Bicyclic stapled peptides based on p53 as dual inhibitors for the interactions of p53 with MDM2 and MDMX

In recent years, the strategy of inhibiting the interactions of p53 with murine double minute 2(MDM2)and murine double minute X(MDMX) has been proved to be a promising approach for tumor therapy.However, the poor proteolytical stability and low intracellular delivery efficiency of peptide inhibitors limit their clinical application. Here, we designed and synthesized the bicyclic stapled peptides based on p53 by combining all-hydrocarbon stapling and lactam stapling strategies. We demonstrated th...     

A Thiol–Ene Coupling Approach to Native Peptide Stapling and Macrocyclization

We report the discovery of a peptide stapling and macrocyclization method using thiol–ene reactions between two cysteine residues and an α,ω‐diene in high yields. This new approach enabled us to selectively modify cysteine residues in native, unprotected peptides with a variety of stapling modifications for helix stabilization or general macrocyclization. We synthesized stapled Axin mimetic analogues and demonstrated increased alpha helicity upon peptide stapling. We then synthesized stapled p53 mimetic analogues using pure hydrocarbon linkers and demonstrated their abilities to block the p53‐MDM2 interaction and selectively kill p53 wild‐type colorectal carcinoma HCT‐116 cells but not p53 null cells. In summary, we demonstrated a robust and versatile peptide stapling method that could be potentially applied to both synthetic and expressed peptides.     

Peptide stapling with the retention of double native side-chains

All-hydrocarbon stapling strategy has been widely applied for enhancing the proteolytic stability of peptides. However, two major technical hurdles to some extent limit the development of stapled peptides for therapeutic usage: rational selection of the stapling sites and the corresponding deletion of the native side chains. Previously we described the development of the olefin-terminated amino acids with the retention of native side chains and successfully applied them in the synthesis of hydrocarbon stapled peptides with single side-chain retention. Here, we explored the feasibility and effectiveness of hydrocarbon stapling strategy characterized as double side-chains retention. Modeled after a lengthy human immunodeficiency virus-1 (HIV-1) fusion inhibitor SC34EK, Leuⁱ, Serⁱ⁺⁴ and Lysⁱ, Leuⁱ⁺⁴ stapled peptides with the retention of double side-chains were effectively obtained. Our complementary study provided a convenient alternative to address where to install the staple in sequence for conventional all-hydrocarbon peptide stapling. Furthermore, this method not only conferred conformational reinforcement for SC34EK with high α-helicity and protease resistance, but also preserved the structural characteristic (key peripheral residues, charge and solubility) of the linear peptide to the maximum, which are crucial for anti-HIV-1 activity.     

Variable-Length Ester-Based Staples for α-Helical Peptides by Using A Double Thiol-ene Reaction

A novel peptide stapling method effected by a double thiol-ene reaction between two cysteine residues and a divinyl diester to access stapled peptides with enhanced cell permeability is reported. This diverse chemical tool kit provides facile access to stapled peptides with varying bridge lengths. Stapled Axin mimetics were synthesised by using this stapling method resulting in improved α-helicity relative to the unstapled peptide. Cell penetrating stapled analogues of the SIGK peptide that targets the protein–protein interaction hotspot of Gβγ proteins were also synthesised that exhibited a moderate increase in α-helicity and were cell permeable. This chemoselective peptide stapling method is highly amenable as a facile method to easily modify synthetic α-helical peptides to target intracellular proteins.     

Tryptophan in Multicomponent Petasis Reactions for Peptide Stapling and Late-Stage Functionalisation

Peptide stapling is a robust strategy for generating enzymatically stable, macrocyclic peptides. The incorporation of biologically relevant tags (such as cell-penetrating motifs or fluorescent dyes) into peptides, while preserving their binding interactions and enhancing their stability, is highly sought after. Despite the unique opportunities offered by tryptophan‘s indole scaffold for targeted functionalisation, its utilisation in peptide stapling has been limited as compared to other amino acids. Herein, we present an approach for peptide stapling using the tryptophan-mediated Petasis reaction. This method enables the synthesis of both stapled and labelled peptides and is applicable to both solution and solid-phase synthesis. Importantly, the use of the Petasis reaction in combination with tryptophan facilitates the formation of stapled peptides in a straightforward, multicomponent fashion, while circumventing the formation of undesired by-products. Furthermore, this approach allows for efficient and diverse late-stage peptide modifications, thereby enabling rapid production of numerous conjugates for biological and medicinal applications.     

A biocompatible stapling reaction for in situ generation of constrained peptides

Constrained peptides are promising next-generation therapeutics. Peptide stapling is a particularly attractive technique to generate constrained macrocycles with improved biological activity and metabolic stability. We introduce a biocompatible two-component stapling approach based on the reagent 2,6-dicyanopyridine and a pseudo-cysteine amino acid. Stapling can proceed either directly on-resin during solid-phase synthesis or following isolation of the linear peptide. The stapling reaction is orthogonal to natural amino acid side chains and completes in aqueous solution at physiological pH, enabling its direct use in biochemical assays. We performed a small screening campaign of short peptides targeting the Zika virus protease NS2B-NS3, allowing the direct comparison of linear with in situ stapled peptides. A stapled screening hit showed over 28-fold stronger inhibition than its linear analogue, demonstrating the successful identification of constrained peptide inhibitors.

A synthetically straightforward and biocompatible peptide-stapling strategy that can be used directly in biochemical assays to identify constrained enzyme inhibitors.     

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

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.     

Double Strain‐Promoted Macrocyclization for the Rapid Selection of Cell‐Active Stapled Peptides

Peptide stapling is a method for designing macrocyclic alpha‐helical inhibitors of protein–protein interactions. However, obtaining a cell‐active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain‐promoted azide–alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell‐active stapled peptides. As a proof of concept, MDM2‐binding peptides were stapled in parallel, directly in cell culture medium in 96‐well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α‐Helicity was confirmed by a crystal structure of the MDM2‐peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high‐throughput biological applications.     

Stapled Peptides with γ‐Methylated Hydrocarbon Chains for the Estrogen Receptor/Coactivator Interaction

“Stapled” peptides are typically designed to replace two non‐interacting residues with a constraining, olefinic staple. To mimic interacting leucine and isoleucine residues, we have created new amino acids that incorporate a methyl group in the γ‐position of the stapling amino acid S5. We have incorporated them into a sequence derived from steroid receptor coactivator 2, which interacts with estrogen receptor α. The best peptide (IC₅₀=89 nm ) replaces isoleucine 689 with an S‐γ‐methyl stapled amino acid, and has significantly higher affinity than unsubstituted peptides (390 and 760 nm ). Through X‐ray crystallography and molecular dynamics studies, we show that the conformation taken up by the S‐γ‐methyl peptide minimizes the syn‐pentane interactions between the α‐ and γ‐methyl groups.     

A Peptide Stapling Strategy with Built-In Fluorescence by Direct Late-Stage C(sp2)−H Olefination of Tryptophan

Fluorescent stapled peptides are important chemical tools for detecting intracellular distribution, protein–protein interactions, and localization of target proteins. These peptides are usually labeled with bulky sized fluorophores through reactive functional groups, which may alter the physical properties and biological activities of peptides. Herein, a unique strategy is developed for synthesizing new stapled peptides with built-in fluorescence. The stapled peptides were prepared through Rh-catalyzed C(sp²)−H olefination in tryptophan (Trp) residues by using pyridine/pyrimidine as the directing groups under mild conditions. This method displays good regioselectivity and high efficiency. Furthermore, as a proof of concept for its biological applications, stapled peptides without additional fluorophore 9 a and 9 b were constructed for a cell imaging study. These peptides displayed strong binding affinity toward integrin αvβ3 in A549 cells by cell imaging experiments. Notably they demonstrated even better anticancer activity than commercial antagonist cyclic (RGDfK). The method will provide robust tools for the peptide macrocyclization field.     

Disulfide Click Reaction for Stapling of S-terminal Peptides

A disulfide click strategy is disclosed for stapling to enhance the metabolic stability and cellular permeability of therapeutic peptides. A 17-membered library of stapling reagents with adjustable lengths and angles was established for rapid double/triple click reactions, bridging S-terminal peptides from 3 to 18 amino acid residues to provide 18- to 48-membered macrocyclic peptides under biocompatible conditions. The constrained peptides exhibited enhanced anti-HCT-116 activity with a locked α-helical conformation (IC₅₀=6.81 μM vs. biological incompetence for acyclic linear peptides), which could be unstapled for rehabilitation of the native peptides under the assistance of tris(2-carboxyethyl)phosphine (TCEP). This protocol assembles linear peptides into cyclic peptides controllably to retain the diverse three-dimensional conformations, enabling their cellular uptake followed by release of the disulfides for peptide delivery.     

Intramolecularly lactam stapled oxyntomodulin analogues inhibit cancer cell proliferation in vitro

As a glucagon (GCG) receptor (GCGR) and glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) dual agonist, oxyntomodulin (OXM) has been attracting scientific attentions due to its efficacies of suppressing appetite, increasing energy expenditure, and inducing body weight loss in obese humans. Based on the scaffold of native OXM, specific helix-favoring amino acids substitutions and the consequent salt bridge formations were believed to offer enhanced and balanced GCGR/GLP-1R activations through increasing α-helical conformation. Novel OXM analogues are obtained by intramolecular lactam stapling of positions [Glu16 & Lys20] or [Lys17 & Glu21] to further strengthen conformationally constrained stabilization. Even though the lactam staple does not provide additional dual GCGR/GLP-1R activations in vitro, the stapled OXM analogues are firstly reported to have higher or lower anti-PANC-1 cell proliferation activity, meanwhile which has no obvious inhibitory effect on the proliferation of HeLa cells. Therefore, it is speculated that the stapled analogues may have the potential to inhibit the proliferation of specific cancer cell types. Among the stapled peptides as well as their precursors, analogue 6 has the most prominent anti-PANC-1 proliferation activity with the IC₅₀ value of 115.1 μmol/L. Its mechanism of actions including effective signal pathways should be worth further investigations in future.     

Stapling of a 3(10)-helix with click chemistry

Short peptides are important as lead compounds and molecular probes in drug discovery and chemical biology, but their well-known drawbacks, such as high conformational flexibility, protease lability, poor bioavailability and short half-lives in vivo, have prevented their potential from being fully realized. Side chain-to-side chain cyclization, e.g., by ring-closing olefin metathesis, known as stapling, is one approach to increase the biological activity of short peptides that has shown promise when applied to 3(10)- and α-helical peptides. However, atomic resolution structural information on the effect of side chain-to-side chain cyclization in 3(10)-helical peptides is scarce, and reported data suggest that there is significant potential for improvement of existing methodologies. Here, we report a novel stapling methodology for 3(10)-helical peptides using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction in a model aminoisobutyric acid (Aib) rich peptide and examine the structural effect of side chain-to-side chain cyclization by NMR, X-ray diffraction, linear IR and femtosecond 2D IR spectroscopy. Our data show that the resulting cyclic peptide represents a more ideal 3(10)-helix than its acyclic precursor and other stapled 3(10)-helical peptides reported to date. Side chain-to-side chain stapling by CuAAC should prove useful when applied to 3(10)-helical peptides and protein segments of interest in biomedicine.     

Targeting the Genome‐Stability Hub Ctf4 by Stapled‐Peptide Design

The exploitation of synthetic lethality by small‐molecule targeting of pathways that maintain genomic stability is an attractive chemotherapeutic approach. The Ctf4/AND‐1 protein hub, which links DNA replication, repair, and chromosome segregation, represents a novel target for the synthetic lethality approach. Herein, we report the design, optimization, and validation of double‐click stapled peptides encoding the Ctf4‐interacting peptide (CIP) of the replicative helicase subunit Sld5. By screening stapling positions in the Sld5 CIP, we identified an unorthodox i,i+6 stapled peptide with improved, submicromolar binding to Ctf4. The mode of interaction with Ctf4 was confirmed by a crystal structure of the stapled Sld5 peptide bound to Ctf4. The stapled Sld5 peptide was able to displace the Ctf4 partner DNA polymerase α from the replisome in yeast extracts. Our study provides proof‐of‐principle evidence for the development of small‐molecule inhibitors of the human CTF4 orthologue AND‐1.     

Design of peptide–dendrimer conjugates with tumor homing and antitumor effects

Development of drug delivery systems for cancer therapy is a crucial issue. Previously, some peptides were designed as tumor homing cell-penetrating peptides with antitumor activities. In this study, dual function dendrimers with tumor targeting activities and antitumor effects were designed using the tumor targeting CPP44 peptide for acute myelogenous leukemia (AML) and the antitumor p16^INK4a peptide. Two types of peptide–dendrimer conjugates were synthesized. One was a CPP44-linked p16^INK4a peptide-conjugated dendrimer (tandem linked dendrimer) and the other was a dendrimer conjugated with separate CPP44 and p16^INK4a peptides (parallel linked dendrimer). In addition, a peptide cathepsin B substrate was linked to the antitumor p16^INK4a peptide to release it from the carriers. These peptide–dendrimer conjugates produced more effective antitumor effects than a CPP44-linked p16^INK4a peptide. The parallel linked dendrimer showed less association with AML cells than the tandem linked dendrimer, but had greater antitumor effects. This suggested that both cellular uptake and antitumor peptide cleavage affected the antitumor activities of dual functional peptide-conjugated dendrimers.     

A Cysteine-Directed Proximity-Driven Crosslinking Method for Native Peptide Bicyclization

Efficient and site-specific modification of native peptides and proteins is desirable for synthesizing antibody-drug conjugates as well as for constructing chemically modified peptide libraries using genetically encoded platforms such as phage display. In particular, there is much interest in efficient multicyclization of native peptides due to the appeals of multicyclic peptides as therapeutics. However, conventional approaches for multicyclic peptide synthesis require orthogonal protecting groups or non-proteinogenic clickable handles. Herein, we report a cysteine-directed proximity-driven strategy for the constructing bicyclic peptides from simple natural peptide precursors. This linear to bicycle transformation initiates with rapid cysteine labeling, which then triggers proximity-driven amine-selective cyclization. This bicyclization proceeds rapidly under physiologic conditions, yielding bicyclic peptides with a Cys-Lys-Cys, Lys-Cys-Lys or N-terminus-Cys-Cys stapling pattern. We demonstrate the utility and power of this strategy by constructing bicyclic peptides fused to proteins as well as to the M13 phage, paving the way to phage display of novel bicyclic peptide libraries.     

Stapled Peptides by Late‐Stage C(sp3)−H Activation

Despite the importance of stapled peptides for drug discovery, only few practical processes to prepare cross‐linked peptides have been described; thus the structural diversity of available staple motifs is currently limited. At the same time, C−H activation has emerged as an efficient approach to functionalize complex molecules. Although there are many reports on the C−H functionalization of amino acids, examples of post‐synthetic peptide C−H modification are rare and comprise almost only C(sp²)−H activation. Herein, we report the development of a palladium‐catalyzed late‐stage C(sp³)−H activation method for peptide stapling, affording an unprecedented hydrocarbon cross‐link. This method was first employed to prepare a library of stapled peptides in solution. The compatibility with various amino acids as well as the influence of the size (i ,i +3 and i ,i +4) and length of the staple were investigated. Finally, a simple solid‐phase procedure was also established.     

Peptide Stapling with Anion‐π Catalysts

We report design, synthesis and evaluation of a series of naphthalenediimides (NDIs) that are bridged with short peptides. Reminiscent of peptide stapling technologies, the macrocycles are conveniently accessible by a chromogenic nucleophilic aromatic substitution of two bromides in the NDI core with two thiols from cysteine sidechains. The dimension of core‐bridged NDIs matches that of one turn of an α helix. NDI‐stapled peptides exist as two, often separable atropisomers. Introduction of tertiary amine bases in amino‐acid sidechains above the π‐acidic NDI surface affords operational anion‐π catalysts. According to an enolate chemistry benchmark reaction, anion‐π catalysis next to peptides occurs with record chemoselectivity but weak enantioselectivity. Catalytic activity drops with increasing distance of the amine base to the NDI surface, looser homocysteine bridges, mismatched, shortened and elongated α‐helix turns, and acyclic peptide controls. Elongation of isolated turns into short α helices significantly increases activity. This increase is consistent with remote control of anion‐π catalysis from the α‐helix macrodipole.     

Rational Design of Self-Assembled Mitochondria-Targeting Lytic Peptide Conjugates with Enhanced Tumor Selectivity

Membrane lytic peptides (MLP) are widely explored as cellular delivery vehicles or antitumor/antibacterial agents. However, the poor selectivity between cancer and normal cells slims their prospects as potential anti-tumor drugs. Herein, we have developed a rationally designed self-assembly strategy to enhance tumor selectivity of MLP-based conjugates, incorporating a hydrophobic triphenylphosphonium (TPP) group for mitochondria targeting, and a hydrophilic arginine-glycine-aspartic acid (RGD) sequence targeting integrins. The self-assembly nanoparticles can enhance the stability of the peptides in vitro plasma and be endocytosed selectively into the cancer cells. The histidine-rich lytic peptide component assists the disruption of endosomal/lysosomal membranes and subsequent the mitochondria membrane, which leads to apoptosis. This rational design of MLP-based conjugates provides a practical strategy to increase the application prospects of lytic peptides in cancer treatment.     

Engineered Histidine-Rich Peptides Enhance Endosomal Escape for Antibody-Targeted Intracellular Delivery of Functional Proteins

Currently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome-to-cytosol delivery based on an extension of the “histidine switch” principle. By substituting the Arg/Lys residues in cationic cell-penetrating peptides (CPPs) with histidine, we obtained peptides with pH-dependent membrane-perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16-residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody-targeted delivery of diverse protein cargoes—including the pro-apoptotic protein BID (BH3-interacting domain death agonist) and Cre recombinase—into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab-hsLMWP-BID fusion conferred strong anti-tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.