Stapled Peptides Research Articles

A Method to Calculate the Relative Binding Free Energy Differences of α-Helical Stapled Peptides.

Hydrocarbon-stapled peptides are a class of bioactive α-helical ligands developed to target protein-protein interactions. Peptide stapling has benefited from the development of several chemical reactions to modulate their membrane permeability and binding affinity. However, in most current programs, choosing the best stapling positions is usually a trial-and-error process. Here, we develop a protocol to obtain optimal stapling positions computationally. Our method is based on molecular dynamics simulations and free energy calculations with nonequilibrium approaches; here, we predict the binding poses, hot-spot residues, and binding affinity differences of a set of perfluoroarene stapled α-helical peptides of the BIM BH3 peptide to the BCLXL receptor. The prediction of the hot-spot residues within the target peptide through computational alanine scanning anticipates not only the key residues for the receptor-peptide complex formation but also which positions should be avoided when applying the stapling groups. The staple moieties introduce local conformational changes not only in the replaced positions but also on their neighbor residues of the template peptide further affecting their binding behavior. Our approach is successful at rank-ordering the binding affinities of these stapled peptides with respect to the BIM BH3 peptide.

Photoisomerization-enhanced 1,3-dipolar cycloaddition of carbon-bridged octocyclic azobenzene with photo-released nitrile imine for peptide stapling and imaging in live cells.

A photo-induced 1,3-dipolar cycloaddition between nitrile imine and highly ring-strained N[double bond, length as m-dash]N double bond as a dipolarophile was discovered. The photo-isomerization of carbon-bridged octocyclic azobenzene (CBOA) into its trans-configuration accelerates the ligation reaction at a very rapid rate (28 400 M-1 s-1). The CBOA-based photo-click reaction was proved to be bioorthogonal. In addition, the NoxaB peptide was successfully cross-linked by a CBOA stapler which plays a dual role: photo-control of the conformation of the peptide and photo-conjugation of probes in live cells.

FlICk (fluorescent isoindole crosslinking) for peptide stapling.

The rigidification of peptide secondary structure via stapling is an important and enduring goal in the development of functional peptides for biochemical and pharmaceutical applications. In addition, the incorporation of fluorophores and chromophores has been a sought-after application for creating peptidic probes of cellular function and localization. The combined application of peptide stapling and fluorescent-readout is featured by the reaction of ortho-phthalaldehydes to create isoindole staples, thus transforming inactive linear and monocyclic precursors into fluorescent or visibly colored monocyclic and bicyclic products with noted biological activity. Given its user-friendliness, we have termed this approach FlICk (fluorescent isoindole crosslink) chemistry and we have featured this application on an array of high-affinity macrocyclic α-MSH derivatives as well as for late-stage intra-annular isoindole stapling furnished a bicyclic peptide mimic of α-amanitin that is cytotoxic to CHO cells. The synthetic methods for preparing substituted ortho-phthalaldehydes along with subsequent applications to FlICk stapling are detailed herein.

Cover Feature: Lipidated Stapled Peptides Targeting the Acyl Binding Protein UNC119 (ChemBioChem 24/2019)

Cover Feature: Lipidated Stapled Peptides Targeting the Acyl Binding Protein UNC119 (ChemBioChem 24/2019)

Lipidated Stapled Peptides Targeting the Acyl Binding Protein UNC119.

The acyl‐binding UNC119 proteins mediate the activation and transport of various N‐myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α‐helical peptides that target UNC119a. By using the hydrocarbon peptide‐stapling approach, cell‐permeable binders of UNC119a were generated that induced the accumulation of cytokinetic and binucleated cells; this suggests UNC119a as a potential target for the inhibition of cytokinesis.

Direct Synthesis of N-Functionalized Dipropargylamine Linkers as Models for Use in Peptide Stapling

N-Substituted dipropargylamines that are suitable as functionalized linkers for peptide stapling can be synthesized in one step under mild conditions from commercially available starting materials (41% to quantitative yield).

A Physical Organic Approach to Tuning Reagents for Selective and Stable Methionine Bioconjugation.

We report a data-driven, physical organic approach to the development of new methionine-selective bioconjugation reagents with tunable adduct stabilities. Statistical modeling of structural features described by intrinsic physical organic parameters was applied to the development of a predictive model and to gain insight into features driving the stability of adducts formed from the chemoselective coupling of oxaziridine and methionine thioether partners through Redox Activated Chemical Tagging (ReACT). From these analyses, a correlation between sulfimide stabilities and sulfimide ν (C═O) stretching frequencies was revealed. We exploited the rational gains in adduct stability exposed by this analysis to achieve the design and synthesis of a bis-oxaziridine reagent for peptide stapling. Indeed, we observed that a macrocyclic peptide formed by ReACT stapling at methionine exhibited improved uptake into live cells compared to an unstapled congener, highlighting the potential utility of this unique chemical tool for thioether modification. This work provides a template for the broader use of data-driven approaches to bioconjugation chemistry and other chemical biology applications.

Multicomponent Reaction Toolbox for Peptide Macrocyclization and Stapling

In the past decade, multicomponent reactions have experienced a renaissance as powerful peptide macrocyclization tools enabling the rapid creation of skeletal complexity and diversity with low synthetic cost. This review provides both a historical and modern overview of the development of the peptide multicomponent macrocyclization as a strategy capable to compete with the classic peptide cyclization methods in terms of chemical efficiency and synthetic scope. We prove that the utilization of multicomponent reactions for cyclizing peptides by either their termini or side chains provides a key advantage over those more established methods; that is, the possibility to explore the cyclic peptide chemotype space not only at the amino acid sequence but also at the ring-forming moiety. Owing to its multicomponent nature, this type of peptide cyclization process is well-suited to generate diversity at both the endo- and exo-cyclic fragments formed during the ring-closing step, which stands as a distinctive and useful characteristic for the creation and screening of cyclic peptide libraries. Examples of the novel multicomponent peptide stapling approach and heterocycle ring-forming macrocyclizations are included, along with multicomponent methods incorporating macrocyclization handles and the one-pot syntheses of macromulticyclic peptide cages. Interesting applications of this strategy in the field of drug discovery and chemical biology are provided.

Manganese-Catalyzed Synthesis of Acylic, Hybrid, and Stapled Peptides

Key words manganese catalysis - C–H activation - allylation - stapled peptides - hybrid peptides

Targeted covalent inhibitors of MDM2 using electrophile-bearing stapled peptides.

Herein, we describe the development of a novel staple with an electrophilic warhead to enable the generation of stapled peptide covalent inhibitors of the p53-MDM2 protein-protein interaction (PPI). The peptide developed showed complete and selective covalent binding resulting in potent inhibition of p53-MDM2 PPI.

The role of olefin geometry in the activity of hydrocarbon stapled peptides targeting eukaryotic translation initiation factor 4E (eIF4E).

Hydrocarbon stapled (HCS) peptides are a class of cross-linked α-helix mimetics. The technology relies on the use of α,α'-disubstituted alkenyl amino acids, which fully contrain the helical region to typically yield peptides with enhanced structural ordering and biological activity. Recently, monosubstituted alkenyl amino acids were disclosed for peptide stapling; however, the impact that this tether has on HCS peptide structure and activity has not yet been fully explored. By applying this HCS to the disordered peptide eIF4E-binding protein 1 (4E-BP1), we discovered that this type of tethering has a dramatic effect on olefin geometry and activity of the resultant stapled peptides, where the putative trans isomer was found to exhibit enhanced in vitro and cellular inhibitory activity against eIF4E protein-protein interactions. We further demonstrated that the metathesis catalyst used for ring-closing metathesis can influence monosubstituted HCS peptide activity, presumably through alteration of the cis/trans olefin ratio. This study represents one of the first in-depth analyses of olefin isomers of a stapled peptide and highlights an additional feature for medicinal chemistry optimization of this class of peptide-based probes.

Macrocyclisation and functionalisation of unprotected peptides via divinyltriazine cysteine stapling

We report a novel divinyltriazine linker for the stapling of two cysteine residues to form macrocyclic peptides from their unprotected linear counterparts. The stapling reaction occurred rapidly under mild conditions on a range of unprotected peptide sequences. The resulting constrained peptides displayed greater stability in a serum stability assay when compared to their linear counterparts.

A Multicomponent Stapling Approach to Exocyclic Functionalized Helical Peptides: Adding Lipids, Sugars, PEGs, Labels, and Handles to the Lactam Bridge.

Peptide stapling is traditionally used to lock peptide conformations into α-helical structures using a variety of macrocyclization chemistries. In an endeavor to add a diversity-generating tool to this repertoire, we introduce a multicomponent stapling approach enabling the simultaneous stabilization of helical secondary structures and the exocyclic N-functionalization of the side chain-tethering lactam bridge. This is accomplished by means of a novel solid-phase methodology comprising, for the first time, the on-resin Ugi reaction-based macrocyclization of peptide side chains bearing amino and carboxylic acid groups. The exocyclic diversity elements arise from the isocyanide component used in the Ugi multicomponent stapling protocol, which allows for the incorporation of relevant fragments such as lipids, sugars, polyethylene glycol, fluorescent labels, and reactive handles. We prove the utility of such exocyclic reactive groups in the bioconjugation of a maleimide-armed lactam-bridged peptide to a carrier protein. The on-resin multicomponent stapling proved efficient for the installation of not only one, but also two consecutive lactam bridges having either identical or dissimilar N-functionalities. The easy access to helical peptides with a diverse set of exocyclic functionalities shows prospect for applications in peptide drug discovery and chemical biology.

Strategy and Effects of Polyproline Peptide Stapling by Copper(I)-Catalyzed Alkyne-Azide Cycloaddition Reaction.

Polyproline is a unique type of peptide that has a stable, robust, and well-defined helical structure in an aqueous environment. These features have allowed polyproline to be used as a nanosized scaffold for applications in chemical biology and related fields. To understand its structural properties and to expand the applications, this secondary structure was tested systematically by stapling the peptide at different locations with staples of various lengths. Using the efficient copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), we successfully prepared stapled polyproline and investigated the impact of this peptide macrocyclization through circular dichroism analysis. Whereas the stapling seems to have no significant effect on polyproline helix II (PPII) conformation in water, the location and the length of the staple affect the transformation of conformation in n-propanol. These results provide valuable information for future research using peptide stapling to manipulate polyproline conformation for various applications.

Two‐Component Stapling of Biologically Active and Conformationally Constrained Peptides: Past, Present, and Future

AbstractPeptides are an emerging class of therapeutics in the pharmaceutical world. Whilst small molecules have dominated the therapeutic landscape for decades, the design and application of peptide drugs is emerging among the pharmaceutical industries and academia. Although highly selective and efficacious, peptides are characterized by poor pharmacokinetic properties and amelioration of their bioavailability remains a major hurdle. Incorporation of conformational constraints within the peptide (such as peptide stapling) has been extensively used to improve the bioavailability of these molecules; consequently, it is not surprising that a plethora of stapling techniques has been developed and has had a significant impact on the development of peptide therapeutics. Among the numerous stapling techniques known, two‐component methodologies allow facile and divergent functionalization of peptides. The authors have pioneered a stapling technique that makes use of the double Cu‐catalyzed azide–alkyne cycloaddition between di‐azido peptides and functionalized di‐alkynyl staples. In recent years, the authors have created biologically active, conformationally constrained peptides functionalized with cell‐penetrating peptides, fluorescent tags, and photo cross‐linking moieties, demonstrating the wide applicability of this methodology. Herein, the impact, advantages, limitations, and future applications of this technology and other two‐component peptide stapling techniques on the development of clinically relevant peptides are highlighted.

SuFEx based click chemistry for peptide stapling

Click chemistry is a chemical synthesis strategy which relies on covalent assembly of functional building blocks using high‐yielding reactions that are broad in scope, chemoselective, and compatible with water and oxygen. The sulfur (VI) fluoride exchange (SuFEx) is the latest addition to the click family due to the unique stability of the SO2F moiety, which confers inertness against acid‐base reactions, resistance to oxidation and reduction, and generally high yields of sulfonylated products. Ethenesulfonyl fluoride (ESF) is a powerful Michael acceptor for installing the SO2F group. We are exploring the use of SuFEx chemistry as a powerful tool in peptide stapling, a technique that locks peptide conformation to improve its pharmacological properties. Specifically, we take advantage of a complementary reactivity of two neighboring nucleophilic side chains of a peptide which can undergo a conjugate addition to ESF, followed by fluoride exchange, thus forming a chemical “staple” and locking a specific conformation of the resulting peptide. Such selective modification can inform the development of chemical probes to improve efficacy of a peptide therapeutic, may improve drug delivery, and facilitate the discovery process. Preliminary studies described herein demonstrate feasibility of this approach.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

David R. Spring

Angewandte Chemie International EditionVolume 57, Issue 27 p. 7936-7936 Author ProfileFree Access David R. Spring First published: 26 January 2018 https://doi.org/10.1002/anie.201800487AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract “If I were not a scientist, I would be a florist. My favorite author (fiction) is J. K. Rowling ...” This and more about David R. Spring can be found on page 7936. David R. Spring The author presented on this page has recently published his 10th article in Angewandte Chemie in the last 10 years: “Targeting the Genome-Stability Hub Ctf4 by Stapled-Peptide Design”: Y. Wu, F. Villa, J. Maman, Y. H. Lau, L. Dobnikar, A. C. Simon, K. Labib, D. R. Spring, L. Pellegrini, Angew. Chem. Int. Ed. 2017, 56, 12866; Angew. Chem. 2017, 129, 13046. Position: University Professor, University of Cambridge E-mail: spring@ch.cam.ac.uk Homepage: http://www-spring.ch.cam.ac.uk ORCID: 0000-0001-7355-2824 Education: 1995 BA in chemistry, University of Oxford 1998 DPhil supervised by Jack E. Baldwin, University of Oxford 1999–2001 Postdoctoral fellow with Stuart L. Schreiber, Harvard University Awards: 2010 Discovery Chemistry and New Technologies Award, Royal Society of Chemistry (RSC); 2011 Norman Heatley Award, RSC; 2013 Félix Serratosa Lecture, IQAC–CSIC Barcelona and RSEQ Tarragona, organized by the Societat Catalana de Química and Real Sociedad Española de Química; 2014 Corday–Morgan Award, RSC Current research interests: Diversity-oriented synthesis; protein–protein interactions; quorum sensing; new antibiotics; next-generation therapeutics If I were not a scientist, I would be a florist. My favorite author (fiction) is J. K. Rowling. My top three films of all time are North by Northwest, The Third Man, and Star Wars. The downside of my job is saying goodbye to amazing co-workers after they have graduated or have been offered jobs in industry. In retrospect I would never again eat the pink blancmange with sweetcorn pudding that my postdoc brought in to group meeting. My favorite food is vegemite. If I won the lottery, I would give the money away (after a nice holiday). My favorite place on earth is Cambridge (UK). My greatest achievement has been helping educate so many talented students in chemistry and research. I hope I have passed on my enthusiasm for chemistry and its potential in understanding and treating disease. The most exciting thing about my research is being able to test small molecules in biological assays, which leads to enhanced understanding of biology and improved medicines. Guaranteed to make me laugh is the news and current-affairs magazine Private Eye. The worst advice I have ever been given was to let my research group provide some of the answers to this Author Profile. I celebrate success by drinking sparkling beverages. I would have liked to have discovered penicillin. My 5 top papers: 1“Double Strain-Promoted Macrocyclization for the Rapid Selection of Cell-Active Stapled Peptides”: Y. H. Lau et al., Angew. Chem. Int. Ed. 2015, 54, 15410; Angew. Chem. 2015, 127, 15630. (Cell-permeable, stapled peptides can have a dramatic biological effect by selectively inhibiting a specific protein–protein interaction.) 2“A strategy for the diversity-oriented synthesis of macrocyclic scaffolds using multidimensional coupling”: H. S. G. Beckmann, F. Nie, C. E. Hagerman, H. Johansson, Y. S. Tan, D. Wilcke, D. R. Spring, Nature Chem. 2013, 5, 861. (The idea of multidimensionality in efficient diversity generation is elegantly illustrated.) 3“Anti-MRSA Agent Discovery Using Diversity-Oriented Synthesis”: G. L. Thomas et al., Angew. Chem. Int. Ed. 2008, 47, 2808; Angew. Chem. 2008, 47, 2808; Angew. Chem. 2008, 120, 2850. (An early illustration of the potential of diversity-oriented synthesis by some of my first group members.) 4“Diversity-Oriented Synthesis Of Biaryl-Containing Medium Rings Using A One Bead/One Stock Solution Platform”: D. R. Spring, S. Krishnan, H. E. Blackwell, S. L. Schreiber, J. Am. Chem. Soc. 2002, 124, 1354. (The main part of my postdoc work with Stuart Schreiber.) 5“Investigations into the Manzamine Alkaloid Biosynthetic Hypothesis”: J. E. Baldwin et al., Angew. Chem. Int. Ed. 1998, 37, 2661; Angew. Chem. 1998, 110, 2806. (This was the main part of my DPhil work with Jack Baldwin.) Volume57, Issue27July 2, 2018Pages 7936-7936 ReferencesRelatedInformation

Different stapling-based peptide drug design: Mimicking α-helix as inhibitors of protein–protein interaction

Peptide stapling strategy has been proven a promising solution in addressing two major pharmacological hurdles, proteolytic stability and membrane permeability, for small peptides as therapeutics. This stapling peptides feature a covalent cross-link of side chains, thus effectively mimicking α-helix as inhibitors of protein–protein interactions. In this review, we category and analyze key examples of various peptide stapling strategies based on different cross-links aligned on the side chain of peptides mainly in the last three years.

Stapling strategy enables improvement of antitumor activity and proteolytic stability of host-defense peptide hymenochirin-1B.

Hymenochirin-1B is a cationic, amphipathic, α-helical host-defense peptide with 29 residues, which was isolated from skin secretions of the Congo clawed frog and showed potent cytotoxic activities against a range of tumor cell lines. However, the application of hymenochirin-1B as a drug is limited due to its conformational flexibility and poor proteolytic stability. In this research, a series of hydrocarbon-stapled analogs of hymenochirin-1B were designed, synthesized, and tested. Some analogs showed remarkable improvement not only in α-helicity, but also in antitumor activity and protease resistance when compared to the parent peptide. The results indicated that most stapled peptide analogues possessed improved activities against a series of tumor cells; in particular, the bicyclic stapled peptide H-10 showed promising prospects for novel anti-tumor drug development. Our data demonstrated the important impacts of the all-hydrocarbon crosslink stapling strategy on the biological activity, proteolytic stability and helicity of hymenochirin-1B.

A study of 2-component i, i + 3 peptide stapling using thioethers

Peptides are promising scaffolds for use as therapeutics, targeting interactions previously considered to be “undruggable” by small molecules. While short peptides are generally unstructured in solution and rapidly degraded by proteases in the cell cytosol, peptide stapling offers an effective method to both stabilize peptides in a helical structure and increase resistance to proteolytic degradation. Most studies of peptide stapling have focused on residues with i, i + 4 and i, i + 7 spacing, while stapling of residues with i, i + 3 spacing has been understudied. Herein, we evaluated a suite of bifunctional linkers for stapling between residues with i, i + 3 spacing, comparing the ability of each compound to react with the peptide and the degree of helicity conferred. Finally, we evaluated the ability of the stapling to increase proteolytic resistance in cell lysates, comparing stapling of i, i + 3 and i, i + 4 spacing, with i, i + 3 spacing resulting in a greater increase in peptide half-life in the model system. This presents an effective stapling strategy, adding to the peptide stapling toolbox.