Peptide stapling, by employing a stable, preformed alpha-helical conformation, results in the production of peptides with improved membrane permeability and enhanced proteolytic stability, compared to the original peptides, and provides an effective solution to accelerate the rapid development of peptide drugs. Various reviews present peptide stapling chemistries, anchoring residues and one- or two-component cyclization, however, therapeutic stapled peptides have not been systematically summarized, especially focusing on various disease-related targets. This review highlights the latest advances in therapeutic peptide drug development facilitated by the application of stapling technology, including different stapling techniques, synthetic accessibility, applicability to biological targets, potential for solving biological problems, as well as the current status of development. Stapled peptides as therapeutic drug candidates have been classified and analysed mainly by receptor- and ligand-based stapled peptide design against various diseases, including cancer, infectious diseases, inflammation, and diabetes. This review is expected to provide a comprehensive reference for the rational design of stapled peptides for different diseases and targets to facilitate the development of therapeutic peptides with enhanced pharmacokinetic and biological properties.

Two major pharmacological hurdles severely limit the widespread use of small peptides as therapeutics: poor proteolytic stability and membrane permeability. Importantly, low aqueous solubility also impedes the development of peptides for clinical use. Various elaborate side chain stapling chemistries have been developed for α-helical peptides to circumvent this problem, with considerable success in spite of inevitable limitations. Here we report a novel peptide stapling strategy based on the dithiocarbamate chemistry linking the side chains of residues Lys(i) and Cys(i + 4) of unprotected peptides and apply it to a series of dodecameric peptide antagonists of the p53-inhibitory oncogenic proteins MDM2 and MDMX. Crystallographic studies of peptide–MDM2/MDMX complexes structurally validated the chemoselectivity of the dithiocarbamate staple bridging Lys and Cys at (i, i + 4) positions. One dithiocarbamate-stapled PMI derivative, ^DTCPMI, showed a 50-fold stronger binding to MDM2 and MDMX than its linear counterpart. Importantly, in contrast to PMI and its linear derivatives, the ^DTCPMI peptide actively traversed the cell membrane and killed HCT116 tumor cells in vitro by activating the tumor suppressor protein p53. Compared with other known stapling techniques, our solution-based DTC stapling chemistry is simple, cost-effective, regio-specific and environmentally friendly, promising an important new tool for the development of peptide therapeutics with improved pharmacological properties including aqueous solubility, proteolytic stability and membrane permeability.

Peptide drugs offer distinct advantages in therapeutics; however, their limited stability and membrane penetration abilities hinder their widespread application. One strategy to overcome these challenges is the hydrocarbon peptide stapling technique, which addresses issues such as poor conformational stability, weak proteolytic resistance, and limited membrane permeability. Nonetheless, while peptide stapling has successfully stabilized α-helical peptides, it has shown limited applicability for most β-sheet peptide motifs. In this study, we present the design of a novel double-stapled peptide capable of simultaneously stabilizing both α-helix and β-sheet structures. Our designed double-stapled peptide, named DSARTC, specifically targets the androgen receptor (AR) DNA binding domain and MDM2 as E3 ligase. Serving as a peptide-based PROTAC (proteolysis-targeting chimera), DSARTC exhibits the ability to degrade both the full-length AR and AR-V7. Molecular dynamics simulations and circular dichroism analysis validate the successful constraint of both secondary structures, demonstrating that DSARTC is a "first-in-class" heterogeneous-conformational double-stapled peptide drug candidate. Compared to its linear counterpart, DSARTC displays enhanced stability and an improved cell penetration ability. In an enzalutamide-resistant prostate cancer animal model, DSARTC effectively inhibits tumor growth and reduces the levels of both AR and AR-V7 proteins. These results highlight the potential of DSARTC as a more potent and specific peptide PROTAC for AR-V7. Furthermore, our findings provide a promising strategy for expanding the design of staple peptide-based PROTAC drugs, targeting a wide range of "undruggable" transcription factors.

Rationale: Antimicrobial peptide LL-37 has been recognized as a favorable alternative to antibiotics due to its broad antibacterial spectrum, low resistance development and diverse biological activities. However, its high manufactory cost, poor proteolytic stability, and unpredictable cytotoxicity seriously hindered its medical translation.