De novo development of proteolytically resistant therapeutic peptides for oral administration

The oral administration of peptide drugs is hampered by their metabolic instability and limited intestinal uptake. Here, we describe a method for the generation of small target-specific peptides (less than 1,600 Da in size) that resist gastrointestinal proteases. By using phage display to screen large libraries of genetically encoded double-bridged peptides on protease-resistant fd bacteriophages, we generated a peptide inhibitor of the coagulation Factor XIa with nanomolar affinity that resisted gastrointestinal proteases in all regions of the gastrointestinal tract of mice after oral administration, enabling more than 30% of the peptide to remain intact, and small quantities of it to reach the blood circulation. We also developed a gastrointestinal-protease-resistant peptide antagonist for the interleukin-23 receptor, which has a role in the pathogenesis of Crohn's disease and ulcerative colitis. The de novo generation of targeted peptides that resist proteolytic degradation in the gastrointestinal tract should help the development of effective peptides for oral delivery.

Switch-peptides: controlling biological function and self-assembly of amyloid ß-derived peptides using enzyme-triggered acyl migrations

Sonia Dos Santos

Conformational 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.

EPFL2005

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

Development of cyclic peptide inhibitors of coagulation factor XIa for safer anticoagulation

Vanessa Carle

Hemostasis is a complex physiological process responsible for the prevention of blood loss caused by vascular injury. The dysregulation of this delicate mechanism can lead to thromboembolic diseases, the leading cause of death worldwide. Anticoagulants are drugs applied for the treatment and prevention of this class of diseases. Their major side effect - severe bleeding, which is a life-threatening condition - strongly limits their potential and leads to events of inappropriate treatment. Thus, there is a strong unmet need for a safer class of anticoagulants. Coagulation factor XI (FXI), a serine protease from the intrinsic pathway of coagulation, has been identified as a promising target for safer anticoagulation. Studies in various animal models and epidemiological data have shown that FXI is involved in the pathogenesis of thrombosis, while not being essential for hemostasis. Various FXI-targeting molecules are currently in development, including small molecules, monoclonal antibodies, and antisense oligonucleotides. Peptide-based FXIa inhibitors have not yet been developed, despite the attractive properties of peptides as a drug format, including high affinity and selectivity, tunable pharmacokinetic properties, a fast onset of action and absence of toxic metabolic products. The aim of my thesis was the development of cyclic peptide-based FXIa inhibitors for safer anticoagulation. In a first project, I have developed a large cyclic peptide phage display library with unprecedented structural diversity. In a proof-of-concept selection performed with the library, peptides displaying long consensus sequences were enriched and potent binders were isolated, confirming the importance of having access to large and structurally diverse libraries. Next, I had applied the new phage display library for selecting cyclic peptide inhibitors of FXIa. Isolated peptides were characterized, and a particularly potent inhibitor was further optimized. The resulting peptide showed a strong anticoagulation effect ex vivo, which was comparable to the effect of heparin at therapeutic concentrations. I then modified the peptide, which improved its plasma stability and prolonged the circulation time in vivo. The modified peptide inhibited plasma coagulation after administration to rabbits and in an ex vivo model of hemodialysis using human blood. Finally, I had stumbled over a cyclic peptide FXIa inhibitor that had a moderate inhibitory constant but a particularly strong anticoagulation activity. In a structure-activity relationship analysis of the peptide, I identified the most important residue for the strong anticoagulation activity, which pointed to an electrostatic interaction and to potentially fast binding kinetics. The peptide indeed bound much faster than other FXIa inhibitors, showing that rapid engagement with the FXIa is important for efficient anticoagulation. Based on these findings, I concluded that it is worth to take into consideration the binding on-rate of inhibitors of coagulation proteases in the future development of anticoagulants.

EPFL2019