"Switch peptides" | EPFL Graph Search

Despite of considerable progress in the research at the interface of Chemistry, Biology and Medicine, neurodegenerative diseases affect more and more seriously our ageing population and remain a real therapeutic challenge for researcher. Nevertheless, advances made in this field allow us to better understand the molecular mechanisms responsible for these disorders and to stress the involvement and the key-role of conformational changes of some proteins. Thought to be at the origin of Alzheimer's disease, amyloid-β peptide undergoes structural modifications leading to β-sheet structures, which further aggregate into toxic fibrils and plaques. However, intrinsic properties of these structures result in problems such as insolubility and self-aggregation limiting their experimental access. Among the main research lines to find an efficient therapeutic agent to fight against Alzheimer's disease, fibrillar inhibition is considered as promising strategy. It requires the use of a molecule, called β-breaker, which would be able to block the misfolding triggered by conformational transitions. Consequently, the present thesis is focusing on the design and chemical synthesis of potential inhibitors of amyloid-β fibrillogenesis. As a specific common feature, the designed compounds integrate in their chemical structure a short sequence ("nucleation site") derived from the central part of Aβ(1-42), allowing to specifically interact with the pathogenic peptide. We first envisaged to design cyclic peptides as potential inhibitors containing proline residues, known for its destabilizing effect upon secondary structures (way A). In collaboration with C. Soto et al., Univ. Galveston, the biological activity of the prototype cyclo(Lys-Leu-Pro-Phe-Phe-Glu) was tested. Most notably, electron microscopy, in collaboration with J. Dubochet and M. Adrian, UNIL, Congo red and thioflavin T staining, and CD studies pointed to the potential of cyclic peptides as Aβ inhibitors. Subsequently, conjugate peptides, assembling the recognition sequence of amyloid-β and organic molecules with potential to block fibrillogenesis were synthesized specifically. (+/-)-Trans-4-cotininecarboxylic acid, 3-indolebutyric acid and a tripeptide β-strand mimic were chosen. The biological assessment of these molecules allowed us to highlight their potential in this therapeutic strategy as well as the advantage given by the intercalation of a proline between the two components of these conjugates. In an effort to introduce in the structure of β-breakers a dynamization element, comparable to the cis/trans isomerization of proline, we designed a new type of β-breaking molecules according to strategy B (Figure). The elaboration of this innovating concept termed "switch-peptides" allows to controll the function of a polypeptide by using intramolecular acyl migration as switch-element for the in situ induction of structure and function. By combining an conformational induction unit σ, a switch element S (cysteine or serine) and an amyloid recognition sequence, we obtained a new generation of dynamic β-breakers. At the Soff state, the σ part was linked to the switch element S via an ester bond, deactivating the structural influence of the conformational induction unit on the target peptide, resulting in the absence of biological activity. By removing the protecting group Y from the amino function of the switch element, intramolecular acyl migration was triggered, restoring the native amide bond, setting off the impact of σ (Son state). In applying pseudo-prolines as β-breaking σ-elements (resulting in a "kink" conformation), the corresponding switch-peptide can adopt a recognition state (Soff) and a β-sheet disrupting state (Son), triggered by controlled acyl migration. We explored the use of cysteine as switch element, with special attention to the chemoselective synthesis, kinetics of acyl migration and potential for β-breaking. Interestingly, the S→N acyl migration proceeded very fast at physiological pH, in comparison to serine derived O→N migrations. Of utmost importance for further extensions of the switch-concept, the design of non peptidic S-elements such as trifunctionalized aromatic compounds would allow us to generate the β-breaking element in situ. As a first step, the intramolecular acyl migration O→N via an intermediate of 5, 6, 7 or 9 membered rings, as well as the reversibility of this reaction, was successfully demonstrated. In addition, enzymatically cleavable protecting groups Y for triggering acyl migrations were established. In conclusion, the present thesis presents some promising concepts in the design of fibril disrupting compounds of considerable therapeutical potential.

Le concept switch-peptides

Karine Murat

Conformational transitions have found broad interest due to their impact on protein misfolding and self-assembly as key events leading to the development of neurodegenerative diseases. However, investigation of these dynamic events has been limited so far mainly due to the intrinsic tendency of the involved polypeptides for self-association and aggregation. Consequently, the elucidation of β-sheet and fibril-forming processes related to degenerative diseases remains a difficult task, often giving contradictory results. In order to overcome these difficulties and to investigate early steps of misfolding and subsequent fibril and plaque formation, a new concept termed "Switch-peptides" is presented in this thesis, opening new perspectives for the rational design of drugs preventing Alzheimer's disease. Figure. Elaboration of the "Switch-peptides" concept via S to N acyl migration. As shown in the Figure, the switch-peptides represent stable, self-contained folding precursors, in which folding and self-assembly is blocked by the presence of the Ser-, Thr-, Cys- derived switch elements S dissecting the regular peptide backbone by an (thio)ester and a flexible C-C bond (Soff). Removal of the protecting group Y by enzymatic cleavage or by change of pH, triggers X,N acyl migration restoring the native peptide backbone in situ, and setting off the folding process in the nascent state ("in statu nascendi", ISN) of the parent molecule. These resulting conformational changes i.e. from a random coil to a folded state, can be used to study the onset of biological activity (A) onset (B) and disruption (C) of secondary and tertiary structures. The work described in the present thesis elaborates the structural and chemical foundations of the concept, notably in the induction of structural and conformational properties of peptides. Special attention is focused on the S to N acyl migration in elaborating an in situ ligation methodology. For potential applications in vitro and in vivo, we explore a new thioester solubilizing group to realize efficient native chemical ligations at physiological conditions (Figure). Besides the synthetic access of switch-peptides thioesters via solid-phase techniques, the impact of internal (steric hindrance) and external pH factors on the kinetics of the intramolecular acyl migration is evaluated by analytical HPLC. The major applications of the switch-peptides concept are explored via three main axis : In order to settle the foundations of strategy B, our study was based on the possibility of inducing in situ the formation of secondary and tertiary structures. For this purpose, an amphiphilic model peptide is designed to mimic conformational transitions relevant in degenerative disease, i.e. transitions of type random coil to β sheet. The role of the switch element S in the disruption of secondary structures due to the conformational decoupling of σ on P (Soff), is demonstrated by CD.The induced conformational transition to β sheet conformations is accompanied by a significant decrease in peptide solubility. Moreover, the controlled release of nucleophile within the S element can modulate the rate of structure transition over a broad time scale, as demonstrated by kinetic studies. The use of orthogonal protecting groups Y allows the sequential and individual triggering of multiple elements S, localized in strategic positions in more complex polypeptides. With this objective in mind, the concept is expanded to the induction of ββα supersecondary structures such as the Zinc finger domain (Zif). Actually, the simultaneous introduction of a chemically (pH) and enzymatically (DPPIV) cleavable group at the N- and C-terminus in a sequence derived of Zif1 268 permited the consecutive onset of secondary and tertiary structure by a hierarchical triggering of acyl migrations. The second part of the thesis is devoted to the elaboration of a new generation of β breaking switch-peptides (Figure, strategy C). For the design of such inhibitors, the concept of "separating" recognition (σ inactivated) and functional (σ activated) states appears essential. To construct molecules able to switch between recognition and functional state, we assemble different pseudoproline (ψ Pro)-containing building blocks as induction units σ for β-sheet breaking and a target peptide P. The impact of the ψ Pro-moiety is decoupled from the peptide chain via a flexible thioester bond (Soff) and set off after acyl migration (Son). As a consequence, the insertion and association into existing β-sheet templates should be promoted. Preliminary results by EM studies indicate that a controlled enzyme-induced acyl migration results in a significant improvement of the β-sheet breaking potential. For example, one of the designed switch-peptides slowed down the fibrillogenesis of an amyloid model, representing a significant step in the developement of therapeutically relevant compounds in Alzheimer's disease. The third part focuses on the in situ creation of structure and function of potentially bioactive peptide ligands (Figure, strategy A) via a novel ligation method ("Switch-ligation"). For the example of an angiotensin II-derived analogue, we explore the potential of chemoselective ligations for the onset of biological function under physiologically relevant conditions. Receptor (AT1) binding studies of [Cys5]Ang II in collaboration with PD. Dr. E. Grouzmann, CHUV reveal a fast and efficient ligation without the need of additives (IC50= 25 nM). As an alternative example, the covalent self-assembling of two precursors results in the in situ creation of an efficient peptide deformylase inhibitor. The perspective to extend these methodologies for in vivo applications has been tentatively explored as a new concept in prodrug design. In conclusion, the results of this thesis contribute substantially to the proof of concept of switch-peptides, opening new perspectives in drug design as well as in the study of peptide and protein misfolding relevant in degenerative diseases.

EPFL2006

Switch peptides

Incorrect folding of proteins, leading to aggregation and amyloid formation, is associated with a group of degenerative diseases including Alzheimer's disease and late onset diabetes. Amyloid forming proteins are believed to be mainly α-helical in their native conformation, but undergo an α-helical to β-sheet conversion paralleled by fibril and plaque formation. As a most challenging experimental hurdle, the transition from helical / rc conformations to β-sheeted structures is followed by dramatic changes in the physicochemical properties, such as self association and insolubility, thus strongly limiting the experimental access of this molecular key process in neurodegenerative diseases. In 2000, M. Mutter and his group proposed a new concept for resolving some of these intrinsic problems of peptide self-assembly. As a basic idea, peptides with a high potential for β-sheet formation, considered as early event in fibrillogenesis, are prepared as 'folding precursors', in which the folding process is blocked by the insertion of a non native bond (see Figure). These modular switch peptides comprise three distinct elements: A conformational induction unit (σ), a switch element (S), and a target peptide (P). A central feature of the concept of switch-peptides, 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 removing the protecting group Y reestablishes the native polyamide backbone via a spontaneous O to N acyl migration reaction, whereupon peptide folding can proceed. The present Ph.D. thesis aims to elaborate this concept in putting conformational transitions from helical or random coil to β-sheet structures in focus. This idea is developed using first potentially β-sheet forming model peptides, and is extended in a second step to amyloid-derived sequences. To this end, a series of model peptides exhibiting high β-sheet potential have been synthesized by SPPS synthesis and the critical chain length for β-sheet formation of oligopeptide (Leu-Ser)n was determined by CD studies. The insertion of a pH inducible S-element allowed for the delineation of the in situ transition from rc (Soff) to β-sheet structure (Son). The kinetic evaluation of these conformational transitions as a function of concentration, pH and temperature served as a base for the host-guest peptides according to the switch concept. Host-guest techniques allow to mimic the structural environment of the peptide segments 'excised' from their polypeptide. Typically, peptide segments (guest sequence) regarded as nucleation centers ('hot spots') for β-sheet and fibril formation are incorporated into a host oligopeptide of high β-sheet potential, e.g. (Leu-Ser)n. The incorporation of S-elements at the C- and N-terminal end of the guest segment results in a rc structure (Soff-state), as monitored by CD and TEM studies. By adjusting the pH to physiological conditions, O,N-acyl migration is triggered (Son) and a well defined rc to β-sheet transition is observed by CD, paralleled by the formation of amyloid-like fibrils. For example, after insertion into the β-sheet promoting host peptide (Leu-Ser)n, the amyloid β (14-24) and amylin derived peptide NFGAIL undergo upon pH induced acyl migration a conformational transition from rc to β-sheet , paralleled by the onset of fibril formation. For evaluating the impact of β-breaking compounds upon the kinetics of β-sheet formation, the potential of the developed host-guest switch peptides to serve as a diagnostic tool is elaborated. As the folding is set off in the moment of creating the bioactive molecule ("in statu nascendi", ISN), the present concept allows for the first time to investigate early steps of protein misfolding as relevant in neurodegenerative diseases, opening new perspectives for the rational design of compounds of therapeutic interest. This is demonstrated for the design and synthesis of a reference host-guest system ('molecular kit for screening amyloid β inhibitors'), in which the kinetics of rc to β-sheet transitions is determined under a variety of experimental conditions, including physiological pH. Finally, as a most challenging goal, the disruption or even reversion of β-sheet formation and aggregation is addressed by applying the concept of switch peptides. To accomplish this formidable task, a series of switch-peptides containing various induction systems are investigated. Preliminary results by CD and TEM studies confirm the potential of host-guest switch-peptides for obtaining quantitative data on the impact of β-breaking compounds. A critical evaluation of known β-breaking peptides point to the need for a reliable screening system as developed in the present thesis. The results obtained firmly establish the capability of employing the concept of switch peptides as a tool to study folding/misfolding events during the dynamic process of structure onset and evolution. Keywords: Switch peptides ; host-guest switch peptides ; screening ; inhibitors ; Aβ ; amylin.

EPFL2006

Self-Assembly of ß-Amyloid Peptides in Cells and on Solid Supports

Enrico Condemi

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease share common mechanisms characterized by protein misfolding and aggregation, including formation of plaques or inclusion bodies. The rapid growing number of patients has motivated intensive research to understand biological phenomena involved in the pathology and hence developing therapeutic strategies. Here, we will focus on β-amyloid peptide (Aβ) involved in Alzheimer's disease. In the first part of this thesis, tissue transglutaminase (tTGase) has been investigated due to its implication in a number of cellular processes and disease states, where the enzymatic actions of tTGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of tTGase in cell survival or cell death mechanisms still remain elusive. tTGase mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that, tTGase induces intramolecular cross-linking of Aβ peptide, resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that, a large population of Aβ monomers contained an increase in mass of 0.984 Da at a glutamine residue, indicating that glutamine Q15 serves as an indispensable substrate in TGase mediated deamidation to glutamate E15. We provide strong analytical evidence on tTGase mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that, tTGase induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation. In the second part of this work, we explore the idea of using synthetic peptides that can switch conformation in a controlled way as a sensor for β-breakers of Aβ. The concept of switch-peptides allows the controlled onset of polypeptides folding and misfolding at physiological pH by in situ O,N-acyl migration. The synthesized sequence consisted in the core sequence responsible for Aβ aggregation, flanked by two β-sheet-inducing template through ester bonds. The triggering of the acyl migration allows the native sequence to be recovered from the ester bonds and structural change from flexible unordered conformation to β-sheets and subsequently fibrils formation were observed in solution. In a more biological environment made of lipid vesicles, the conformation of the peptide has been shown to act comparably to wild type Aβ peptide by circular dichroism (CD), namely increased negative charges over the surface led to increased helical propensity followed by a transition to β-sheets and accelerated fibrils formation. Thereafter, the conformation of the cysteine analogue of the switch-peptide covalently linked to gold surface was characterized by Fourier-transform infrared spectroscopy (FTIR). In monolayer of switch-peptide tethered to a planar gold surface, the peptide already adopts most likely β-turn conformation and no changes in the infrared spectra were detected after the triggering of the switch. On gold nanoparticles, the switch-peptide folds spontaneously into β-sheet conformation. These unwanted foldings limit the efficiency of the sensor and has driven us to an alternative configuration, where gold nanoparticles were coated with a 95:5 mixture of two peptide sequences able to stabilize the nanoparticles and to act as recognition sequence, respectively. In this configuration, the switch-peptide is added in the gold nanoparticles solution and the aggregation triggered. Here, we want to take advantage of a particular feature of gold nanoparticles that consists in a change of solution color from red to blue as the nanoparticles come closer to each other during aggregation. Preliminary results have shown that a discrimination between aggregated and soluble samples is possible. However, further improvements are necessary before a potential biosensor could be expected.

EPFL2011

Le concept switch-peptides

Karine Murat

EPFL2006

Switch peptides

EPFL2006

Self-Assembly of ß-Amyloid Peptides in Cells and on Solid Supports

Enrico Condemi

EPFL2011