Phage selection of chemically stabilized alpha-helical peptide ligands

Short alpha-helical peptides stabilized by linkages between constituent amino acids offer an attractive format for ligand development. In recent years, a range of excellent ligands based on stabilized alpha-helices were generated by rational design using alpha-helical peptides of natural proteins as templates. Herein, we developed a method to engineer chemically stabilized alpha-helical ligands in a combinatorial fashion. In brief, peptides containing cysteines in position i and i + 4 are genetically encoded by phage display, the cysteines are modified with chemical bridges to impose alpha-helical conformations, and binders are isolated by affinity selection. We applied the strategy to affinity mature an alpha-helical peptide binding beta-catenin. We succeeded in developing ligands with K-d's as low as 5.2 nM, having >200-fold improved affinity. The strategy is generally applicable for affinity maturation of any alpha-helical peptide. Compared to hydrocarbon stapled peptides, the herein evolved thioether-bridged peptide ligands can be synthesized more easily, unnatural amino acids are required and the cyclization reaction is more efficient and yields no stereoisomers. A further advantage of the thioether-bridged peptide ligands is that they can be expressed recombinantly as fusion proteins.

New methods for developing (bi)cyclic peptides by phage display

Camille Villequey

Cyclic peptide ligands are a promising molecular format for the development of therapeutics. They combine some of the advantages of large protein therapeutics (high affinity and specificity) and of small molecule drugs (accessibility to chemical synthesis and low immunogenicity). With phage display, large combinatorial libraries of more than one billion mono- and polycyclic peptides can be developed and screened against virtually any target. During phage affinity selections, the physical link between phenotype (encoded peptide) and genotype (phage DNA) allows the identification of enriched peptide binders by sequencing of the phage vector DNA. Based on these concepts, our laboratory has established a robust chemistry in which phage displayed peptides containing two or three cysteines are cyclized or bi-cyclized with thiol-reactive linkers. This procedure has been generally applied for the isolation of potent and selective (bi)cyclic peptide ligands with therapeutic potential. The isolation of binders with optimal properties is highly dependent on the possibility to generate (bi)cyclic peptide libraries with high structural diversity and on the ability to thoroughly decode the phage selection output. In my PhD work, I developed new methods to generate large and structurally diverse libraries and to decode more efficiently phage selected peptides. In my first project, I explore the cyclization of peptides with two chemical bridges as a method to provide rapid access to phage-encoded libraries with high scaffold diversity. To this end, a range of twelve structurally diverse chemicals were tested for thiol-reactivity and phage compatibility. Subsequently, peptide libraries of the format XCXnCXnCX (X = random amino acid, C = cysteine, n = 3 or 4) were cyclized with six of these chemical linkers and panned against the protease plasma kallikrein. The selection yielded inhibitors with remarkable affinity (sub-nM KI), target selectivity (>1000-fold) and proteolytic stability (t1/2 in plasma > 3 days) despite a relatively small molecular mass (~ 1200 Da). In the second project, I developed phage-encoded libraries of small cyclic peptides in order to identify ligands that have the potential to be orally absorbed. A major challenge for the generation of such library is the usually low diversity of compounds due to the limited number of amino acids. In this work, we addressed this limitation by taking advantage of a recently discovered peptide cyclization reaction in which the thiol of a cysteine is ligated with the N-terminal amino group using a chemical reagent. We applied this strategies to two phage-encoded peptide libraries of four and five amino acids (XXXC and XXXXC) that were cyclized with eight chemical linkers yielding a collection of more than 1.3 million compounds. Panning the libraries against the two disease targets plasma kallikrein and coagulation factor XIa yielded ligands with single-digit micromolar affinity. In a third project, I developed a strategy to bypass the bacterial infection step in phage display. Bacterial infection is usually crucial for the propagation and identification of enriched clones by DNA sequencing. However, mutations or chemical modifications of the phage coat proteins, sometimes lead to decreased infection rates and therefore compromise the entire procedure. Consequently, there is an interest for developing methods in which this step could be completely omitted.

EPFL2017

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