développé une méthode pour introduire des α-amino γ-lactames β-substituées dans des séquences peptidiques. Appliquer cette stratégie au peptide 101.10 a permis d’améliorer la compréhension de la relation structure-activité pour la modulation allostérique du récepteur IL-1R. Les composés peptidomimétiques ont le potentiel de mimer la conformation et l’activité des peptides bioactifs. Ils offrent le potentiel d’améliorer la reconnaissance moléculaire, d’optimiser le transport à travers des différentes membranes biologiques et d’augmenter la résistance métabolique. Parmi les différentes classes de composés peptidomimétiques, les α-amino γ-lactames (Agl) permet de rigidifier par des liens covalents la chaine peptidique principale favorisant les structures secondaires de type tour β. Les analogues Agl β-substituées mimer et rigidifier la chaine latérale des acides aminés. Cette thèse introduit des méthodes efficaces pour la synthèse stéréocontrolée des résidus α-amino γ-lactames β-substituées possédant diverses fonctionnalités. L’introduction de ces résidus dans différents peptides bioactifs a été effectuée pour étudier leur relation structure-activité. En utilisant le peptide modulateur du récepteur IL-1R 101.10 comme peptide représentatif, la présente recherche a permis d’identifier de nouveaux agents tocolytiques qui peuvent prolonger la gestation et améliorer le pronostique néonatal. Dans le chapitre 2, des stéréo-isomères de (Agl) et β-hydroxy-α-amino-γ-lactam (Hgl) ont été utilisés pour étudier l’influence de la configuration du groupement hydroxyle sur la conformation et l’activité du peptide 101.10. L’orientation de ce groupement dans les peptides Agl et Hgl s’est avéré avoir une influence conformationnelle et sur l’activité. La spectroscopie par dichroïsme circulaire (CD) a illustré la propension de certains analogues, comme le [(3R,4S)-Hgl3]-101.10, à adopter des tours β. Les analogues Agl et Hgl ont été examinés dans une série d’essais in vitro et in vivo modélisant les accouchements prématurés. Dépendant de leur structure et configuration, les analogues lactames ont démontré une sélectivité fonctionnelle différente dans diverse processus biologiques, démontrant les particularités de divers phénotypes. Par exemple, l’inhibition des JNK et ROCK kinases s’est avérée importante respectivement dans leurs effets tocolytiques et dans la diminution de la vaso-oblitération. Notamment, parmi les douze analogues testés, [(3R,4S)-Hgl3]-101.10 s’est avéré démontrer la même activité in vitro et in vitro que le peptide parent. Dans le chapitre 3, des méthodes de déplacement du groupement hydroxyle des résidus Hgl ont permis l’introduction stéréosélective de substituent en position β des résidus Agl. Une combinaison de réaction de Mitsunobu sur les résidus trans Hgl et une ouverture nucléophile les sulfamidates cycliques dérivés des lactames cis, ont mené à l’obtention de mime rigidifiés de résidus Ser, Thr, Cys, Dap, Dab, His et Met. Dans le chapitre 4, différentes lactames β-substitués ont été introduits dans la séquence du peptide 101.10 par une combinaison de chimie en solution et sur support solide pour étudier d’avantage les éléments structurels nécessaire pour réguler l’activité et la signalisation de cette cytokine clef dans la médiation de l’inflammation. Considérant l’activité de l’analogue [(3R,4S)-Hgl3]-101.10, plusieurs analogues β-substitués possédant une orientation similaire pour la chaine principale et latérale ont été synthétisés. Certains analogues ont démontré une activité biologique prometteuse dans des modèles de rétinopathie et seront étudiés dans le futur. En conclusion, des méthodes de synthèse d’α-amino-γ-lactames et de leur contrepartie β-substitués et leur introduction dans des peptides d’intérêt pour étudier leur relation structure-activité ont été développés. En utilisant ces méthodologies sur le modulateur allostérique du récepteur IL-1R 101.10, le conformère actif in vivo responsable de l’activité tocolytique et protectrice contre la rétinopathie associée aux accouchements prématures ont été identifiés. Considérant l’utilité de la synthèse de lactames pour le développement d’agents susceptibles de prolonger la gestation et d’améliorer le prognostique associé aux accouchements prématurés, cette thèse a permis la conception de prototypes de médicaments pour traiter les accouchements prématurés ainsi que l’évaluation des contraintes structurelles pertinentes pour la biologie des peptides.
Preterm birth (PTB) is an unmet biomedical need. Despite efforts to counter the onset of preterm labor, the rate of premature birth has increased steadily in developed countries. The interleukin-1 receptor (IL-1R) has been pursued as a target for designing agents which can prolong labor and improve neonatal outcomes. Towards these goals, a lead peptide 101.10 had been shown to modulate the IL-1R, to delay PTB and to mitigate associated retinopathy of prematurity (ROP) by an allosteric mechanism featuring biased signaling. With the goals of understanding the active conformers and improving the activity of 101.10, methods were conceived for the synthesis and introduction of β-substituted α-amino γ-lactams into peptides. Applying such methods on 101.10 has provided insight into the structure-activity relationships required for allosteric modulation of the IL-1R. Peptidomimetics are promising structures that replicate peptide function and conformation. They offer the potential to improve molecular-recognition, to enhance transport across biological membranes, and to resist metabolism. Among peptidomimetic classes, α-amino γ-lactam (Agl) residues introduce covalent constraint to rigidify the peptide backbone and have been employed to favor turn secondary structures. β-Substituted Agl analogs offer additional potential to mimic and restrict peptide side-chain geometry. This thesis introduces effective methods for the stereo-controlled synthesis of β-substituted α-amino γ-lactams residues having various side chain functionality. Introduction of the parent Agl residue and β-substituted counterparts into biologically active peptides has been explored to study structure-activity relationships. Employing the IL-1R modulator 101.10 as a representative peptide, the described research has furnished novel labor delaying agents that can improve neonatal outcomes. In chapter 2, α-amino-γ-lactam (Agl) and β-hydroxy-α-amino-γ-lactam (Hgl) stereoisomers were employed to study the influence of configuration and hydroxyl group side chain on conformation and activity of the interleukin-1 receptor modulator peptide 101.10. The configuration and hydroxyl group side chain influenced the conformation and biological activity of Agl and Hgl-101.10 analogs. Circular dichroism (CD) spectroscopy illustrated β-turn conformers for specific analogs, such as [(3R,4S)-Hgl3]-101.10. The Agl and Hgl analogs were examined in a series of in vitro assays and in vivo models of PTB. Contingent on their structure vi and configuration, the lactam analogs exhibited different functional selectivity in the various biological pathways, and indicated the requirement for specific phenotypes. For example, inhibition of the JNK and ROCK kinase pathways were respectively shown to be important for delaying labor and diminishing vaso-obliteration in the PTB and ROP models. Notably, among the twelve analogs, [(3R,4S)-Hgl3]-101.10 was found to exhibit identical in vitro and in vivo activity as the parent peptide. In chapter 3, methods were developed for displacement of the β-hydroxy-α-amino-γ-lactam (Hgl) residue alcohol to introduce stereo-selectively different β-substituents on Agl residues. A combination of Mitsunobu chemistry on the trans Hgl residue, and nucleophilic ring opening of the cyclic sulfamidate derived from the cis lactam counterpart provided constrained mimics of Ser, Thr, Cys, Dap, Dab, His and Met residues. In chapter 4, various β-substituted lactams were introduced into the sequence of 101.10 by combination of solution and solid phases chemistry to further study the structural requirements for regulating the activity and signaling of this key cytokine mediator of inflammasome activation. Considering the activity of [(3R,4S)-Hgl3]-101.10, the β-substituted Agl analogs were synthesized possessing similar backbone and side chain configurations. Certain analogs exhibited promising biological activity in the ROP model meriting further study. In sum, methods were conceived for the synthesis and application of α-amino-γ-lactams and their β-substituted analogs to study peptide structure-activity relationships. Employing this chemistry on the IL-1R allosteric modulator 101.10 has identified the active conformer and in vitro activity responsible for ability to delay labor and mitigate retinopathy of prematurity. Considering the utility of the lactam synthesis methods for the development of improved agents for delaying labor and improving neonatal outcomes, this thesis has conceived useful prototypes for drugs to treat PTB, as well as useful methods for dissecting the structural requirements for peptide chemical biology.

The fibrinolytic system plays a vital role in maintaining vital organ homeostasis. Fibrinolysis, defined as the dissolution of fibrin (the major scaffold for intravascular thrombus), is the process that regulates thrombus growth after hemostasis has been achieved, thus preserving tissue perfusion. An understanding of fibrinolysis has led to the development of pharmacologic agents that can be used in the treatment of arterial and venous thrombotic disorders, including acute myocardial infarction, acute ischemic stroke, and pulmonary embolism. Fibrinolytic therapy makes use of the vascular system’s intrinsic defense mechanism by accelerating and amplifying the conversion of an inactive enzyme precursor (zymogen), plasminogen, to the active enzyme plasmin. In turn, plasmin hydrolyzes several key bonds in the fibrin (clot) matrix, causing dissolution (lysis). A single-chain glycoprotein consisting of 790 amino acids, plasminogen is converted to plasmin by cleavage of the Arg560–Val561 peptide bond. The plasminogen molecule also contains specific lysine binding sites, which mediate its interaction with fibrin and α2-plasmin inhibitor. A serine protease with trypsinlike activity, plasmin attacks lysyl and arginyl bonds of fibrin at two principal sites: (1) the carboxyterminal portion α-chain (polar region) and (2) the coiled coil connectors containing α-, β-, and γ-chains. The ability of a fibrinolytic agent to dissolve an occlusive thrombus is determined by several factors. After administration the agent must be delivered to, perfuse, and ultimately infiltrate the thrombus while concomitantly being provided with an adequate amount of substrate (plasminogen) and the appropriate metabolic environment for an enzymatic reaction (conversion of plasminogen to plasmin) to take place. The intrinsic composition or ultrastructure of a thrombus also affects its lysability. Changes in the total amount and distribution of blood flow determine oxygen delivery to metabolically active tissues. They also determine the delivery of enzymatic substrate and plasminogen activators to the occlusive thrombus. In the heart, coronary blood flow correlates directly with mean arterial pressure. The flow-pressure curve is relatively flat above 65 to 70 mmHg, but becomes steeper as the mean arterial pressure decreases below this point. The relationship within the brain is more complex.

Fibrinogen interacts with platelets and endothelial cells via specific binding sites. While the platelet fibrinogen receptor has been identified and was found to be associated with GPIIb-IIIa, the binding site on endothelial cells has not been characterized yet. The platelet GPIIb-IIIa belongs to the newly identified cytoadhesin family which includes immunologicaly related receptors interacting with RGD containing proteins. A cytoadhesin has recently been described on endothelial cells and the possibility that fibrinogen might interact with this glycoprotein was examined. Peptides corresponding to γ and α chains sequences were synthesized and their capacity to inhibit fibrinogen binding to endothelial cells and platelets were compared. Analogues of the γ chain from His 400 to Val 411 produced an inhibition similar to that observed for fibrinogen platelet interaction, suggesting that the structure function relationship of γ peptides is identical in both systems. In contrast synthetic analogues corresponding to the a chain and including RGD yielded slightly different results. While RGD alone was inactive on platelet, this tripeptide was active on endothelial cells. RGDS and RGDF corresponding to α 572-575 and α 95-98 partially or fully inhibited fibrinogen binding to endothelial cells but the structure activity relationship was different when compared to that observed for platelets. Addition of the N and C sequences adjacent to RGDS reduced the activity of this peptide whereas the activity of RGDF analogues was not modified by addition of N and C-sequences. In contrast platelet fibrinogen binding decreased with these RGDF analogues. These results suggest that fibrinogen endothelial cell binding is mediated by an RGD adhesion receptor with subtle differences in the recognition selectivity of the ligand, which is controled by the surrounding sequence.

Factor XIIIa catalysis the formation of isopeptide bonds Between γ-carbamoyl groups of peptide-bound glutamines and ε-amino groups of lysines or lysine analogues. During fibrin crosslinking two such bonds are rapidly formed between the C-termini of two γ-chains in adjacent molecules and then several bonds are more slowly formed between several α-chains. The crosslinking sites in the γ-chain were identified already 15 years ago, those in the α-chain are still only tentatively or partially identified,, However, by determining the incorporation of lysine analogues in the α-chain it could be shown that the glutamines in positions 328, 366 and possibly also 237 may participate in crosslinking reactions. Analyses of cyanogen bromide fragments isolated from crosslinked fibrin indicated the segments 271-776 and 518-587 to contain the primary crosslinking sites.In the present study factor XHI-containing fibrinogen was incubated over night with thrombin in presence of calcium ions and cysteine or, as a control, in presence of EDTA. The fibrin material was cleaved with cyanogen bromide, mercaptolysed, pyri-dylethylated and then subjected to Sephacryl S-300 chromatography. The early protein fractions were tested by reversed-phase high-performance liquid chromatography (HPLC) using fibrinogen fragments as reference. In the control sample Aa-chain fragment 271-776 eluted first but in the crosslinked sample it was missing and instead a heterogeneous mixture of higher-molecular weight components was observed. N-Terminal sequence analysis showed the mixture to contain not only the expected fragments 241-476 and 518-584,but in fact all glutamine- or lysine-containing Aα-chain fragments between positions 208 and 611. In the corresponding 6 fragments a total of 6 glutamines and 21 lysines as potential crosslinking sites are present. Two fragments contain only one each of these residues which therefore must be true crosslinking sites. Remaining sites and the actual linkages were identified after reversed-phase HPLC of the tryptic peptide mixture by N-terminal sequence and total amino acid analyses.The linkage pattern will provide information about the localisation and conformation of the C-terminal part of the α-chain and its contribution to the fibrin polymer structure.

The molecular basis of platelet-fibrin interactions has been investigated by using synthetic peptides as potential inhibitors of binding fibrin protofibrils and fibrinogen to ADP-stimulated platelets, adhesion of fibrin fibers to the platelet surface, and platelet-mediated clot retraction. Synthetic peptides RGDS and HHLGGAKQAGDV, corresponding to regions of the fibrinogen α and γ chains previously identified as platelet recognition sites, inhibited the binding of radiolabelled soluble fibrin oligomers to ADP-stimulated platelets with IC50 values of 12 and 40 μM, respectively. The IC50 values obtained with fibrinogen as the ligand were 3-fold higher. Synthetic GPRP and GHRP, corresponding to the N-terminal sequences of the fibrin α and β chains, were minimally effective in blocking soluble fibrin oligomer binding to ADP-stimulated platelets. The extent of fibrin:platelet adhesion was determined with a microfluorimetric technique which measures the quantity of fluorescein-labelled fibrin attached to the surface of platelets. The signal obtained from the brightly fluorescent platelet:fibrin adducts was time- and concentration-dependent, and was fully inhibited by a monoclonal antibody directed against the glycoprotein II:IIIa complex (HP1-1D, kindly provided by Dr. W. Nichols). Inhibition of fibrin:platelet adhesion by RGDS, HHGGAKQAGDV, and GHRP all exhibited a similar, linear dependence on the peptide concentration, reaching 1/2 maximum at about 200 μM, suggesting nonspecific effects. GPRP inhibited fibrin assembly but did not appear to have specific effects on fibrin:platelet adhesion. The time course of clot retraction was followed by right angle light scattering intensity measurements. Only RGDS affected clot retraction, causing a 4-fold decrease in rate at 230 μM. These results indicate that fibrinogen and fibrin protofibrils, which are obligatory intermediates in the fibrin assembly pathway, share a set of common platelet recognition sites located at specific regions of the α and γ chains of the multinodular fibrin(ogen) molecules. The RGDS site is also involved in mediating interactions between the three dimensional fibrin network and ADP-stimulated platelets.

Autolysis or tryptic hydrolysis converts β-thrombin (α-T) t β-thrombin ( β -T), and subsequently β-T to y-thrombin (γ-T). Human β-T differs from native α-T by the loss of a unique ll-re-sidues peptide arising from the B chain. Unlike its bovine counterpart, human β-Tisa transient intermediate and its enzymatic properties had not yet been investigated using purified materiaL After 3 min incubation of human β-T with trypsin-sepharose, the resulting β-T was separated from α- and γ-T by chromatography on Biorex 70 with a gradient from 10 mM to 500 mM phosphate at pH 8. No major differences were found between human α- and β-T regarding the kinetic parameters (Km, kcat, kcat/Km) on S 2238, nor the rate of inactivation by TLCK. In contrast, inhibition of β-T by DFP was slower (k = 426 ±[; 10.8 M-1 min−1) compared to α-T (764.5 ± 19.5 M−11min−1and the inhibition constant for benzami-dine was higher with β-T (Ki = 11.2 ± x00B1;.2 10−4 M) compared to α-T (Ki = 2.86± 0,06 10−4 M). The drastic reduction in the clotting activity of β-T (25 u mg−1 versus 3000 u mg−1 for α-T) was further explored by measuring the affinity of β-T for fibrinogen and fibrin. Human fibrinogen was used as a competitor in the inactivation of thrombin by DFP : 10 μM fibrinogen prevented the inhibition of α-T by DFP but failed to modify the inactivation rate of α-T. Binding of thrombin to fibrin was studied using fibrin monomers covalently linked to sepharose 4B, equilibrated in 50mM Tris, pH 7.5, 50 mM NaCl : β -T did not bind to the resin, whereas α-T was retained and eluted upon application of a NaCl gradient.In conclusion, the loss of the peptide extending from lie (63) to Arg (73) in the thrombin B chain is responsiblefor multiple defects in thrombin enzymatic activity. Although, the three active site residues Ser (205), His (43), Asp (99) remain in an active configuration, subtle changes are induced in the microenvironment of the catalytic Ser (205), and in particular, in the primary binding pocket. In addition, the results presented in this study indicate thatthe loss of clotting activity is mainly the result of a decreased affinity for fibrinogen and fibrin, suggesting that the structural changesaffect both the fibrinopeptide groove and the anionic binding site involved in fibrinogen/fibrin recognition.

Fibrin polymerization is competitively inhibited by the te-trapeptide GlyProArgPro. This peptide corresponds to the N-terminal sequence of the fibrin α-polypeptide chain, which is exposed upon release of fibrinopeptide A by thrombin. A binding site for GlyProArgPro was suggested to be located in the C-terminal end of the 411 amino acids containing γ-chain (Varadi and Scheraga, Biochemistry, 25, 519, 1986). In order to characterize the polymerization domain, GlyProArgProLys-azidoazobenzene, a photoactivable derivative of GlyProArgPro was synthesized. Photoaffinity label was bound to fibrinogen in the dark and photolysis was carried out at 0°C. After reduction and S-carboxymethy-lation of the photoaffinity labeled fibrinogen, the polypeptide chains (Aα, Bβ,γ ) were separated by ion-exchange chromatography. Photolabel binding was monitored imnunologically with anti-azo-benzene antibodies (ELISA, Western blot). Selective labeling of the γ-chain was observed. Labeled γ-chains were further digested with CNBr, and the resulting fragments were separated by reversed phase HPIC, immunologically characterized and identified by Edman degradation. GlyProArgProLys-azidoazobenzene was incorporated in the 18 kD CNBr-fragment (γ95-264). The CNBr-fragments arising from C-terminal end of the γ-chain were not labeled.Our results indicate that the binding site of GlyProArgPro is localized exclusively on γ-chain, within the sequence γ95-264.

It is a well established fact that fibrinogen occurs not only in blood plasma but also in the α-granules of the platelets. Recently, it has been shown that fibrinogen is synthesised in the megakaryocytes as well as in the liver. Plasma fibrinogen is derived from the liver, but platelet fibrinogen either exclusively or partially from the megakaryocytes. Conclusive, proteinche-mical evidence for the identity of the fibrinogens from the two biosynthetic sources has previously not been produced. However, the two fibrinogen preparations have been shown to have the same overall peptide chain composition, to be thrombin-clottable and release fibrinopeptides of A- and B-type, and to react with antibodies against plasma fibrinogen. The two preparations differ in the way that platelet fibrinogen lacks the higher-molecular-mass γ-chain variant.The aim of the present investigation was to conduct a detailed proteinchemical comparison between human plasma and platelet fibrinogen. For this purpose, fibrin(ogen)s from the two sources were mercaptolysed, alkylated and the three peptide chains isolated by reversed-phase high-performance liquid chromatography (HPLC). The peptide chains were then analysed directly for amino-terminal sequence and for carboxyterminal sequence by isolation of a terminal fragment. The HPLC-fingerprint patterns of the cyanogen bromide-cleaved chains were compared. The native fibrinogens were also treated with thrombin and the fibrinopeptide type distribution determined by reversed-phase HPLC. The carbohydrate side chain compositions were established by ion-exchange-chromatographic methods after acid hydrolysis. So far no previously unrecognized differences have been observed.

Prolonged thrombin time (partially corrected by calcium chloride) and normal reptilase time were found in the plasma of two siblings with arterial thrombosis. Their purified fibrinogen showed similar abnormalities as well as impaired fibrino-peptide release in response to thrombin, delayed polymerization of pre-formed fibrin monomers and normal sialic content. Binding of radiolabelled thrombin by patient's fibrin was 30% of normal. Supernatants from patients' fibrin clots contained abnormal amounts of thrombin (not adsorbed by fibrin) and caused abnormal enhancement of platelet aggregation and ATP secretion from platelets exposed to sub-threshold concentrations of ADP or epinephrine. Hirudin suppressed the enhancing effect of the supernatant and substitution of γ-thrombin for α-thrombin led to normalization of platelet response. Studies on fibrinolysis showed that the abnormal fibrinogen from these patients as well as its naturally occurring derivative fibrin are highly resistant to lysis by plasmin. Thus our data support the concept that, in addition to the enhanced activation of platelets by residual free thrombin, thrombosis in these patients is the result of an impaired sensitivity of fibrinogen the lytic effect of plasmin.

Maximal 3- to 5-fold increases in platelet cGMP levels as measured by specific radioimmunoassay are observed on stimulation of aspirin-treated platelet-rich plasma with saturating doses of ADP, adrenaline, 5HT, PAF, thrombin, 9,11-epoxymethano-PGH2 (U44069), collagen, ristocetin and the Ca2+-ionophore ionomycin. The dose/response curves for the elevation in cGMP induced by these agents are either superimposable on, or lie to the right of, those describing the rate or extent of the aggregatory response as measured by an increase in light transmittance. The increase in cGMP induced by ADP is totally inhibited by addition of PGI2 or forskolin with dose/response relationships superimposable on those observed for inhibition of the aggregatory response. No increase in cGMP is observed if platelets are stimulated by PAF or ionomycin in an unstirred system or when aggregation induced by ADP is prevented by addition of a monoclonal antibody which recognises the glycoprotein IIb/IIIa complex.Addition of the fibrinogen γ-chain C-terminal decapeptide (γ402-411) or α-Chain tetrapeptide ARG-GLY-ASP-SER prevents aggregation and the increase in cGMP induced by PAF. The γ-chain decapeptide also completely prevents the increase in cGMP induced by ristocetin, but the a-chain tetrapeptide is ineffective in this respect. Both peptides inhibit to some extent aggregate formation induced by ristocetin.The data demonstrates a strong correlation between aggregate formaticfn and the increase in the platelet cGMP levels and support the previous postulate that platelet-platelet contact causes, activation of guanylate cyclase. No relationship is apparent between the effects of the various agents tested on cGMP levels and their known ability to increase cytosolic Ca2+ concentration. (Supported by SERC and Ciba-Geigy.)