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Dynamic combinatorial libraries in protein mimicry applying the template assembled synthetic protein concept
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Amyloid ß-derived switch-peptides as tool to study conformational changes relevant in degenerative diseases
Get PDFThe rapid growing number of patients diagnosed with a neurodegenerative disease and more particularly with Alzheimer's disease (AD) has stimulated intensive research in determining and understanding biological phenomena causing such devastating diseases and hence allowing for the elaboration of adapted therapeutic treatments. These diseases are also commonly called "conformational" diseases because they result from the misfolding of a protein leading to the formation of self-associated β-sheets, which in turn give rise to the formation of oligomers, protofibrils as well as insoluble fibrils characterizing the plaques found in the brain of affected patients. Consequently, the investigation of such proteins, in particular of Amyloid β (Aβ) in the case of AD, is a limited and difficult task to achieve, which often leads to contradictory results. To overcome these difficulties and to be able to study the key steps of conformational transitions and misfolding of such peptides and proteins, our research group has developed a new tool, called switch-peptides, enabling to block (Soff state) and trigger (Son state) peptide folding at will (Figure). The introduction of a switch element S built from Ser, Thr or Cys residues disrupts the regular polypeptide chain by the insertion of an ester and a flexible C-C bond resulting in a conformational disconnection of P1 and P2 (Figure), i.e. in an unordered (random coil), non-folded conformation. Each S element is protected by a protecting group Y (Soff state) that can be cleaved independently by adding a base, an enzyme or by light, depending on the chemical nature of Y. The cleavage of the different protecting groups Y triggers a spontaneous O to N acyl migration, re-establishing the regular amide backbone of the peptide chain, hence enabling the peptide to fold "in statu nascendi" and to adopt a well-defined secondary structure. The present thesis explores the potential of this novel concept for the example of conformational transitions relevant in amyloid β misfolding. In the first part we investigate the chemical stability of the S element in aqueous media, exposing a number of switch-peptides to various experimental conditions. Most notably, the ester bond proved to be stable at acidic as well as physiological conditions for several hours, opening a broad range of biological applications. The second part of the work is dedicated to the study of conformational transitions of switch-peptides derived from Aβ(1-42). By incorporating one or several switch elements disposing orthogonal protecting groups Y, the impact of different fragments of the peptide as nucleation site for the process of β-sheet formation, self-assembly and aggregation has been revealed as monitored by CD, TEM studies and ThT (pathway B, Figure). For the first time, the orthogonal triggering of the two switch elements, i.e. S26 and S37 allowed to delineate the important role of the C-terminal part of Aβ in the early step of misfolding. Subsequently, one of the nucleation sites for aggregation, i.e. segment Aβ(14-24) was excised from the native sequence and transformed to a switch-peptide applying the host-guest technique. Detailed CD studies were applied for investigating conformational transitions of type random-coil (Soff) to β-sheet structure (Son), serving as proof of concept for the use of Aβ-derived nucleation sites as guest sequence in combination with β-sheet promoting host peptides for the screening of potential inhibitors of fibril formation as early molecular event in the context of AD. This has been demonstrated in applying the elaborated host-guest peptides to evaluate the β-sheet breaking potential of pseudo-proline (ψPro)-containing switch-peptides derived from Aβ (pathway C, Figure). Preliminary results indicate that the in situ formation of kink-conformations may exert a β-sheet destabilizing effect, confirming previous observations from the Soto group. Finally, the use of switch-peptides as β-sheet and fibril breaking molecules by in situ α-helix nucleation (pathway A, Figure) has been explored. To this end, the potentially β-sheet forming segment Aβ(14-24) was linked via S element to a helix nucleating peptidomimetic ("N-Cap"). In the Soff state (pH ≤ 4), CD and TEM studies point to the onset of a β-sheet, fibril forming structure. In triggering O,N-acyl migration (pH ≈ 7), a so far unprecedented transition of type β-sheet to α-helix is observed, paralleled by a drastic increase in solubility and a complete disappearance of fibrils. The reversibility of β-sheet and fibril formation by α-helix nucleation in situ represents a most interesting observation and deserves further exploration as potential tool in the study of folding processes. In conclusion, the concept of "switch-peptides" has been successfully applied to biologically relevant molecular events of utmost therapeutic interest.LCB
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Switch peptides : in situ nucleation of conformational transitions relevant to protein misfolding diseases
No full textAspects of conformational transitions, folding, and misfolding of peptides and proteins have recently taken center stage in various domains at the interface of chemistry, biology, and medicine because of their impact on protein misfolding diseases. Due to the intimate relationship between sequence, secondary structure, and physicochemical properties, studies on oligo- and polypeptides exhibiting high propensity for β-sheet formation are severely limited by their intrinsic tendency toward self-association and irreversible aggregation. Consequently, β-sheet and fibril-forming processes related to protein misfolding diseases remain elusive, as delineation of structure nucleation or inhibition is a formidable task, often yielding contradictory observations. With the aim of surmounting these experimental difficulties and of inducing conformational transitions in situ, the present thesis elaborates the novel concept of switch-peptides (see figure). Modular switch-peptides are synthesized comprising three distinct elements: a conformation induction unit (σ), a switch-element (S), and a peptide (P). Of central importance to the switch-peptide concept, the switch-element S dissects the native polyamide backbone of the peptide P by an ester and a flexible C-C bond, thereby preventing the peptide from folding and separating the conformational impact of σ. The triggering of the switch-element by removal of the protecting group Y reestablishes the native polyamide backbone via a spontaneous O → N acyl migration reaction, whereupon peptide folding can then proceed. Detailed herein is the development of the key elements of the concept, σ and S, and their subsequent application to various model peptides and peptides of biological interest (P). In particular, two synthetic routes to S-elements were demonstrated, i.e. the stepwise solid phase synthesis of depsipeptides using a minimal protection strategy and the preparation of protected depsidipeptides for use as building blocks in solution or solid-phase synthesis. Kinetic investigations of their acyl rearrangements reveal half-lives that range from less than one minute to several hours. The possibility to tailor the half-lives by manipulation of experimental conditions is also elaborated. Orthogonal protection strategies including photolytically and enzymatically labile groups were developed. This enabled the incorporation of multiple S-elements into a peptide that could be triggered sequentially. In this way, the mutual conformational impact of peptide segments as well as their role in folding and self-assembly was explored. The design and synthesis of novel, helix-nucleating N-cap compounds (a type of σ element) is described and applied for the in situ nucleation of helical conformations according to the figure. For the first time, a detailed study of a wide variety of original N-caps is realized in which their helix inducing abilities are quantified by CD spectroscopy. When applied to model β-sheet-forming switch-peptides (random coil → β-sheet), the use of an N-cap is shown to overcome the intrinsic β-sheet propensity and instead induce a conformational transition to an α-helix. Furthermore, an hitherto unobserved conformational transition of type β-sheet → α-helix in an amyloid beta-derived switch-peptide is demonstrated and shown by electron microscopy to inhibit its fibrillization. Through a series of designed switch-peptides, the S-element is shown to indeed conformationally decouple segments σ and P and, upon triggering, to induce conformational transitions relevant to protein misfolding. The results obtained firmly establish the capability of employing the switch-peptide concept as a tool to study folding events during the dynamic process of structure onset and evolution. With the methodologies elaborated herein, the stage is set for its further application to the study of structure-function relationships, prodrug design, the design of diagnostic systems for evaluating inhibitors of secondary structure, and to β-breaker peptides.LCB
Synthèse, structure et propriétés physicochimiques des cyclosporines
No full textCyclosporin is a cyclic undecapeptide useful as drug for organ transplantation due to its immunosuppressive properties. Ten years ago, an anti-viral activity was discovered, incompatible with immunosuppression. These two activities originate in the presence of two receptors, i.e. cyclophiline A, which interferes in two processes and calcineurine, regulating immunosuppression. Consequently, the synthesis of modified analogues at different positions represents a research domain of high importance for elucidating structure-activity relationships of these receptors. Position 2 of cyclosporin is in close contact to cyclophiline and, consequently, of particular interest. cyclosporin C (CsC) differs from the well-known immunosuppressive analogue cyclosporin A (CsA) by the presence of a trifunctional amino acid at position 2, i.e. a threonine residue replacing 2-amino butyric acid (Abu2) in CsA. While the biological and pharmacokinetic properties of CsC and CsA are very similar, the presence of the threonine makes CsC a most attractive candidate for structure-activity relationship studies in applying the pseudo-proline concept (ΨPro) developed in the group of Prof. M. Mutter. The advantage of this concept relies in the direct incorporation of the pseudoproline moiety into CsC by cyclocondensation of ketals with yields reaching up to 60%, depending on the substituents R1 et R2 (figure 1). Figure 1: Direct insertion of the pseudo-proline system into CsC NMR analysis showed that monosubstitutions at C2 strongly effect the conformational properties. For example, unique conformations were found in weakly polar (CDCl3) and polar solvents (DMSO), depending on R1/R2. In contrast, the simultaneous presence of R1 and R2 result in intermediate effects. The drastic conformational impact on the Cs backbone by the invertion of ΨPro-systems is manifested by the loss of biological activity. Based on these results, the ΨPro concept was applied for the synthesis of so-called soft prodrugs of CsC. As prototype, a derivative containing a phosphate group (R1 = 3,5-Dimethoxy-4-phosphonooxyphenyle; R2 = H) improved the water solubility of cyclosporin by a factor higher than 500. The developed strategies can be applied to any peptide featuring a threonine or serine in its primary sequence, paving the way to a new class of prodrugs. With the goal to obtain analogues of higher receptor selectivity, the second part of the thesis focuses on the modification of position 8 of cyclosporin. Most notably, located in the effector domain of calcineurin, modifications of residue D-Ala8 should prevent the binding of the dimeric complex Cs-cyclophiline A to calcineurin, a prerequisite for antiviral and non-immunosuppressive activity. Based on experiments on the alkylation of CsA at D-NAla8, a selective ring-opening procedure of CsA between residues Ala7 and D-Ala8 was elaborated in three major steps: Selective alkylation of the nitrogen of D-Ala8 in 43% yield (5 steps), N —> O acyl transfer of Ala7 (>70%) and a solvolysis followed by Edman degradation to the linear decapeptide in 24.5% yield. Starting from CsA, the linear decapeptide was obtained on 10.5% overall yield for 10 steps (figure 2). Figure 2: Retrosynthesis of the 7-8 ring-opening of CsA While the reported ring opening procedures allow the study of the influence of modifications on positions 11 to 5 of Cs, the elaborated strategy gives access to the efficient modification of key position 8, opening interesting perspectives for structureactivity relationship studies of high therapeutical potential.LCB
Cyclosporin analogues : potential HIV inhibitors and water-soluble prodrug systems
No full textCyclosporins are a family of hydrophobic cyclic undecapeptides produced by the fungus Tolypocladium niveum. The main metabolite cyclosporin A (CsA, see Scheme, R = R' = H) is the first line drug currently used to prevent rejection of organ transplants. Its strong immunosuppressive activity is achieved through the formation of a cyclosporin–cyclophilin A (CypA) complex inhibiting the calcium- and calmoduline-dependent protein phosphatase calcineurin (Cn). This trimeric complex prevents translocation of the transcription enhancer NFAT into the nucleus, inhibiting T-cell activation. It was also shown that, in the presence of CsA, chronically infected cells produce non-infectious HIV particles. By binding with the HIV capsid gag polyprotein pr55, CypA is recruited in stoichiometric amounts in the nascent HIV virion. The formation of the CsA-CypA complex disrupts this interaction at the maturation stage of the HIV infection and blocks the replication of HIV in cells. Based on SAR studies, the present thesis aims to develop novel analogues and prodrugs of CsA exhibiting antiviral, non-immunosuppressive activities. The first part of this work describes the regioselective synthesis and the pharmacological properties of a novel series of dimers of cyclosporin analogs modified at position 8 (see Scheme, in blue). Our findings demonstrate that the dimerization at position 8 of cyclosporin derivatives strongly increase their affinity to CypA and drastically alter their binding to Cn, thus representing the first derivatives of cyclosporin exhibiting non-immunosuppressive (loss of Cn binding), anti-viral (CypA binding) activities by the exclusive modification at position 8. By designing linkers of tailored length to permit the cooperative interaction with both immunophilin receptors, bivalent ligands have been developed. As indicated by receptor binding studies, these dimers represent the most potent non-immunosuppressive cyclophilin binders reported up to now, potential anti-HIV-1 inhibitors. The goal of the second part of this thesis was to use various concepts for prodrug design for modulating the pharmacokinetic properties and for increasing the low water-solubility of cyclosporin analogs that is responsible for undesirable pharmaceutical properties of cyclosporins such as erratic oral absorption profile, poor oral bioavailability and complications in formulating. A prodrug is a pharmacologically inactive derivative of a parent drug molecule that requires spontaneous or enzymatic transformation within the body in order to release the active drug, exhibiting improved delivery properties compared to the parent drug molecule. The elaborated chemical modifications of the cyclosporin derivatives allowed us (1) to mask the bioactive conformation of the cyclosporine, leading to the loss of binding to CypA and Cn, and consequently in a loss of the immunosuppressive activity and (2) to increase the solubility of the cyclosporins through the introduction of a solubilizing group (SG). As a first chemical modification, the hydroxyl group of the residue MeBmt-1 of cyclosporins was masked by a chemical unit that can be selectively removed enzymatically (Scheme, in green). The double prodrugs showed an increase in water-solubility of factors up to 1000 compared to their parent drug and a specific enzymatic controlled drug release. Alternatively, a novel strategy for the selective introduction of an acyl-function at different positions of hydroxyl-containing cyclosporins (Scheme, blue and red) was explored. The corresponding O-acyl isopeptides undergo spontaneous O,N-acyl migration (serving as chemical switch), resulting in the recovery of the parent compound under physiologically conditions (pH controlled drug release). The investigated derivatives showed high thermodynamic stability, differential conversion rates and strongly increased water solubility, offering novel chemoreversible prodrugs of cyclosporin analogs. In extending this concept, O-acyl iso-cyclosporins stabilized by a pro-moiety were developed, setting the stage for a novel class of double prodrugs exhibiting improved thermodynamic stability and tailored chemical and enzymatic conversion rates. With the increasing challenges in drug delivery of complex therapeutic agents, the elaborated systems provide versatile, generally applicable strategies for hydroxyl group containing drug candidates.LCB
Switch-peptides: controlling biological function and self-assembly of amyloid ß-derived peptides using enzyme-triggered acyl migrations
No full textConformational transitions of peptides and proteins have recently moved to the center of interest in various domains of research at the interface of chemistry, biology, and medicine due to their implication in an increasing number of diseases in which the pathogenesis is causally linked to the misfolding of a protein. For example, the amyloid β protein linked to Alzheimer's disease undergoes various conformational transitions, eventually leading to the formation of self-associating β-sheets, which in turn give rise to insoluble, fibrillar aggregates. Consequently, studies of such proteins are hampered by problems of solubility and often yield contradictory results. With the aim of surmounting these difficulties in the study of conformational transitions of peptides and proteins, a novel tool based on the concept of "Switch-Peptides" enabling the in situ induction of peptide folding will be elaborated in the present thesis. As shown in the Scheme, N(Y)-protected switch-peptides serve as stable, self-contained precursor molecules, in which folding and self-assembly is blocked by the presence of Ser or Thr derived switch-elements S, dissecting the regular peptide backbone by an ester and a flexible C-C bond. Removal of the protecting group Y triggers a spontaneous acyl migration that restores the native polypeptide chain ("statu nascendi") setting off the folding process. The body of work presented herein consists of two main themes: With the goal of developing a system amenable to physiological conditions, the introduction and evaluation of different, enzymatically cleavable Y groups was conceived. For proof of concept, the biologically active peptides angiotensin and neuropeptide Y were used, allowing the study of the impact of the S-element on their biological activities. Investigations of the analogue [Thr]5-Ang II containing a pH-triggered switch (Y = H+) reveal that the acyl migrations are very fast, exhibiting half-lives of less than one minute. With the introduction of enzymatically labile Y groups, specifically an Arg-Pro dipeptide (cleavable by dipeptidyl aminopeptidase) or the amino acid pGlu (cleavable by pyroglutamate aminopeptidase), rates of conversion ranging from minutes to hours are observed as monitored by HPLC, NMR and CD. Problems of steric interference associated with the action of enzymes such as esterases and penicillin amidase were overcome by developing linkers of different structural features. D-aminopeptidase (Y = D-Ala) was shown to extend the rates of enzymatic cleavage to half-lives of several days. The enzymes studied display high specificity for the switch-peptide substrates and a quantitative conversion from the Soff to the Son state. We took advantage of the affinity of Ang II and NPY for their respective receptors (AT2 and Y1) to show that their switch-peptide analogs are inactive in the Soff state where as after enzymatically triggered acyl migration, the peptides display high receptor affinity (IC50 = 45 nM for Ang II and IC50 = 5 nM for NPY). In the case of NPY, a conformational transition from a random coil to an α-helix as bioactive conformation is observed by CD. The second part of the work is dedicated to conformational transitions of type random coil → β-sheet in different model peptides or peptides derived from Aβ(1-42). All peptides studied show high potential for self-association and subsequent precipitation. However, in the Soff state, the switch-peptides display a high degree of stability and strongly enhanced aqueous solubility. After enzymatic triggering of the acyl migration, a conformational transition to a β-sheet accompanied by the formation of fibrils and spontaneous precipitation are observed by CD and EM studies. Based on the elaborated systems, the introduction of several orthogonal S-elements in Aβ derived peptides was realized. The incorporation of two different S-elements into the Aβ(1-42) sequence was found to greatly facilitate the normally difficult synthesis, and enabled the study of the influence of the individual peptide segments on the overall folding process. It was shown for the first time that the hexamer Aβ(37-42) is essential for the formation of β-sheets and subsequent fibrillization. As a final aspect, the elaboration of switch-peptides exhibiting β-breaking properties was investigated. Preliminary results indicate that one of the designed switch-peptides substantially slows down the formation of fibrils, representing a significant step in the development of therapeutically relevant compounds for the treatment of Alzheimer's disease. In conclusion, the elaborated methodologies allow for a general application of the switch concept as diagnostic tool in the study of fibrillogenesis.LCB
Elaboration d'un vaccin anti-nicotine : développement et synthèse de conjugués immunogéniques de dérivés
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Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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