Stapled Peptides: Locking in Helical Shape
Peptide Engineering
ALRN-6924
Sulanemadlin (ALRN-6924) is the first stapled peptide to reach clinical trials, targeting the p53-MDM2/MDMX interaction in cancer. It demonstrates that stapled peptides can permeate cells and disrupt intracellular protein-protein interactions.
Guerlavais et al., Journal of Medicinal Chemistry, 2023
Guerlavais et al., Journal of Medicinal Chemistry, 2023
View as imageMost peptide drugs fail for the same three reasons: they are degraded by proteases within minutes, they cannot cross cell membranes to reach intracellular targets, and they lose their bioactive shape when removed from the protein surface they were designed to mimic. Stapled peptides address all three problems simultaneously. By installing a covalent hydrocarbon bridge across one face of an alpha-helical peptide, the helix is locked into its bioactive conformation, protease cleavage sites are shielded, and a hydrophobic surface is created that facilitates membrane penetration.
The concept was developed in the early 2000s by Gregory Verdine and colleagues at Harvard and has since generated a clinical-stage drug candidate (sulanemadlin/ALRN-6924), hundreds of research-stage molecules, and a growing body of structure-activity data that informs peptide drug design across oncology, infectious disease, and metabolic disorders. For the broader landscape of engineered peptide variants, see Peptoids: The Peptide Cousins That Resist Protease Degradation.
Key Takeaways
- Stapled peptides use all-hydrocarbon cross-links (typically via olefin metathesis between non-natural amino acids at i, i+4 or i, i+7 positions) to constrain alpha-helical conformation, improving binding affinity, protease resistance, and cell permeability simultaneously (Li et al., Chemical Society Reviews, 2025)
- Sulanemadlin (ALRN-6924) is the first stapled peptide in clinical trials, targeting the p53-MDM2/MDMX protein-protein interaction; phase 1 data showed a 45% disease control rate in p53 wild-type solid tumors (Guerlavais et al., Journal of Medicinal Chemistry, 2023)
- Li et al. (2025) systematically dissected the geometric and hydrophobic constraints of stapled peptides, finding that staple position relative to the binding interface is more critical than overall helicity for biological activity
- Shi et al. (2025) demonstrated that all-hydrocarbon stapling of the antimicrobial peptide cecropin A generated variants with 16-fold improved proteolytic stability while maintaining or enhancing antimicrobial potency
- Yang et al. (2025) developed double biaryl-stapled GLP-1R/GIPR dual agonist peptides, showing that stapling technology can be applied to metabolic disease targets, not just oncology
- Computational approaches are now enabling rational stapled peptide design: Schofield et al. (2026) used computational methods to design, synthesize, and evaluate stapled peptide antagonists without relying on large empirical screening campaigns
The chemistry of peptide stapling
Why peptides lose their shape
In solution, short peptides (fewer than approximately 20 amino acids) rarely maintain stable secondary structures. An alpha helix that exists on the surface of a folded protein unravels when that sequence is excised and dissolved in water. The entropic cost of maintaining helical order in a short, unconstrained peptide is too high. Since many protein-protein interactions are mediated by helical surfaces, a peptide drug designed to mimic one face of a protein-protein interface will often fail because it cannot maintain the helical shape needed for binding.
How stapling works
Peptide stapling introduces a covalent cross-link between two non-natural amino acids positioned on the same face of a designed alpha helix. The most common approach uses ring-closing olefin metathesis (RCM):
- Two non-natural amino acids bearing terminal olefin (alkene) side chains are incorporated at positions i and i+4 (one helical turn apart) or i and i+7 (two helical turns apart)
- A Grubbs-type ruthenium catalyst catalyzes a metathesis reaction between the two olefin groups, forming a covalent carbon-carbon bond
- The resulting hydrocarbon bridge spans one face of the helix, physically preventing it from unfolding
Li et al. (2025) reviewed advances in hydrocarbon stapled peptides via ring-closing metathesis, cataloguing the synthetic strategies, common staple lengths, and the relationship between cross-link chemistry and biological function. They noted that i,i+7 staples provide greater conformational constraint than i,i+4 staples but are more synthetically challenging.[1]
Beyond hydrocarbon staples
The field has expanded beyond the original all-hydrocarbon approach. Perdriau et al. (2025) developed guanidinium-stapled helical peptides that use a positively charged cross-linker instead of a hydrophobic one, enabling targeting of protein-protein interactions where the binding interface requires cationic character.[2]
Neuville et al. (2025) took a different approach entirely, using foldamer helix mimetics to create cell-permeable peptide inhibitors of the p53-hDM2 interaction. Instead of stapling a natural alpha helix, they built a synthetic backbone that naturally adopts a helical conformation, sidestepping the need for a cross-linker.[3]
For other approaches to improving peptide stability, see D-Amino Acid Peptides: Mirror-Image Molecules That Resist Degradation and Cyclic vs Linear Peptides: Why Shape Matters for Function.
What stapling accomplishes
Protease resistance
The hydrocarbon bridge physically blocks access to the peptide backbone at the staple site. Proteases that would normally cleave the peptide at these positions are sterically excluded. Shi et al. (2025) demonstrated this directly by performing systematic all-hydrocarbon stapling analysis on cecropin A, an antimicrobial peptide that is potent in vitro but rapidly degraded in biological fluids. Stapled variants showed up to 16-fold improvement in proteolytic stability in human serum while maintaining or enhancing antimicrobial activity against gram-negative bacteria.[4]
Yang et al. (2025) applied hydrocarbon stapling to antimicrobial peptides more broadly, finding that the position and length of the staple determined whether proteolytic resistance was gained at the expense of membrane-disrupting activity or whether both properties were preserved simultaneously.[5]
Cell permeability
Unstapled peptides are generally membrane-impermeable. The hydrocarbon staple creates a hydrophobic patch on the peptide surface that enables interaction with the lipid bilayer and facilitates cellular uptake, primarily through endocytic pathways followed by endosomal escape. This property is particularly valuable for targeting intracellular protein-protein interactions, which represent a vast category of disease-relevant targets that conventional peptides and small molecules struggle to reach.
Li et al. (2025) dissected the geometric and hydrophobic constraints that determine stapled peptide cell permeability. They found that the position of the staple relative to the binding interface is critical: staples placed on the non-binding face maximize both cell permeability (by presenting the hydrophobic staple to the membrane during uptake) and target engagement (by leaving the binding face unobstructed).[6]
Binding affinity
Constraining the helix pre-organizes the peptide into the shape complementary to its target. This reduces the entropic penalty of binding (the peptide doesn't need to fold upon contact) and typically increases binding affinity by 2- to 100-fold compared to the unconstrained linear peptide. Babych et al. (2026) probed the effect of different stapling strategies on peptide-protein interactions, finding that the improvement in affinity depends on whether the native interaction already involves a pre-formed helix (modest improvement) or requires folding upon binding (large improvement).[7]
Clinical applications
Cancer: the p53-MDM2/MDMX axis
The most advanced clinical application of stapled peptides is in oncology, specifically targeting the protein-protein interaction between the tumor suppressor p53 and its negative regulators MDM2 and MDMX. In approximately half of human cancers, p53 is wild-type but functionally suppressed by overexpression of MDM2 or MDMX. Disrupting this interaction should reactivate p53 and trigger apoptosis in tumor cells.
Sulanemadlin (ALRN-6924) is a stapled peptide designed to mimic the alpha-helical domain of p53 that binds to MDM2 and MDMX. It enters cells, binds both MDM2 and MDMX with high affinity, displaces endogenous p53, and reactivates p53-dependent gene expression. Phase 1 clinical trial data showed a 45% disease control rate (2 complete responses, 2 partial responses) in patients with p53 wild-type solid tumors and lymphomas.
Han et al. (2025) designed stapled peptide inhibitors targeting the p53-MDM2 interaction using alternative stapling chemistries, expanding the toolkit for this validated oncology target.[8] Morgan et al. (2025) developed conformationally constrained p53 stapled peptides using a diyne-girder approach that allowed real-time monitoring of target engagement via Raman spectroscopy.[9]
Metabolic disease: stapled multi-agonists
Stapling is not limited to oncology targets. Yang et al. (2025) developed double biaryl-stapled GLP-1R/GIPR dual agonist peptides for obesity and diabetes. The double staple improved both protease resistance and receptor binding, demonstrating that stapled peptide technology can be applied to GPCR-targeting peptides in metabolic disease.[10]
Zhou et al. (2025) applied stapling to a GLP-1R/GIPR/GCGR triple agonist, creating a long-acting stapled triple agonist for obesity treatment. The stapled variant maintained activity at all three receptors while dramatically improving half-life, addressing a key limitation of unmodified multi-agonist peptides.[11]
Infectious disease: antiviral and antimicrobial stapled peptides
Wang et al. (2025) developed a short double-stapled peptide that potently inhibits human betacoronaviruses by blocking viral membrane fusion. The stapled peptide mimics a helical region of the viral fusion machinery and prevents the conformational change required for viral entry into host cells.[12]
Patil et al. (2025) reviewed the broader potential of stapled peptides in antiviral applications, noting that viral entry and replication involve numerous protein-protein interactions mediated by helical interfaces, all of which are potential stapled peptide targets.[13]
Saxena et al. (2025) designed a stapled peptide targeting the Ebola virus matrix protein dimer interface, demonstrating activity against one of the most dangerous viral pathogens and expanding the infectious disease applications of stapled peptide technology.
Wound healing and regenerative medicine
Chen et al. (2026) developed stapled peptide inhibitors targeting the VGLL4/TEAD4 protein interaction to accelerate cutaneous wound healing. By disrupting a transcriptional complex that suppresses tissue regeneration, the stapled peptides promoted faster wound closure in preclinical models.[14]
Computational design: the new frontier
Traditionally, stapled peptide development required synthesizing and testing dozens of variants with different staple positions, lengths, and chemistries. Schofield et al. (2026) demonstrated a computational alternative: using molecular dynamics simulations and binding free energy calculations to design stapled peptide antagonists in silico before synthesis. Their computationally designed candidates showed activity comparable to empirically optimized ones, dramatically reducing the time and cost of development.[15]
This computational approach is particularly valuable because the structure-activity relationships for stapled peptides are complex. The optimal staple position depends on the target protein's surface topology, the peptide's intrinsic helical propensity, the desired cell permeability, and the intended route of administration. Predicting the combined effect of these factors experimentally requires large synthesis campaigns. Predicting them computationally requires sophisticated models but only modest bench work to validate.
For the broader topic of computational peptide design, see Peptidomimetics: Molecules That Mimic Peptides but Aren't. For backbone modifications that achieve some of the same goals through different chemistry, see Beta-Peptides and Gamma-Peptides: Beyond Natural Amino Acid Backbones.
Limitations and open questions
Stapled peptides are not a universal solution. Several limitations remain.
Cost of synthesis. Non-natural amino acids, Grubbs catalysts, and the multi-step synthesis required for stapled peptides make them substantially more expensive to manufacture than standard peptides. This limits their current application to high-value targets (oncology, rare diseases) where the cost per dose is acceptable.
Immunogenicity uncertainty. The hydrocarbon staple introduces a non-natural structural element. Whether this triggers immune responses upon repeated dosing in humans is not fully resolved, as ALRN-6924 clinical data is still limited in duration. Long-term immunogenicity data will be needed before stapled peptides are considered for chronic conditions.
Target selectivity. Cell-permeable stapled peptides access the entire intracellular proteome. Off-target binding to unintended protein surfaces is a concern, particularly for heavily hydrophobic stapled peptides that may interact nonspecifically with multiple intracellular proteins.
Oral bioavailability remains elusive. While stapling improves protease resistance and membrane permeability, oral delivery of stapled peptides has not been convincingly demonstrated. Current clinical candidates require parenteral administration.
The Bottom Line
Stapled peptides solve three fundamental problems of peptide drug design in one chemical modification: a covalent hydrocarbon bridge locks the alpha helix, blocks protease access, and creates a hydrophobic surface for cell entry. Sulanemadlin (ALRN-6924) proved the concept in clinical trials by reaching intracellular p53-MDM2/MDMX targets. The technology has expanded beyond oncology into metabolic disease (stapled GLP-1/GIP/glucagon agonists), infectious disease (coronavirus and Ebola inhibitors), and wound healing. Computational design is beginning to replace empirical screening. Manufacturing cost, immunogenicity, and oral delivery remain unsolved challenges.