Rozprawy doktorskie na temat „Α/β Peptide”

Synthetic construction of protein molecules has been widely pursued over the last two decades. A primary goal behind de novo protein design has been to build minimal systems by capturing the essential features of protein structures. Such minimal models can be used to understand underlying principles governing folding, structure, and function of proteins molecules. Several approaches envisioning successful construction of synthetic proteins have been described over the years, some of them being admirably successful (DeGrado et al, 1999; Richardson et al> 1992; Baltzer, 1998). Specific patterning of polar and apolar residues in synthetic sequences has been widely used to achieve designed polypeptide structures like helix bundles (DeGrado et ah, 1999) and (3-sheets (Smith and Regan, 1997; Lacroix et a/., 1998), with reliance on hydrophobic driving forces for folding. Our laboratory has been pursuing a distinctly alternative approach, that employs stereochemically constrained amino acids to generate specific secondary structures which can then be assembled into composite structures by appropriately chosen linking segments. This approach, which involves linking prefabricated modules of secondary structures can be termed as a "Meccano set" approach to protein design (Balaram, 1992). The studies embodied in the present thesis describe attempts at construction of synthetic polypeptide motifs using the stereochemically directing influence of conformationally constrained amino acid residues, such as DPro or Aib (α-aminoisobutyric acid). This thesis is subdivided into 8 chapters, with Chapter 1 providing a perspective of the field of protein design. Subsequent chapters (2-8) describe studies directed towards the specific goal of construction of polypeptide motifs. Chapter 2 describes synthesis and conformational characterization of two octapeptides Boc-Leu-Val-Val-DPro-LAla-Leu-Val-Val-OMe (1) and Boc-Leu-Val-Val-DPro-DAla-Leu-Val-Val-OMe (2), designed to investigate the effect of specific β-turn stereochemistry on β-hairpin structures. 500 MHz NMR studies establish that both peptides 1 and 2 adopt predominantly β-hairpin conformations in chloroform and methanol solutions, with interstrand registry established by observation of long-range nuclear Overhauser effects (NOEs). Specific NOEs provide evidence for a type II' β-turn conformation for the DPro-LAla segment in 1, while the NMR data suggest that a type I' DPro-DAla β-turn conformation predominates in the peptide 2. The crystal structure of 1 reveals two independent molecules in the crystallographic asymmetric unit, both of which adopt β-hairpin conformations nucleated by a type II’ β-turn across DPro-LAla and stabilized by 3 cross strand hydrogen bonds. These designed β-hairpins with defined tight turns produce characteristic vibrational circular dichroism (VCD) patterns, demonstrating the utility of VCD as a probe for conformational analysis of β-hairpins. In Chapter 3, we present conformational analysis on designed β-hairpin sequences incorporating a 'Phe-Phe' residue pair at a non-hydrogen bonding position. Two octapeptides Boc-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe and Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe were synthesized and conformationally characterized by 500 MHz NMR spectroscopy. Specific NOEs observed in solution provide conclusive evidence favoring specific orientation effects pertaining to the 'Phe-Phe' pair. The peptides exhibited anomalous electronic CD, which has been explained in terms of aromatic contributions by the side chain chromophores. Interestingly, the VCD patterns obtained for these peptides were almost identical to those obtained for other β-hairpins, described in Chapter 2. Chapter 4 describes the synthesis and conformational analysis of designed decapeptide sequences with centrally located DPro-Xxx β-trun segments. Two sequences Boc-Met-Leu»Phe-Val'DPro-Ala-Leu-Val-Val-Phe-OMe (1) and Boc-Met-Leu-Val-Val-^ro-Gly-Leu-Val-Val-Phe-OMe (2) were designed to study the effect of chain length elongation, of β-strands, on designed β-hairpin structures. 500 MHz NMR studies establish β-hairpin folds in both these sequences, with strand segments aligned even at the termini of the structures. Multi-stranded, antiparallel β-sheet structures can be generated by successive placement of β-hairpin sequences in a single polypeptide chain. The successful construction of three stranded β-sheet structures is described in Chapter 5 of this dissertation. A 14-residue peptide Boc-Leu-Phe-Val-DPro-Gly-Leu-Val-Leu-Ala-DPro-Gly-Phe-Val-Leu-OMe (LFV14) was designed such that it is composed of three strand segments linked by two DPro-Gly turn segments. The peptide showed excellent solubility in apolar media, permitting detailed conformational analysis by 500 MHz NMR spectroscopy in organic solvents. Observation of long-range, interstrand NOEs, diagnostic of multiple hairpin structures, provides conclusive evidence for a predominantly populated three stranded β-sheet structure in solution. Extension of this strategy has been described in which an 18-residue peptide, Arg-Gly-Thr-Ile-Lys-DPro-Gly-Val-Thr-Phe-Ala-DPro-Ala-Thr-Lys-Tyr-Gly-Arg, was designed with enhanced solutility in water to probe (β-sheet structure formation in aqueous and mixed aqueous-methanol systems. NMR data provided conclusive evidence in favor of the desired structure being significantly populated in methanol and methanol-water mixtures (50 %, v/v). In water, spectroscopic evidence suggests that the long-range order expected of a three-stranded structure is lost, possibly due to water invading the interstrand hydrogen bonds. Successful construction of a four-stranded antiparallel β-sheet structure has been demonstrated in Chapter 6. A 26-residue peptide Arg-Gly-Thr-Ile-Lys»DPro-Gly-Ile-Thr- Phe-Ala-DPro-Ala-Thr-Val-Leu-Phe-Ala-Val-DPro-Gly-Lys-Thr-Leu-Tyr-Arg was designed to have four strand segments linked by three DPro-Xxx turn segments. The peptide exhibited excellent NMR properties permitting structure determination by analysis of NOE data, which revealed that a four stranded β-sheet structure is indeed populated in methanol. Structural studies on this peptide in mixed methanol-water established that the four stranded β-sheet is appreciably populated at a composition of 50 % (v/v) methanol-water mixture, with the β-sheet structure still detectable even at a composition of 70 % water-30 % methanol. In a completely aqueous environment, the β-sheet structures is significantly disrupted, presumably due to solvent invasion. The nucleating β-turns, however, appear to have retained their structural integrity even in this competitive environment. Chapter 7 describes the insertion of L-Lactic acid (Lac), a hydroxy acid, into polypeptide helices stabilized by a-aminoisobutyricacid (Aib). This study was undertaken to investigate the effect of hydrogen bond deletion on peptide helices. Crystal structure determination of three oligopeptides containing Lac residues has been performed. Peptide 1, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe, and peptide 2, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Leu-OMe adopt completely helical conformations in the crystalline state, with the Lac(6) residue comfortably accommodated in the center of a helix. NMR studies of peptide 1 and its all amide analog 4, Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe, provide firm evidence for a continuous helical segment in both the cases. In a 14-residue peptide 3, Boc-Val-Ala-Leu-Aib- Val- Ala-Leu- Val- Ala-Leu- Aib-Val-Lac-Leu-OMe, residues Val( 1 )-Leu( 10) adopt a helical conformation, which is terminated by formation of a Schellman motif, with Aib(ll) as the site of chiral reversal. The loss of the hydrogen bond at the C-terminus appears to facilitate the chiral reversal at Aib(l 1). In the final section of this thesis, Chapter 8, successful construction of a synthetic motif containing two distinct elements of secondary structure, a (β-hairpin and a helix, has been described. The design of a 17-residue peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Gly-Gly-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe, BH17, is based on a modular approach, in which previously characterized β-hairpin (Leu-Phe-Val-DPro-Gly-Leu-Phe-Val) and helix (Val-Ala-Leu-Aib-Val-Ala-Leu) modules are linked by a Gly-Gly linker. The positioning of the achiral Gly residue at position 8 facilitates termination of the potential helical segment (residues 1-7) by formation of a Schellman motif. Gly(9) is anticipated to be the sole conformationally flexible residue. NMR studies on BH17 indicated the presence of both the helix (residues 1-7) and the β-hairpin (residues 10-17) structures in the sequence, with four major conformational possibilities at the linking segment. Crystal structure determination of BH17 revealed that the two elements of structure are approximately arranged in an orthogonal fashion. The crystal structure validates the original premise that a modular assembly strategy may be viable for the construction of larger synthetic structures. Chapter 9 summarises the major results of this thesis. (For formulae, please refer "pdf" format)

The protein-protein interactions (PPIs) occur when two or more proteins are bound together. Also, this protein-protein interactions (PPIs) cause the various biological processes in the body. Due to this reason, abilities of controlling or inhibiting PPIs can give us promising advantages like (1) better understanding of biological systems, (2) development of new diagnostic approaches for health or disease, and (3) establishment of novel molecular therapeutics. Many proteins adopt the secondary structures, where most of protein-protein interactions take place. -Helices and -sheets are the prevalent secondary conformations, but there are extended secondary structures such as -hairpins, -turns, 310 helix, and so on. As a result, construction of molecules mimicking these protein secondary structures is tractable target for drug design. Moreover, in drug discovery, designing peptidomimetics or non-peptidic mimetics is a popular strategy instead using peptides or truncated peptides because peptides or truncated peptides are prone to proteolysis and degraded in the body. Also, peptidomimetics and non-peptidic mimetics have not only the similar topology as peptides but also resistance to proteolysis. Due to these advantages, in this study, peptidomimetics or non-peptidic mimetics were synthesized and tested for different targets: (1) synthesis of non-peptidic -helical mimetics for p53-MDM2 inhibition, (2) solution-phase synthesis of -hairpin peptide for the inhibition of multiple myeloma cells (MM) adhesion, and (3) synthesis of -hairpin peptoid-peptide hybrids. The synthesis in all three different studies was succeeded, but they still need some improvements. For instance, non-peptidic -helical mimetics, terpyrimidyl derivatives, were synthesized successfully, but they did not show any bioactivity against p53-MDM2. Also, they have a solubility problem. Based on these results, it is necessary to improve the pharmacokinetic properties and bioactivity by changing the substituents on the rings or structures. The -hairpin peptide for the second case already showed good bioactivity against multiple myeloma (MM). For the next level of bio-study, the considerable amount of a -hairpin peptide was demanded. In order to make the substantial -hairpin peptide, the solution phase peptide synthesis was chosen instead of the solid phase peptide synthesis because of the cost-effect. Two methodology were tried for the solution-phase peptide synthesis: (1) segment ligation and (2) continuous synthesis. In the former case, the -hairpin peptide synthesis was successful, but, in the latter case, it is necessary to investigate the appropriate coupling reagents for each step. Peptoid-peptide hybrids has been one of the popular peptidomimetics in the last two decades. Also, mimicking the peptide secondary structure in peptoids has been studied extensively these days. The combination of these two factors was the goal for the third case. Because peptoid-peptide hybrids with a secondary structure can be recognizable by native proteins and resistant to proteolysis. So far, three sets of peptoid-peptide hybrids were synthesize and checked the secondary structure formation by using NMR. However, there was no indication of the secondary structure formation in the three sets of peptoid-peptide hybrids. This result suggests that it is necessary to introduce the more constrained components in peptoid-peptide hybrids. In the above three chapters, it has been tried to find the new drug candidates by synthesizing peptidomimetics or non-peptidic mimetics. Even though the synthesis was successful, some intended results such as the bioactivity or the secondary structure formation were not obtained. However, these results can give us the inspirations to improve properties of peptidomimetics or non-peptidic mimetics for a certain purpose, which leads to earn the intended results and eventually find new drug candidates.

La maladie d'Alzheimer est un syndrome neurodégénératif caractérisé par la présence de plaques séniles et de dégénérescences neurofibrillaires. Le constituant majeur des plaques séniles est le peptide β-amyloi͏̈de (Aβ) qui est issu de la maturation protéolytique de la protéine précurseur du peptide β-amyloi͏̈de ( βAPP). Deux activités enzymatiques, les β- et γ- sécrétases libèrent respectivement les extrémités N- et C-terminales du peptide Aβ. La βAPP peut subir une maturation protéolytique alternative dans la séquence du peptide Aβ par une α-sécrétase, prévenant ainsi la production du peptide amyloi͏̈de. De plus, le clivage α-sécrétase génère un fragment soluble (APPα) qui est neurotrophique et citoprotecteur. La maturation de la βAPP est sous le contôle de voies de phosphorylation, comme la voie régulée par la protéine kinase C (PKC). (. . . )

Les N-tert-butanesulfinamides, des auxiliaires chiraux développés par Ellman il y a une vingtaine d’années, sont à l’heure actuelle de plus en plus sollicités pour la préparation d’amines chirales fonctionnalisées, et ce, du fait de la disponibilité des deux énantiomères à faible coût et de leurs conditions de déprotection faciles à mettre en œuvre. Cependant, leur utilisation dans le cadre de la synthèse de dérivés trifluorométhylés quaternaires à partir des N-tert-butanesulfinylalkyl- ou arylcétimines correspondantes, connues pour leur faible hydrostabilité, reste à ce jour très limitée. Les éthers d’hémiaminals trifluorométhylés dérivés de l’auxiliaire d’Ellman constituent des analogues stables de ces cétimines. Ils permettent de générer in situ la cétimine correspondante, qui peut subir l’addition de l’espèce nucléophile présente. Ce manuscrit relate les différentes réactions étudiées sur ces éthers d’hémiaminals, donnant ainsi accès à des synthons azotés α-trifluorométhylés quaternaires de façon énantiopure : d’une part, les amines homoallyliques obtenues par l’addition d’allylalanes, qui, après quelques étapes, permettent de fournir les azétidines trifluorométhylées correspondantes, et d’autre part, les β3,3-aminoacides trifluorométhylés, préparés via une réaction de Reformatsky hautement diastéréosélective. Les acides β-aminés ainsi préparés ont été réinvestis dans des couplages peptidiques en solution pour la préparation d’une gamme d’α/β- et de β-di- et tripeptides, sur lesquels des études conformationnelles préliminaires ont été menées à l’état solide et/ou en solution.Mots-clés : Auxiliaire d’Ellman, Addition nucléophile, Couplage peptidique en solution, Dérivé azoté α-trifluorométhylé, Éther d’hémiaminal, Synthèse asymétrique
Chiral N-tert-butansulfinamides, developped by Ellman 20 years ago, have been increasingly applied for the preparation of chiral functionnalized amines, because of the affordability of both enantiomers and of their mild conditions of cleavage. However, the use of theses auxiliaries for the synthesis of quaternary trifluoromethyl derivatives remains quite limited, the corresponding trifluoromethyl ketoimines being highly unstable.Chiral N-tert-butanesulfinyl alkyl(aryl) trifluoromethyl hemiaminal ethers have been developped to be used as bench-stable surrogates of these ketoimines : once under reaction conditions, they afford the corresponding ketoimine in situ, which can be subject to a nucleophilic addition.In this manuscript, different reactions led on these hemiaminal ethers are described, affording valuable and optically pure trifluoromethylated quaternary building-blocks : on the one hand, homoallylic amines, obtained by the addition of allylalane species, and which can afford, after a few steps, teh corresponding trifluoromethyl azetidines, and on the other hand, chiral trifluoromethyl β3,3-amino acids, afforded by a highly diastereoselective Reformatsky reaction.These β 3,3-amino acids have been then involved in solution-phase peptide couplings in order to synthetise a wide range of α/β- and β-di- and tripeptides, whose conformation have been the object of preliminary studies in the solid state and/or in solution.Key-words : Ellman auxiliary, Nucleophilic addition, Solution-phase peptide coupling, α-trifluoromethylated nitrogen derivative, Hemiaminal ether, asymmetric synthesis

Les polyoxines, peptidonucléosides d'origine naturelle connus pour leurs propriétés antifongiques en tant qu'inhibiteurs de la biosynthèse de la chitine, sont des composés très actifs in vitro, mais présentant une faible biodisponibilité in vivo. Le but de cette thèse a donc été de trouver des méthodes de synthèse d'analogues C2'-désoxy-C2'-fluorés de la thymine polyoxine C, constituant nucléosidique de la polyoxine J (la plus active), ainsi que d'obtenir, d'une manière originale, la partie peptidique non naturelle de la polyoxine J, à savoir l'acide 5-O-carbamoyle polyoxamique. Après une présentation de la cible biologique et de l'intérêt de l'introduction d'un atome de fluor, ce travail s'est divisé en deux grandes parties. Tout d'abord, deux approches de la synthèse de la partie nucléosidique fluorée par passage par des 2-désoxy-2-fluorobutyrolactones, puis une optimisation de la synthèse de la partie peptidique via la formation d'un intermédiaire oxazolidinone
Polyoxins, which form an important class of peptidyl nucleosidic antibiotics that selectively and competitively inhibit chitin synthase, are very active components in vitro, but weakly bioavailable in vivo. The subject of this thesis has been to found new synthetic methods to access to C2'-fluoro-C2'-deoxy analogues of thymine polyoxine C, nucleosidic part of polyoxine J (the more active one), and to obtain, by an original way, the non natural peptidic moiety of polyoxine J, which is the 5-O-carbamoyl polyoxamic acid. After a presentation of the biological target and the interest of the introduction of a fluorine atom, this work is divided into two parts. Fisrt, two approaches of the fluorinated nucleosodic part using 2-deoxy-2-fluorobutyrolactones as intermediates, and then, an optimization of the synthesis of the peptidic part using the formation of an oxazolidonone intermediate

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

Dans le système nerveux central, l'α-mélanocyte-stimulating hormone (α-MSH) et la β-endorphine sont les deux peptides majeurs issus de la molécule précurseur proopiomélanocortine. Notre travail a consisté d'une part à localiser et caractériser ces deux peptides dans le cerveau de différentes espèces de vertébrés et d'autre part à étudier les mécanismes de régulation de l'α-MSH neuronale dans l'hypothalamus du rat. En combinant les techniques de localisation immunocytochimique et de quantification radioimmunologique, nous montrons que les neurones synthétisant les deux neuropeptides sont essentiellement intrahypothalamiques. La purification de l'α-MSH hypothalamique par chromatographie liquide à haute performance (HPLC) démontre que le peptide est présent sous la forme déacétylée biologiquement inactive. L'étape d'acétylation semble étroitement associée à la libération du neuropeptide. La concentration de l'α-MSH dans les vésicules sécrétoires et les caractéristiques de libération in vitro du peptide par l'hypothalamus indiquent que l'α-MSH peut être assimilée à un neurotransmetteur ou neuromodulateur. Nous avons étudié l'influence de divers facteurs sur l'activité des neurones hypothalamiques à α-MSH chez le rat. Par des traitements in vivo avec des drogues appropriées et un système de perifusion in vitro de tranches de tissu hypothalamique, nous montrons que les neurones à α-MSH ne sont pas régulés par la dopamine. Par contre, la libération du neuropeptide est inhibée par l'acide aminobutyrique (GABA) via les recepteurs GABAa. Son action est potentialisée par les benzodiazépines de type central

Costimulation blockade protocols are promising alternatives to the use of chronic immunosuppression for promoting long-term allograft survival. However, the efficacy of costimulation blockade-based protocols is decreased by environmental insults such as viral infections. For example, lymphocytic choriomeningitis virus (LCMV) infection at the time of costimulation blockade treatment abrogates skin allograft survival in mice. In this dissertation, we test the hypothesis that viruses shorten allograft survival by activating the innate immune system through pattern-recognition receptors (PRRs), such as toll-like receptors (TLRs). To investigate the role of innate immunity in shortening allograft survival, costimulation blockade-treated mice were co-injected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid, poly(I:C)), TLR4 (lipopolysaccharide, LPS), or TLR9 (CpG DNA) agonists, followed by transplantation with skin allografts 7 days later. Costimulation blockade prolonged skin allograft survival that was shortened in mice coinjected with TLR agonists. To investigate the underlying mechanisms of this observation, we used synchimeric mice, which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, co-administration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) upregulated CD44, and 3) failed to undergo apoptosis. We also demonstrate that costimulation blockade-treated CD8α-deficient mice exhibit prolonged allograft survival when co-injected with LPS. These data suggest that TLR agonists shorten allograft survival by impairing the apoptosis of alloreactive CD8+T cells. We further delineate the mechanism by which TLR agonists shorten allograft survival by demonstrating that LPS and poly(I:C) fail to shorten allograft survival in IFNRI- deficient mice. Interestingly, the ability of poly(I:C) to more potently induce IFN-α/β than LPS correlates with its superior abilities to shorten islet allograft survival and induce allo-specific CTL activity as measured by an in vivo cytotoxicity assay. The ability to shorten allograft survival and induce IFN-α/β is a TLR-dependent process for LPS, but is a TLR-independent process for poly(I:C). Strikingly, the injection of IFN-β impairs alloreactive CD8+T cell deletion and shortens allograft survival, similar to LPS and poly(I:C). These data suggest that LPS and poly(I:C) shorten allograft survival by inducing IFN-α/β through two different mechanisms. Finally, we present data showing that viruses (LCMV, Pichinde virus, murine cytomegalovirus and vaccinia virus) impair alloreactive CD8+T cell deletion and shorten allograft survival, in a manner comparable to LPS and poly(I:C). Similar to LPS, LCMV and MCMV exhibit an impaired ability to shorten allograft survival in MyD88-deficient mice. These data suggest that the MyD88 pathway is required for certain viruses and TLR-agonists to shorten allograft survival. In this dissertation, we present data supporting an important role for TLRs and IFN- α/β in shortening allograft induced by costimulation blockade. Our findings suggest that targeting these pathways during the peri-transplant period may enhance the efficacy of costimulation blockade protocols in the clinic.

Les maladies à prions sont des maladies neurodégénératives caractérisées par l'accumulation dans le système nerveux central d'une protéine anormalement conformée et neurotoxique, la protéine prion Scrapie (PrPSc). La PrPSc est l'isoforme transconformationnelle d'une protéine normale de l'hôte, la protéine prion cellulaire (PrPC). Il est établi que la toxicité de la PrPSc est restreinte aux neurones et que la neurodégénérescence résulte d'une corruption de la/des fonction(s) physiologique(s) de la PrPC par la PrPSc. Néanmoins, nul ne sait s'il s'agit d'une perte de fonction protectrice ou d'un gain de fonction toxique de la PrPC, ou d'une combinaison des 2 événements, en partie parce que les fonctions de la PrPC restent difficiles à cerner. C'est pourquoi, identifier la/les fonction(s) de la PrPC est un prérequis pour comprendre comment la PrPSc exerce sa neurotoxicité. Mes travaux de thèse montrent pour la première fois une fonction protectrice de la PrPC vis-à-vis de la cytokine pro-inflammatoire sTNF-alpha Nous démontrons que la PrPC ajuste la sensibilité des cellules au sTNF-alpha en contrôlant le clivage des récepteurs au sTNF-alpha (TNFR1) par TACE. Au niveau mécanistique, la PrPC exerce un double contrôle sur TACE en gouvernant (i) son activité enzymatique, via le couplage de la PrPC à la NADPH oxydase/production de dérivés réactifs de l'oxygène, et (ii) sa localisation, en modulant négativement la voie de signalisation intégrines bêta-1/ROCK/PDK1, ce qui assure le maintien de TACE sous forme active à la membrane plasmique. La déplétion en PrPC provoque la micro-agrégation des intégrines bêta-1, la suractivation du duo de kinases ROCK/PDK1, et l'internalisation subséquente de l'alpha-secrétase TACE dans des microvésicules enrichies en Cavéoline-1. TACE est alors découplée de son substrat TNFR1, qui s'accumule à la membrane plasmique et rend les neurones déplétés en PrPC hypersensibles au stress inflammatoire de type sTNF-alpha. Ces mêmes défauts ont été retrouvés, avec des intensités comparables, dans les neurones infectés par les prions, ce qui appuie l'idée que la perte de la fonction cytoprotectrice de la PrPC dans les neurones vis-à-vis du sTNF-alpha participe à la progression des maladies à prions. Concernant l'infection à prions, un travail en collaboration révèle que les cellules de Purkinje du cervelet qui n'expriment pas les zébrines sont plus sensibles à la toxicité de 2 souches de prions, 22L et ME7, que celles qui expriment les zébrines. Cette étude suggère un rôle protecteur des zébrines vis-à-vis des prions. Un axe majeur de ma thèse identifie une nouvelle cible déréglée en aval du module de signalisation PDK1/TACE dans les maladies à prions, la protéine précurseur des amyloïdes (APP) avant tout connue pour son rôle dans la maladie d'Alzheimer. En abrogeant le clivage non-amyloïdogène d'APP par TACE, la PrPSc est à l'origine d'une surproduction d'Abêta40/42. Les peptides Abêta40/42 sont majoritairement sous forme monomérique, mais des formes multimériques (trimères et tétramères) d'Abêta40/42 sont aussi générées. Cette production d'Abêta40/42 dépend de la suractivation de PDK1 puisque l'inhibition pharmacologique de la kinase permet de réduire la production des peptides Abêta40/42 monomériques et rend les multimères indétectables. Il est à noter que les peptides Abêta produits ne modifient ni la réplication ni l'infectiosité de la PrPSc. Toutefois, l'Abêta40/42 généré par l'infection à prions est capable de former des dépôts dans les cerveaux de souris si, et seulement si un « seed » d'Abêta exogène a été co-transmis avec la PrPSc. De manière importante, la conjonction infection à prions/dépôts d'Abêta accélère la mort des souris infectées par les prions. Ces travaux définissent les conditions qui permettent la formation de plaques Abêta et mettent en exergue l'émergence d'une pathologie mixte causée par la présence de PrPSc et des dépôts d'Abêta dans un contexte infection à prions
Prion diseases are neurodegenerative disorders characterized by the accumulation into the central nervous system of an abnormally folded protein called Scrapie prion protein (PrPSc). PrPSc is the transconformational isoform of a ubiquitous protein of the host named cellular prion protein (PrPC). It is well established that the toxicity of PrPSc is restricted to neurons and arise from a corruption of the physiological function(s) of PrPC. However, the mechanisms by which PrPSc exerts its neurotoxicity remain poorly understood, partly because the physiological function(s) of PrPC is/are still elusive. Currently, no one knows if PrPC loses a protective role or acquires a toxic function upon its conversion into PrPSc, a combination of both events is also possible. Identifying PrPC-associated function(s) is thus a prerequisite to understand how PrPSc provokes neurodegeneration. The present work reports for the first time a protective role of PrPC towards the pro-inflammatory cytokine sTNF-alpha-associated toxicity. We show that PrPC adjusts cell sensitivity to sTNF-alpha by controlling TACE-dependent TNFR1 shedding. Mecanistically, PrPC governs both (i) TACE activity, through PrPC coupling to NADPH oxidase/Reactive Oxygen Species production, and (ii) TACE localization, by downregulating the beta-1 integrins/ROCK/PDK1 signaling pathway, thus PrPC ensures the bioavailability of an active TACE at the plasma membrane. PrPC depletion provokes the micro-aggregation of beta-1 integrins, the overactivation of ROCK and PDK1 kinases, and the subsequent internalization of TACE into Caveolin-1 enriched micro-vesicles. This leads to a defect of TNFR1 shedding, which accumulates at the plasma membrane and renders PrPC-depleted neurons highly vulnerable to sTNF-alpha insult. These alterations have also been reported in prion-infected neurons with the same intensities, supporting the view that a loss-of-the protective function of PrPC towards sTNF-alpha likely occur along prion diseases. Within a prion infectious context, a collaborative work revealed that the cerebellar Purkinje cells that do not express zebrins are highly vulnerable to the toxicity of two prion strains, 22L and ME7, compared to Purkinje cells that express zebrins. This suggest a protective role of zebrins against PrPSc-associated toxicity. A major part of my thesis identifies a new target deregulated downstream from the PDK1/TACE signaling module, the amyloid precursor protein (APP), well-known for its implication in Alzheimer's disease. By abrogating the non-amyloidogenic cleavage of APP by TACE, PrPSc provokes the overproduction of Abeta40/42 peptides. Abeta40/42 predominates as monomers but are also found as multimeric assemblies, i.e. trimers and tetramers. PrPSc-induced Abeta40/42 overproduction relates to PDK1 overactivation as pharmacological inhibition of PDK1 attenuates production of Abeta monomers and renders multimers undetectable. Of note, our work reveals that Abeta peptides do not impact on PrPSc replication nor infectivity. Nevertheless, Abeta40/42 peptides generated upon prion infection can deposit in mice brains only if an exogenous Abeta seed is co-transmitted with PrPSc. Importantly, Abeta deposition leads to early death of prion-infected mice. This work delineates the conditions that allow Abeta plaques formation and highlights the onset of a mixed-pathology caused by the co-occurrence of PrPSc and Abeta deposition within a prion infectious context

La protéine prion cellulaire (PrPC) est une protéine majoritairement exprimée à la surface des neurones, dont la conversion transconformationnelle en prion pathogène PrPSc, est à l'origine des maladies à prions. Il est clairement établi que la neurodégénérescence induite par la PrPSc dépend de l'expression de la PrPC dans les neurones et résulte d'une déviation de la/des fonction(s) de la PrPC par la PrPSc. Identifier le rôle de la PrPC est donc un pré-requis pour aborder les mécanismes de neurodégénérescence dans les maladies à prions. Une partie de mes travaux de thèse a permis de montrer que la PrPC exerce un rôle cytoprotecteur vis-à-vis de la cytokine inflammatoire TNFalpha. L'extinction de la PrPC dans les neurones (neurones PrPnull) rend ces cellules hypersensibles au TNFalpha en raison de l'accumulation membranaire des récepteurs au TNFalpha (TNFR). Mes travaux démontrent que la perte de la fonction régulatrice de la PrPC sur l'agrégation et la signalisation des intégrines bêta 1 dans les neurones PrPnull provoque la suractivation de la kinase PDK1, l'internalisation subséquente de l'alpha-sécrétase TACE, et un découplage de TACE vis-à-vis de l'un de ses substrats, TNFR. Étant donné la proximité phénotypique entre les neurones PrPnull (Ezpeleta et al. 2017) et les neurones infectés par la PrPSc (Pietri et al. 2013 ; Alleaume-Butaux et al. 2015), mes travaux plaident en faveur d'une perte de fonction cytoprotectrice de la PrPC dans les maladies à prions. Concernant l'infection à prions, mes travaux montrent que TACE internalisée en réponse à la suractivation de PDK1 est découplée d'un autre substrat, la protéine précurseur des peptides amyloïdes (APP), ce qui mène à l'accumulation des peptides neurotoxiques Abêta 40 et Abêta 42 caractéristiques de la maladie d'Alzheimer. Dans un contexte « infection à prions », les peptides Abêta 40/42 sont présents majoritairement sous une forme monomérique, et de façon plus discrète sous forme trimérique et tétramérique. Par des approches in vitro et in vivo, nous montrons que les peptides Abêta générés par les cellules infectées par les prions ne modifient ni la réplication ni l'infectiosité des prions. Néanmoins, nous démontrons que les formes oligomérisées d'Abêta sont capables de se déposer sous forme de plaques amyloïdes dans le cerveau des souris transgéniques APP23 infectées par les prions. Dans ces souris, les dépôts d'Abêta accélèrent la pathogenèse des prions. Le dernier axe de mon travail de thèse concerne les nanoparticules, des matériaux de taille nanométrique couramment utilisés dans de nombreux produits et procédés industriels. Mes travaux mettent en évidence que, à l'instar de la PrPSc et d'Abêta, des assemblages de nanoparticules de dioxyde de titane ou de noir de carbone se lient à la PrPC exprimée à la surface des neurones et dévient sa fonction de signalisation. Cette interaction PrPC/nanoparticules provoque, entre autres, la suractivation de PDK1, l'internalisation de TACE, et l'accumulation membranaire de TNFR. Les cellules neuronales exposées aux nanoparticules deviennent alors hypersensibles au stress inflammatoire TNFalpha. Le découplage de TACE à APP induit par les nanoparticules augmente aussi la production de peptides Abêta par les neurones. Même si aucune donnée épidémiologique n'associe une exposition aux nanoparticules à la maladie d'Alzheimer, mes travaux suggèrent une implication causale des nanoparticules dans l'initiation voire l'amplification de cette maladie
The cellular prion protein (PrPC) is a protein mostly expressed at the plasma membrane of neurons. Its transconformation into the pathogenic prion PrPSc is at the root of prion diseases. It is clearly established that the PrPSc-induced neurodegeneration depends on the expression of PrPC in neurons and results from the corruption of PrPC function(s) by PrPSc. Unravelling the role of PrPC is thus a prerequisite to grasp neurodegeneration mechanisms in prion diseases. Part of my work shows that PrPC exerts a cytoprotective function against TNFalpha inflammatory cytokine. PrPC silencing in neurons (PrPnull-neurons) renders these cells highly sensitive to TNFalpha due to surface accumulation of TNFalpha receptor (TNFR). My work demonstrates that the loss of PrPC regulatory function on the clustering and signaling downstream of bêta 1 integrins in PrPnull neurons provokes the overactivation of the kinase PDK1, subsequent internalization of TACE alpha-secretase, and uncoupling of TACE from TNFR substrate. Because of the phenotypic proximity between PrPnull neurons (Ezpeleta et al. 2017) and PrPSc-infected neurons (Pietri et al. 2013; Alleaume-Butaux et al. 2015), my work supports the view of a loss of PrPC protective function in prion diseases. As concerns prion infection, my work shows that after PDK1 overactivation, internalized TACE is uncoupled from another substrate, the amyloid peptides precursor protein (APP), leading to the accumulation of neurotoxic peptides Abêta 40 and Abêta 42, hallmarks of Alzheimer's disease. Within a prion infectious context, Abêta 40/42 peptides are predominantly present as monomers, and to a lesser extent, as trimers and tetramers. By combining in vitro and in vivo approaches, we show that Abêta peptides produced by infected neurons do not alter replication nor the infectivity of prions. Nevertheless, we demonstrate that oligomerized Abêta is able to form amyloid plaques in the brain of transgenic APP23 mice infected by prions. In these mice, Abêta deposits accelerate prion pathogenesis. The last axis of my work deals with nanoparticles, that is, nanometric materials commonly found in manufactured products and industrial processes. My work shows that, as PrPSc and Abêta, titanium dioxide or carbon black assemblies interact with PrPC at the surface of neurons and deviate its signaling function, which leads, inter alia, to PDK1 overactivation, TACE internalization, TNFR accumulation at the plasma membrane, and neuronal cells hypersensitivity to TNFalpha inflammatory stress. We also found that nanoparticle-induced TACE uncoupling from APP increases Abêta peptide production by neurons. Even if no epidemiological study has demonstrated to date a link between nanoparticle exposure and Alzheimer's disease, my work suggests an causal implication of nanoparticles in the initiation or amplification of this disease

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.

This thesis describes NMR studies which probe weak interactions between amino acid side chains in folded peptide structures. Aromatic/aromatic interactions between facing phenylalanine residues have been probed in antiparallel β-sheets, while aromatic/proline interactions have been examined using cyclic peptide disulfides that occur in the venom of marine cone snails. Novel intramolecular hydrogen bonded structures in hybrid peptides containing backbone homologated residues, specifically γ-amino acids, are also described. Chapter 1 provides a brief background to the principles involved in the design of antiparallel β-sheet structures and an introduction to previous studies on aromatic/aromatic and aromatic/proline interactions in influencing peptide conformations. A summary of the NMR methods used is also presented. Chapter 2 discusses the structural characterisation of a designed 14 residue, three stranded β-sheet peptide, Boc-LFVDP-PLFVADP-PLFV-OMe (LFV14). The results described in this Chapter support the presence of multiple conformational states about the χ1 (Cα-Cβ) torsional degree of freedom for the interacting aromatic pairs in solution. Chapter 3 presents the structural characterisation of a designed 19 residue three stranded hybrid β-sheet peptide, Boc-LVβFVDPGLβFVVLDPGLVLβFVV-OMe (BBH19). β-amino acid residues (β-phenylalanine, βPhe) were incorporated at facing positions on antiparallel β-sheets. The BBH19 structure provides an example of interaction between the N and C-terminal strands in a three stranded structure with an α/β hybrid backbone. Chapter 4 focuses on studies of the conformations of the contryphan In936 (GCVDLYPWC*) from Conus inscriptus and the related peptide Lo959 (GCPDWDPWC*) from Conus loroissi. Both peptides possess a macrocyclic 23 membered ring, with multiple accessible conformational states. Chapter 5 describes conformational analysis of a novel 20 membered cyclic peptide disulfide, CIWPWC (Vi804), from Conus virgo. NMR structures were calculated for Vi804 and an analog peptide, CIDWPWC, DW3-Vi804. Chapter 6 explores the solution conformation of hybrid sequences containing α and γ residues. Oligopeptides of the type (αγ)n and (αγγ)n have been studied in solution by NMR methods. Chapter 7 provides a summary of the results described in this thesis and highlights the major conclusions.

The insertion of ω- amino acid residues as guests into host α-peptide sequences permits expansion of the range of polypeptide secondary structures. The term ω- amino acid is used to refer to the entire family of residues generated by homologation of the backbone of α - amino acid residues. This explores the consequences of insertion of substituted β-residues (β3) , unsubstituted β-residues , unsubstituted γ-residues (gamma aminobutyric acid) and unsubstituted δ-residues (delta aminovaleric acid) into host α -peptide sequences. Chapter 1 provides an introduction to the conformational properties of β-peptides and reviews current literature on the structural features of peptides containing ω-amino acid residues. The available crystallographic information is summarized. The conformational properties of β- residues may be described by three degrees of torsional freedom : φ (N – Cβ) , θ (Cβ -Cα) and ψ (Cα-CO). Similarly, the conformational properties of γ -residues is based on four torsional parameters ( φ , θ1 , θ2, ψ) and the conformational properties of δ - residues is based on five degrees of freedom ( φ , θ1 , θ2, θ3,ψ). The rational use of β -residues in peptide design requires an understanding of the nature of local conformations, which are readily accessible. The conformational space for β -residues can be represented in a three dimensional plot. The observed distribution of φ , θ and ψ values for β -residues in peptide crystal structures presented in this section permits a correlation - between the torsion angle θ and the secondary structure context. The gauche (g+ and g ) conformations induce helical folding and the trans conformation is generally observed in the strands of a hairpin. The most striking feature of hybrid sequences is the observation of novel hydrogen bonded rings in peptide structures. Chapter 2 describes the effects of insertion of β-residues into specific positions in the strand segments of designed peptide hairpins. Insertion of β -residues into the strands of a hairpin changes the orientation of peptide bonds, resulting in a “polar sheet” arrangement. The conformational analysis of three designed peptide hairpins composed of α/β - hybrid segments are described: Boc-Leu-βPhe-Val-DPro-Gly- Leu-βPhe-Val-OMe (BBH8) , Boc-βLeu- Phe-βVal-DPro-Gly-βLeu-Phe-βVal-OMe (BAB8) and CF3COO-H3N+-Leu-Val-Val-βPhe-DPro-Gly-βPhe-Leu-Val-Val-OMe (BHFF10). All the peptides have been characterized by 500 MHz 1H-NMR spectroscopy and several crucial long range NOEs confirm a predominant population of β-hairpin conformations in CD3OH. X-ray diffraction studies on single crystals of peptide BBH8 reveal a β-hairpin conformation, stabilized by three cross-strand hydrogen bonds and a Type II′β-turn at the DPro-Gly turn segment. Designed β-hairpin peptide scaffolds may be used to probe cross-strand sidechain interactions in β-sheet structures. A previously reported peptide β-hairpin, Boc-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe exhibited an anomalous far UV CD spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe 7 (Zhao, C.; Polavarapu, P.L.; Das,C. and Balaram, P. J. Am. Chem. Soc., 2000, 122, 8228-8231). In BBH8 and BHFF10 the two cross-strand βPhe residues are at non-hydrogen bonding positions, with the benzyl sidechains pointing on opposite faces of the β- sheet. BBH8 yields a “hairpin –like” CD spectrum, with a minimum at 224 nm. The CD spectrum of BAB8 reveals a negative band at 234 nm and a positive band at 221 nm suggestive of an exciton split doublet. BHFF10 yields a “hairpine-like” CD spectrum, with a negative band at 220 nm. Chapter 3 describes the synthesis and conformational characterization of three hybrid decapeptides : Boc-Leu-Val-βGly-Val-DPro-Gly- Leu-βGly -Val-Val-OMe (BHB10), Boc-Leu-Val-γAbu-Val-DPro-Gly- Leu-γAbu -Val-Val-OMe (BHC10) and Boc- Leu-Val-δAva-Val-DPro-Gly- Leu-δAva -Val-Val-OMe (BHD10). These peptides were designed to systematically investigate the effect of insertion of additional methylene groups into the strands of a hairpin. The incorporation of additional carbon atoms changes the local polarity of the strands. 500 MHz NMR studies establish that BHB10 and BHD10 adopt predominantly β- hairpin conformations in methanol, with interstrand registry established by observation of long range NOEs. The observation of both DPro 4 (CαH) ↔ Gly 5 (NH) and Gly 5 (NH) ↔ Leu 6 (NH) NOEs provides evidence for a Type II ′β - turn for all the hairpins. In BHC10, no long range NOEs were observed. However, X-ray diffraction studies in single crystals reveal a β- hairpin conformation, nucleated by a DPro-Gly Type II′β-turn. Chapter 4 describes an attempt to incorporate one or two ω amino acid residues in the turn region of a potential hairpin, in order to assess the effect of expansion of the nucleating turn. The DPro-LPro segment has been shown to stabilize β-hairpin conformations in both cyclic (Shankaramma,S.C.; Moehle, K. ; James, S.; Vrijbloed, J.W.; Obrecht,D and Robinson, J.A. Chem Commun. 2003,1842-1843) and acyclic sequences ( Raj Kishore Rai ; S.Raghothama and P. Balaram , unpublished results) . In the present study the following turn segments have been considered: βDPro -αLPro , βLPro -αLPro and βLPro -αDPro. The synthesis and conformational analysis of three octapeptide sequences -Boc-Leu-Phe-Val-βDPro-αLPro-Leu-Phe-Val-OMe (βDPαLP8), Boc-Leu-Phe-Val-βLPro-αLPro-Leu-Phe-Val-OMe (βLPαLP8)and Boc-Leu-Phe-Val-βLPro-αDPro-Leu-Phe-Val-OMe (βLPαDP8) are described. In the βDPro-αLPro peptide, NMR evidence clearly supports a β-hairpin conformation, with a nucleating hybrid βα turn stabilized by a C11 (4 →1) hydrogen bond. In the other two octapeptides, no evidence for folded structures was obtained. These results suggest that nucleating turn formation is facilitated only in the heterochiral βD-αL case. Further expansion of the turn segment in potential hairpins has been investigated by inserting two contiguous β-residues into the center of a host α-peptide sequence. The conformational studies on two synthetic hexapeptides, Boc-Leu-Phe-βDPhe-βLPro-Phe-Leu-OMe (βDFβLP6) and Boc-Leu-Phe-βLPhe-βLPro-Phe-Leu-OMe (βLFβLP6) suggest that the βDPhe-βLPro segment is capable of forming a C12 turn in methanol. Two octapeptide sequences, Boc-Leu-Val-Leu-βDPhe-βLPro-Leu-Phe-Val-OMe (βDFβLP8N) and Boc-Leu-Val-Val-βDPhe-βLPro-Leu-Val-Val-OMe (βDFβLP8V) have also been investigated to probe the possible formation of hairpin structures. In these cases, spectroscopic analysis is hampered by the presence of multiple conformations, because of the tendency of the βDPhe-βLPro bond to exist in both cis and trans conformations. NMR studies on the conformational properties of a hexapeptide Boc-Leu-Val-βDPro-βLPro-Leu-Phe-OMe (βDPβLP6) in CDCl3 reveal that in the major conformer the Val 2(NH) ↔ Leu 5 (NH) NOE is observed, suggesting the presence of a 12-membered hydrogen bonded turn. A ββ - segment can give rise to two types of hydrogen bonded rings , 10 – membered (C10) and 12- membered (C12). In an attempt to generate C10 turns, an N-methylamino acid has been inserted next to a ββ - segment, preventing the formation of the 12 – membered turn. In such a situation formation of a 10-membered turn, with reverse hydrogen bond directionality, may be facilitated. The conformational properties of Boc-Leu-Val-βDPhe-βLPro-(N-Me) Leu- Phe-OMe (βDFβLPNMeL6) has been studied by 500 MHz NMR spectroscopy. The data suggests the formation of a C11 turn at the βLPro- (N-Me) Leu segment in CDCl3-DMSO mixtures, instead of formation of a C10 turn at the βDPhe -βLPro segment. Studies on the peptide Boc-Leu-Phe-Val-βLPro-(N-Me) Leu-Leu-Phe-Val-OMe (βLPNMeL8) also suggest the absence of turn formation and folded structures. In hybrid sequences, an important question to be addressed is whether ω amino acids can be accommodated into helical structures. Two contiguous β- residues have been inserted into a helical sequence. The conformational properties of a 11- residue peptide, Boc-Val-Ala-Phe-Aib-βVal-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA11) are described in Chapter 5. This sequence was based on the parent α- peptide Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe, which adopted a complete helical conformation in crystals (Aravinda, S.; Shamala, N.; Das, C .; Sriranjini, A.; Karle, I.L. and Balaram, P. J. Am. Chem. Soc. 2003, 125, 5308-5315). 500 MHz 1H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl3 solution , as evidenced by diagnostic nuclear Overhauser effects. The molecular conformation in crystals reveals a continuous helical fold, stabilized by seven intramolecular hydrogen bonds. The characterization of two synthetic octapeptides Boc-Val-Ala-βPhe-Aib-Val-Ala-βPhe-Aib-OMe (VAβFU8) (βPhe residues have been incorporated at (i /i+4 positions) and Boc-Val-Ala-βPhe-Aib-βPhe-Ala-Val-Aib-OMe (βFUβF8) (βPhe residues have been incorporated at (i /i+2 positions) is also presented. NMR data suggests the retention of helical conformation in both the peptides. In order to delineate the conformations of hybrid peptides with three contiguous β-residues, two peptides have been synthesized Boc-Phe-Aib-βGly-βLeu-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA10) and Boc-Val-Ala-Phe-Aib-βGly-βLeu-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA12). NMR studies in chloroform support continuous helical conformation in the decapeptide.

The effects of peptides derived from β-lactoglobulin and α-lactalbumin on metabolic syndrome were studied. α-lactalbumin and β-lactoglobulin were hydrolyzed with trypsin, alcalase, flavourzyme, or a combination of alcalase and flavourzyme and fractionated. Angiotensin coverting enzyme inhibition of the < 1 kDa fraction of alcalase hydrolyzed β-lactoglobulin was 95 %. Antioxidant activity of the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme was 18 %. Stimulated adipocytes incubated with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with either trypsin or alcalase produced 30 pg/mL of interleukin 6. Adiponectin and glucose transporter type 4 secretions increased 1.1 and 0.86 fold respectively during incubation with the < 1 kDa fraction of β-lactoglobulin hydrolyzed with a combination of alcalase and flavourzyme. Results indicate that β-lactoglobulin peptides formed with alcalase and a combination of alcalase and flavourzyme influence markers associated with metabolic syndrome and may be useful as functional foods or nutraceuticals.

博士
國立中興大學
化學系所
101
Protein backbone oxidation was investigated by studying the α-H abstraction reaction in a s-hairpin peptide, called Chignolin (PDB ID 1UAO), with density functional theory calculation at B3LYP/6-31G(d,p) without any constraint. In order to stabilize the zwitterionic form of Chignolin with the salt bridges, the effects of aqueous solution were implemented by using microsolvation combined with a conductor-like polarizable continuum model (CPCM). Comparison between three glycine residues located at three different sites in Chignolin was used to examine the possible site specificity of this backbone oxidation. To construct the reaction profile of these α-H abstraction reactions, the pre- and post-reactive complexes along with their associated transition states were located and verified with the intrinsic reaction coordinate (IRC) method. The bond dissociation energy and reaction rates of these OH α-H abstraction reactions were calculated with transition state theory. The differences in this abstraction reaction between the neutral and zwitterionic forms of Chignolin were also compared. A molecular dynamics simulation was implemented to study the explicit solvation effect on the abstracted Chignolin structure. The range of the simulation time scale covers from fs to ns, i.e., from onset of the abstraction to the abstracted products reaching thermal equilibrium. Our results show that there are three kinds of site-specificity in this abstraction reaction. The reactivity and stability of the abstraction products and their abstraction modes are all dependent on the location where OH attacks. Furthermore the free energy landscapes of these abstraction products are distinctively different. This may imply the pathological disorders or diseases caused by this type of radicals are also dependent on the abstraction location.

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.

ΔPhe an analogue of a coded amino acid phenylalanine (Phe) residue but with double bond between Cα and Cβ atoms, is one of the well studied residue among all the dehydro amino acids, as a conformation constraining amino acid. Due to the presence of double bond Cα=Cβ, and consequent conjugation of ΔPhe ring electrons with Cα=Cβ double bond, ΔPhe gains conformation restricting (constraining) characteristics compared to coded amino acid Phe. ΔPhe which assumes an achiral residue has all its atoms restricted to an approximate plane. Apart from the conformation constraining property, the designer friendly ΔPhe residue has its ability to i) engage in side chain aromatic interactions ii) act as nuclei for C-HLO/N-HLπ weak interactions involving the side chain and/ or backbone atoms, and iii) acquire ambidextrous conformation as observed in many model peptides. It is these properties, which makes ΔPhe, a residue of intense research in the field of de novo protein secondary and super secondary design. Analysis of solid state and solution state structures of containing ΔPhe residues suggests that ΔPhe, in general induces β-bend in short peptides and 310-helical conformation in longer peptides (>4).

碩士
國立臺灣大學
化學研究所
101
β-Sheet is a common secondary structure. The structure can be stabilized by cross strand interactions such as backbone hydrogen bonds and side chain electrostatic interactions. Ion pairing interactions can form between two oppositely charged amino acids, such as Lys, Arg, Glu, and Asp. However, some unnatural amino acids contain different number of methylene groups in the side chain are not incorporated in natural proteins. Accordingly, the ion pairing interaction involving Asp-Lys, Asp-Orn, and AspDab were investigated in a β-hairpin system. The 2D-NMR spectra including TOCSY, ROESY, and COSY were acquired for the peptides. The peptide β-hairpin structure as confirmed by chemical shift deviations, 3JNHα coupling constants, and NOE connectivities. The β-hairpin population and stability were determined by comparing the chemical shifts with the reference peptides. The peptide stability followed the trend HPTAspLys ~ HPTAspOrn > HPTAspDab. The ion pairing interactions were derived from double mutant cycles. The corresponding ion pairing interactions followed the trend Asp-Lys ~ Asp-Orn ~ Asp-Dab. Calmodulin (CaM) is a highly conserved and abundant protein in eukaryotic cells. Calmodulin participates in many physiological processes, such as muscle contraction, insulin and pituitary secretion, and neurotransmission. CaM can bind to basic amphiphilic α-helical peptides. To control the 3D-arrangement of side chains and minimize proteolytic degradation, β-amino acids were incorporated into peptides. β-Amino acids are unnatural amino acids containing one extra methylene group in the backbone compared to natural α-amino acids. In this study, the side chain sequences of peptides were the same as the basic amphiphilic α-helical peptide, but the backbone patterns were varied by mixing α- and β-amino acids, including αβ, ααβ, αααβ, and ααβαααβ. The binding affinities were determined by CD spectroscopy and fluorescence anisotropy. The proteolytic susceptibilities were tested against trypsin.

Ribosome Inactivating Proteins (RIPs) are protein or glycoprotein toxins that bring about the arrest of protein synthesis by directly interacting with and inactivating the ribosomes. Such toxins are in general, of plant origin and differ from bacterial toxins that inhibit protein synthesis by mechanisms other than ribosome inactivation. After the toxins had been in the centre of interest in biomedical research for a couple of decades in the end of 19th century, the scientific community largely lost interest in the plant toxins. Interest in these toxins was revived when it was found that they are more toxic to tumor cells when compared to normal cells. Based on their structure RIPs can be classified into three types: Type I RIPs – They consist exclusively of a single RNA-N-glycosidase chain of ~30kDa. Type II RIPs – They consists of chain-A comparable to type I RIPs linked by a disulfide bridge to an unrelated chain-B, which has carbohydrate binding activity. The molecular weight of the type II RIPs is ~60kDa. Type III RIPs – Besides the classical type II RIPs a 60kDa RIP (called JIP60) has been identified in barley (Hordeum vulgare) that consists of chain-A resembling type I RIPs linked to an unrelated chain-B with unknown function. In addition to these classes of RIPs there is another group of toxins called four subunit toxins, whose structure is almost similar to type II RIPs, but are made up of two such subunits linked by non-covalent interactions forming tetramers having two A- and two B-chains. The definition and classification of these toxins is not so clear as they are frequently referred to as agglutinins or lectins (e.g Abrus precatorius agglutinins I and II, Ricinus communis agglutinin etc.), having red blood cell (RBC) agglutinating activity. However they have been found to be less toxic and better agglutinins when compared with type II RIPs. The present thesis reports the crystal structure of a type II RIP, Abrus precatorius agglutinin-I (APA-I) from the seeds of Abrus precatorius plant. The protein was purified from the plant seed and crystallized. The crystal structure was solved by molecular replacement method. Preliminary crystals of abrus agglutinin were obtained almost thirty years ago and unsuccessful attempts to solve the crystal Structure of APA-I were made almost five years ago by other groups. The structure solution of API-I was obtained at 3.5 Å using synchrotron data set collected at room temperature from a single crystal. Crystal structure is already known for Abrin, another type II RIP isolated from the same seeds. Abrin and APA-I have similar therapeutic indices for the treatment of experimental mice with tumors, but APA-I has much lower toxicity, with lethal dose (LD50) being 5mg/kg of body weight when compared with Abrin-a (LD50 = 20 μg/kg of body weight). The striking difference in the toxicity shown by Abrin and its agglutinin (APA-I) encouraged us to look at the structure function relationship of these proteins, which might prove to be useful in the design and construction of immunotoxins. As apparent from the comparative study, the reduced toxicity of APA-I can be attributed to fewer interactions it can possibly have with the substrate due to the presence of Pro199 at the binding site and not due to any kink formed in the helix due to the presence of praline as reported by other groups. In recent years, these plant RIPs which inhibit protein synthesis have become a subject of intense investigation not only because of the possible role played by them in synthesizing immunotoxins that are used in cancer therapy but also because they serve as model system for studying the molecular mechanism of transmembrane translocation of proteins. In silico docking studies were carried out in search of inhibitors that could modulate the toxicity of RIPs. Many adenine like ringed compounds were studied in order to identify them as novel inhibitors of Abrin-a molecule and facilitate detailed analyis of protein ligand complex in various ways to ascertain their potential as ligands. In addition, the structural analysis of conformationally constrained, α β-dehydrophenylalanine containing dipeptides is carried out. While there are several studies of molecular self assembly of peptides containing coded amino acids, not much work has been done on molecular assembly formation utilizing non-coded amino acids. The non-coded amino acid used in the analysis is a member of α β-dehydroamino acids. These are the derivatives of protein amino acids with a double bond between Cα And Cβ atoms and are represented by a prefix symbol ‘Δ’. They are frequently found in natural peptides of microbial and fungal origins. The presence of α , β-dehydroamino acid residues in bioactive peptides confers altered bioactivity as well as an increased resistance to enzymatic degradation. Thus, α, β-dehydroamino acid residues, in particular α, β-dehydrophenylalaine(ΔPhe) has become one of the most promising residues in the study of structure-activity relationships of biologically important peptides. The utilization of in the molecular self assembly ΔPhe in the molecular self assembly offers in added benfit in terms of variey and stability. Taking advantage of the conformation constraining property of the ΔPhe residue, its incorporation in three dipeptide molecules has been probed. In this thesis the crystal structures of the following designed dipeptide are reported.(I). +H3N-Phe-ΔPhe-COO˙ (FΔF); (II). +H3N-Val-ΔPhe- COO˙ (VΔF); +H3N-Ala-ΔPhe-COO˙ (AΔF). The peptides were found to be in the zwitterionic conformation and two (I, II) of the three dipeptides have resulted in tubular structures of dimensions in the nanoscale range. Chapter 1 starts with a brief introduction of RIPs, their classification and overall fold, with Abrin-a as example. A brief mention is made about how the protein is translocated in the cell and the depurination mechanism. Chapter 2 presents the purification of APA-I from the seeds of Abrus precatorius plant, the crystallization of APA-I, X-ray intensity data collection on these crystals and processing of data sets for APA-I. Chapter 3 details the structure determination of tetramer Abrus precatorius agglutinin-I,(APA-I), using the molecular replacement method, iterative model building and refinement and the quality of final protein structure model. Chapter 4 details the crystal structure of Abrus precatorius agglutinin-I (APA-I), the comparison of primary and secondary structure of APA-I with Abrin-a and the structural insights into the reduced toxicity in relation to Abrin-a and future prospects. Chapter 5 deals with the in-silico modeling of Abrin-a inhibitors using the docking method. Abrin-a is being tested extensively for the design of therapeutic immunotoxins. Chapter 6 deals with the self-assembly of dipeptides containing conformationally constrained amino acid, α. β -dehydrophenylalanine (ΔF).