Unleashing the Antiviral Potential of Stapled Peptides: A New Frontier in Combating Human Neurotropic Viral Infections

ABSTRACT

Neurotropic viral infections continue to pose significant global health challenges, with pathogens such as herpes simplex virus (HSV), varicella‐zoster virus, human immunodeficiency virus, poliovirus, enteroviruses, parechovirus, West Nile virus and Japanese encephalitis virus driving the search for more effective therapeutic interventions. Current antiviral strategies, including small molecules and monoclonal antibodies, often face limitations such as drug resistance, narrow spectrum activity and adverse side effects, underscoring the need for alternative approaches. Antiviral peptides are emerging as potential therapeutic agents against these viral infections as entry and fusion inhibitors. However, their clinical development is limited by poor stability, low bioavailability and insufficient cellular penetration. To address these limitations, peptide stapling, a chemical modification that stabilises peptide α‐helices through covalent linkage, has emerged as a transformative technique to enhance the therapeutic potential of peptides, especially in antiviral drug development. Stapling techniques, including hydrocarbon staples, lactam bridges and metal‐coordination bonds, are explored for their ability to improve peptide stability, bioavailability and target binding affinity. This review examines the application of stapling in the development of antiviral peptides with a focus on stapled peptides targeting viral fusion and entry mechanisms, highlighting their potential against neurotropic viruses such as HSV and influenza. By integrating the structural rigidity conferred by stapling, these constructs promise to overcome delivery barriers and achieve superior antiviral efficacy. This paper underscores the pivotal role of peptide stapling by highlighting recent advancements in antiviral therapeutics and presents a roadmap for future research into multifunctional stapled peptides.

Human neurotropic viruses like SARS‐CoV‐2, HSV, HOPV and RSV pose significant risks due to their ability to invade and persist in the nervous system. Stapled peptides represent a promising therapeutic approach with enhanced stability, target affinity and bioavailability, emerging as an effective strategy for neutralising human neurotropic viruses.

1. Introduction

Numerous viruses are associated with neurological disorders globally, impacting the morphology and physiology of the central nervous system (CNS), leading to malformations and elevated mortality rates. Neurotropic viruses are defined as viruses capable of inducing diseases in the CNS, characterised by their neuroinvasive and neurovirulence properties. The group includes various viruses such as Herpes Simplex Virus (HSV), Varicella‐Zoster Virus (VZV), Human Immunodeficiency Virus (HIV), Poliovirus, Enteroviruses, Parechovirus, West Nile Virus, Japanese Encephalitis Virus, as well as measles and mumps viruses, among others (Lulla and Sridhar 2024). Once they enter the nervous system, these viruses can establish latency or persist in host cells, leading to recurrent infections and potential long‐term neurological complications. The central nervous system, however, presents a challenging environment for therapeutic interventions due to the protective blood–brain barrier and the highly specialised cell types involved (Wiley et al. 2015) (Abdullahi et al. 2020). The epidemiology of infections caused by these viruses varies geographically due to differences in vector distribution, human activities and climate conditions (Ludlow et al. 2016). Emerging infections such as Zika and Nipah and the resurgence of vaccine‐preventable diseases like measles and polio highlight evolving epidemiological trends influenced by urbanisation, deforestation and climate change (Montgomery et al. 2008; Rubin et al. 2015; Palacios and Oberste 2005). Over the past few decades, developing antiviral drugs has been pivotal in addressing life‐threatening viral diseases despite the ongoing challenges of emerging pathogens and the limited therapeutic options available (Wu et al. 2024).

During the preceding decades, advances in molecular biology and drug development have led to the emergence of a diverse array of antiviral agents, broadly categorised into small molecules and biologics. Small‐molecule antivirals often target specific viral enzymes or processes, offering oral bioavailability and ease of synthesis, while biologics, including monoclonal antibodies, recombinant proteins and antiviral peptides, provide highly specific mechanisms of action, such as neutralising viral particles or blocking host‐virus interactions. The development and deployment of these therapeutics have transformed the treatment landscape for diseases such as influenza, HIV, Ebola and most recently, COVID‐19, underscoring the importance of both classes of agents in combating existing and emerging viral threats. Among small‐molecule antivirals, notable examples include remdesivir, a nucleotide analogue that inhibits the RNA‐dependent RNA polymerase of RNA viruses and has been used to treat COVID‐19 and Ebola; molnupiravir, another nucleoside analogue that induces lethal mutagenesis in viral RNA; and nirmatrelvir/ritonavir (Paxlovid), a protease inhibitor combination effective against SARS‐CoV‐2 (Zhong et al. 2022). Ribavirin, originally developed for hepatitis C and respiratory syncytial virus, have been repurposed for emerging viruses like SARS‐CoV‐2 and Ebola, though their clinical effectiveness and safety profiles vary. In contrast, biologic antivirals include virus‐neutralising monoclonal antibodies (mAbs), which bind to viral surface proteins and prevent viral entry into host cells. Examples of such biologics are convalescent plasma‐derived antibodies and recombinant monoclonal antibodies developed against viruses like SARS‐CoV‐2, Ebola and Zika. Additionally, recombinant soluble human receptors, such as soluble ACE2, have been engineered to block viral entry, while CRISPR/Cas‐based therapies are being explored for their ability to target and degrade viral genomes in infected cells (Ianevski et al. 2022).

The protein biotherapeutics approach presents an effective avenue for combating neurotropic viral infection. While current antiviral strategies, such as vaccines and small molecule inhibitors, have contributed significantly to controlling viral infection, their constraints emphasise the need for substitution therapy (Kainov et al. 2025). Antiviral peptides (AVPs) present a compelling solution, offering diverse mechanisms to inhibit viral replication, disrupt entry pathways and overcome drug resistance through their high specificity and adaptability. Antiviral peptides (AVPs) have emerged as promising therapeutic agents against neurotropic viruses due to target specificity and lower toxicity. Enhancing the stability, bioavailability and antiviral capacity of AVPs to traverse biological barriers, such as the blood–brain barrier, has significantly improved their therapeutic efficacy against neurotropic infections (Lourenço et al. 2023). Peptidomimetic methods play a crucial role in overcoming the peptide stability limitation. The primary peptidomimetic methods include D‐amino acid incorporation, PEGylation, acetylation, dimerization, amino acid mutation, lipidation, cyclization and stapling (Monroe et al. 2022).

Stapling is an emerging advanced peptidomimetic method that forms covalent crosslinks between side chains to stabilise the α‐helical structure of peptides (Kim and Kang 2024). This modification locks the peptide in its required conformation and improves stapled peptides' proteolytic stability, cell permeability and binding affinity, making them promising therapeutic candidates. While initially developed to enhance the properties of anticancer and antimicrobial peptides, stapling techniques have also been successfully applied to antiviral peptides, including the development of fusion inhibitors targeting viral proteins, making this approach particularly beneficial for producing peptides with increased bioactivity for intracellular targeting in both antiviral and anticancer applications (Lourenço et al. 2023; Shen, Zhang, et al. 2023). Researchers addressed the limitations of the antimicrobial peptide SAAP‐148 by developing a novel peptide, SLP‐51, using a combination of silver‐catalysed arginine N‐glycosylation and all‐hydrocarbon double‐stapling. SLP‐51 demonstrated enhanced antimicrobial activity, improved stability against proteolysis, reduced hemolytic toxicity and strong in vivo efficacy against drug‐resistant bacterial infections and associated inflammation (Li et al. 2025).

A comprehensive perspective on the role of stapled antiviral peptides in blocking the neurotropic viral cycle has not been reported. Thus, the present review discusses the potential of stapling as a novel, promising approach to restrict neurotropic viruses.

1.1. Life Cycle and Latency of Human Neurotropic Viruses: Mechanism and Pathogenic Insights

Human neurotropic viruses can infect the nervous system, encompassing both the central nervous system (CNS) and peripheral nervous system (PNS). The life cycle of neurotropic viruses unfolds through a series of well‐defined stages: attachment, entry, fusion, genome replication, gene expression (transcription and translation), assembly, envelopment and egress (Figure 1 provides a schematic overview of these stages and highlights key host‐virus interactions). The process begins when viruses bind to specific receptors on the surface of host cells, initiating primary infection. HSV uses nectin‐1, HVEM and 3‐OS‐HS as receptors (Rahangdale et al. 2023), whereas SARS‐CoV‐2 interacts with angiotensin‐converting enzyme 2 (ACE2) (Jayaprakash and Surolia 2022). HIV targets CD4 receptors (Wilen et al. 2012), while EBOV utilises T‐cell immunoglobulin and mucin domain 1 (TIM‐1) (Kuroda et al. 2015). The influenza virus binds to sialic acid‐terminated glycoconjugates (Overeem et al. 2021). Following attachment, the virus penetrates the host cell and the fusion process facilitates the transport of the viral capsid into the cell.

FIGURE 1.

The viral lifecycle of neurotropic viruses includes adsorption, entry, replication and egress during primary infection. These viruses invade the nervous system by crossing the BBB, establishing latency in neurons. Factors such as genetic variation, stress and ageing can trigger reactivation, making attachment, entry, replication and latency potential antiviral peptide targets.

Upon entering the host cell, neurotropic viruses exploit the cellular machinery to replicate their genome via transcription and translation processes. The replication site depends on the virus type: DNA viruses such as HSV replicate in the nucleus, whereas RNA viruses, including SARS‐CoV‐2 and rabies virus, primarily replicate in the cytoplasm. After genome replication, newly synthesised virions are assembled and obtain an envelope sourced from the host cell membrane. Mature virions are subsequently released via egress, enabling the infection of neighbouring cells and further propagation of the virus during the primary infection phase.

A distinctive feature of neurotropic viruses is their ability to establish latency, especially after invading the CNS. During this phase, the virus remains transcriptionally silent, effectively evading immune surveillance. Neurotropic viruses infiltrate the CNS via multiple pathways. This includes retrograde axonal transport along with motor or olfactory neurons, hematogenous spread across the blood–brain barrier (BBB) or blood‐cerebrospinal fluid barrier (Ludlow et al. 2016), Additionally invasion of the BBB via infected leukocytes (Trojan horse). Once within the CNS, many neurotropic viruses enter a latent state, characterised by dormancy and the potential for later reactivation. HSV and VZV establish latency in the trigeminal and dorsal root ganglia (Steiner et al. 2007), whereas HIV and RSV persist in astrocytes, microglia and perivascular macrophages (Churchill et al. 2015; Chatzidimitriou et al. 2020; Bohmwald et al. 2021). EBOV can persist in a latent state within macrophages in the brain's ventricular system, choroid plexuses and CD68+ cells (Liu et al. 2022; Zeng et al. 2017).

The host immune response plays a crucial role in controlling acute infection and maintaining latency, but immune evasion strategies employed by these viruses often complicate clearance and contribute to disease recurrence. Reactivation of latent viruses is often triggered by stress or immunosuppression, leading to renewed viral replication and potential disease recurrence. Therefore, a comprehensive understanding of the life cycle and latency mechanisms of neurotropic viruses is essential for the development of effective therapeutic strategies. AVPs can target attachment, entry, uncoating, replication, transcription, translation, assembly, release and post‐release maturation to mitigate the early phase of viral infection (Figure 1). Despite advances in our understanding, many aspects of neurotropic virus latency and reactivation remain elusive, underscoring the need for continued research in this field.

2. Overview of Peptide Stapling

Why staple the peptides? Stapled peptides represent a promising class of therapeutics designed using receptor‐ or ligand‐based strategies to target diseases such as cancer, infectious diseases, inflammation and diabetes. This advanced peptide stapling technique improves the stability, bioavailability and overall therapeutic potential of peptides (Li et al. 2024). The alpha‐helix is a standard secondary protein structure crucial in many functions. Molecules that mimic alpha helices at protein–protein interaction (PPI) binding interfaces are of great interest, as they can act as competitive PPI inhibitors. Selective PPI inhibition could enable the development of therapeutics targeting a broad range of previously ‘undruggable’ proteins (Lau et al. 2015). The efficiency of the stapling strictly depends on the position of the staple and the nature of the cross‐link. While the choice of the position of the staple mainly depends on the structure of the peptide substrate and cannot be assessed without considering the target of application (Kim and Kang 2024), the evaluation of the best kind of cross‐link could be performed based on previous studies that show the different properties of various types of stapling (Moiola et al. 2019).

2.1. α‐Helix Stapling

Stapling, a form of chemical modification, addresses these challenges by introducing covalent constraints that stabilise the peptide's α‐helical conformation, thus improving its overall pharmacokinetic profile (You et al. 2023; Tran et al. 2017). The term ‘stapled peptides’ was initially introduced by Verdine and colleagues, building upon pioneering work by Schafmeister and team, who developed techniques for linking the side chains of two non‐natural amino acids (Schafmeister et al. 2000). The defined concept involved the insertion of a hydrocarbon or other synthetic linker between two amino acid side chains, typically on i and i + 4 or i and i + 7 positions of the peptide sequence (Walensky and Bird 2014). The numbering starts at the N‐terminus of the peptide, with ‘i’ denoting the position of a specific residue in the peptide sequence. Peptide stapling introduces covalent crosslinks, typically between side chains at positions i and i + 4 or i and i + 7, stabilising the α‐helical conformation (Figure 2a). The i, i + 4 stapling mimics a single helical turn, enhancing stability and cell permeability, while i, i + 7 stapling spans two helical turns, offering additional rigidity and structural support for specific functional requirements. These configurations are tailored based on the peptide's intended application and target interaction (Li et al. 2024; Lau et al. 2015; Xie et al. 2016; Sabale et al. 2021). This innovation allows the formation of a staple within the peptide structure, a chemical bridge that locks the peptide in a specific, biologically active conformation. This cross‐linking locks the peptide in its bioactive α‐helical form. Peptide stapling resists enzymatic degradation and promotes cell penetration by stabilising the structure, improving membrane interaction and resistance to degradation. These properties make them a valuable strategy in drug design and therapeutic application, and have significantly advanced peptide drug development, making it possible to design peptides that act as potent modulators of PPI (Shi et al. 2022).

FIGURE 2.

(a) Amino Acid Spatial Arrangement in Stapled Peptide following i i + 4, i + 7 spacing pattern, forming a helical confirmation. This pattern stabilises the structure, enhancing the bioactivity of the peptide. (b) Schematic representation of single‐stapled (i, i + 7) and double‐stapled (i, i + 4 and i + 7, i + 11) peptides. The figure was created using https://www.biorender.com/.

Stapled peptides have shown significant therapeutic advantages over conventional peptides. These include enhanced stability, as they are more resistant to enzymatic degradation, and improved bioavailability due to better pharmacokinetics and resistance to rapid metabolism (Xie et al. 2016). Their stapled conformation maintains an optimal structure for binding to target proteins, increasing target‐binding efficiency. Furthermore, the stapling process enhances cell permeability by making peptides more lipophilic, aiding their ability to cross cellular membranes (Tian et al. 2017). Stapled peptides have demonstrated promise in several therapeutic areas. In cancer therapy, they have been used to target oncoproteins such as MDM2‐p53 interactions, as seen in ALRN‐6924 (Xu et al. 2024). In infectious diseases, they inhibit viral and bacterial protein interactions, with applications such as HIV‐1 fusion inhibitors and other emerging antiviral therapies. Additionally, they hold potential in autoimmune disorders by modulating immune pathways through selective interaction targets (Huang et al. 2023).

The rational design of more potent and effective antiviral peptides (AVPs) requires knowledge of their mechanisms of action, which are not yet fully understood. A key advantage of stapled peptides in protein interaction research is their capacity for precise specificity testing through mutagenesis that deliberately disrupts bioactivity (Lee et al. 2022). This can be achieved through alanine scanning, which sequentially replaces each natural residue with alanine, or staple scanning, which systematically explores staple positions along the helical surface. The ability to develop negative control mutants makes stapled peptides especially valuable in biological and therapeutic targeting studies (Bird et al. 2011).

2.2. Comparative Analysis of Single‐Stapled and Double‐Stapled Peptides

Single‐stapled peptide, which contains a single hydrocarbon brace, primarily stabilises α‐helical structure and provides moderate improvements in proteolytic resistance and cell permeability. In contrast, double‐stapled peptides incorporate two staples, which not only reinforce the α‐helical conformation but also can stabilise more complex secondary structures, such as β‐sheets. This dual stabilisation results in significantly greater resistance to the enzymatic degradation, markedly higher binding affinity due to more effective prepayment of the entropy penalty, and enhanced cellular uptake. Moreover, double‐stapled peptides have demonstrated superior oral bioavailability and the capacity to target intracellular protein–protein interactions, which are often considered undruggable. However, the synthesis and design of double‐stapled peptides are more challenging, frequently requiring computational modelling to ensure that the stapled placements do not bind with the key interfaces. Collectively, these features position double‐stapled peptides as superior candidates for therapeutic applications demanding robust stability and efficient cellular delivery, surpassing the capabilities of single‐stapled analogues. Table 1 provides a comparative overview of the key distinctions between the two mainstays based on experimental and computational data in a few areas, including stability, binding and therapeutic usefulness. (Figure 2b describes a schematic representation of single‐stapled (i, i + 7) and double‐stapled (i, i + 4 and i + 7, i + 11) peptides).

TABLE 1.

Comparative analysis of single‐stapled and double‐stapled peptides.

S. No.	Parameters	Single‐stapled peptide	Double‐stapled peptide	References
1.	Proteolytic stability	Improvement over linear peptide	Significantly enhanced and robust	(Bird et al. 2010)
2.	Structural scope	Stabilises α‐ helical motifs only	Concurrently stabilises α‐helix and β‐sheet structures	(Ma et al. 2024)
3.	Binding affinity	Increased compared to linear peptide	Markedly higher than single stapled	(Zheng et al. 2021)
4.	Cell permeability	Varicable and may require optimization	Consistently improved	(Ma et al. 2024)
5.	Oral bioavailability	Limited absorption in Gastro‐intestinal tract	Potential for oral delivery	(Bird et al. 2010)
6.	Therapeutic efficacy	Effective for extracellular targets	Enables intracellular targeting	(Gaillard et al. 2017)
7.	Target versatility	Suitable for well‐defined α‐helix interfaces	Engages undruggable targets	(Zhang et al. 2024)
8.	Synthetic complexity	Straightforward staple placement	Requires computational design to avoid binding disruption	(Gaillard et al. 2017)

2.3. Diversity of Stapled Peptide Structures Beyond the α‐Helix

Stapled peptides were originally developed to stabilise α‐helical structures, but advances in peptide chemistry have led to a diverse array of stapling strategies that extend beyond the α‐helix. These alternative approaches enable the stabilisation of different secondary structures, expanding the functional and therapeutic scope of stapled peptides.

2.3.1. β‐Sheet and β‐Hairpin Stapled Peptides

β‐stapled peptides are designed to stabilise β‐sheet or β‐hairpin conformations, which are important for many protein–protein interactions but are inherently less stable in solution than α‐helices. These peptides are engineered by using staples such as 4‐mercaptoproline or hydrocarbon crosslinks. Stapling in these peptides can be achieved using template‐assisted olefin metathesis or other chemistries to covalently link side chains across the β‐strands, thereby locking the peptide into its bioactive β‐sheet arrangement. This approach has been shown to yield peptides with enhanced structural rigidity and resistance to proteolytic degradation, as well as improved specificity for their targets (Pace et al. 2021). In the year 2023, Venita and the team have developed innovative all‐hydrocarbon‐stapled β‐hairpin anti60 peptides (AMPs) that exhibit strong structural integrity and functional efficacy against resistant pathogens. The results of this study serve as a valuable foundation for future efforts in designing new β‐hairpin AMPs (Selvarajan et al. 2023).

2.3.2. Cross‐Stitched and Stitched Peptides

Cross‐stitched (also called ‘stitched’) peptides contain multiple staples or crosslinks within a single peptide chain. These staples may connect different regions of the peptide, such as linking both α‐helical and β‐sheet elements, or locking together distant segments to create a highly constrained macrocyclic structure. Cross‐stitching provides even greater conformational rigidity and can further enhance stability, binding affinity and cell permeability compared to single staples (Hilinski et al. 2014). This strategy is particularly useful for targeting large or complex protein–protein interfaces and for developing peptides with prolonged biological activity. A study was performed in 2018 by Thomas and team that further supports this approach, demonstrating how dual‐stapling strategies can enhance peptide conformational rigidity and bioactivity, aligning well with our findings on helicity and stability improvements (Speltz et al. 2018).

2.3.3. Loop‐Stapled and Turn‐Stapled Peptides

Few stapling techniques focus on stabilising loop or turn regions within peptides, which are often critical for molecular recognition but are typically flexible and susceptible to degradation. Loop‐ or turn‐stapling can be accomplished through cysteine alkylation, lactam bridges or other covalent linkages, effectively locking these motifs into their functional conformations. Such stabilised loops and turns can mimic key recognition elements of natural proteins and improve the pharmacological properties of the peptide (Pace et al. 2021).

These alternative stapling strategies demonstrate the versatility of peptide stapling chemistry, allowing for the stabilisation of a wide range of peptide secondary structures beyond just α‐helices.

3. Types of Peptide Stapling

The classification of peptide stapling techniques depends on several factors, reflecting the diversity in their design and application. Key criteria include the type of cross‐linking reactions, such as covalent bond formation or click chemistry methods. The location of stapling residues, typically positioned to stabilise specific secondary structures, also plays a critical role. Additionally, the nature of functional groups, whether derived from natural amino acid residues or non‐natural modifications, contributes to the classification. These classifications provide a framework for selecting and optimising stapling strategies tailored to therapeutic and research needs. Based on all these criteria, some major stapling techniques are in practice, such as hydrocarbon stapling (Bird et al. 2011), lactam stapling (Klein et al. 2017), disulphide bridging (Forte et al. 2018), photocrosslinking (Wan et al. 2023) and others, each tailored to optimise the therapeutic potential of peptides in different contexts (Figure 3).

FIGURE 3.

Macrocyclization Approaches in Stapled Peptide Design representing hydrocarbon stapling, lactam bridges, thioether linkages and disulphide bonds. These techniques create conformational constraints that reinforce the alpha‐helical shape, improve resistance to enzymatic degradation, enhance cell permeability and strengthen binding affinity to target molecules, making them highly effective for therapeutic applications. The figure was created using https://www.biorender.com/.

3.1. Hydrocarbon Stapling

Hydrocarbon stapling is the most prevalent stapling technique, involving the incorporation of α,α‐disubstituted non‐natural amino acids, such as olefin‐containing amino acids, into the peptide sequence (Walensky and Bird 2014). Through ring‐closing metathesis (RCM), these side chains form a hydrocarbon bond, stabilising the helical structure. Hydrocarbon stapling has been used successfully to stabilise peptides targeting various viral proteins, including those involved in viral entry and replication. Studies have shown that stapled peptides exhibit improved pharmacokinetic profiles, including increased serum stability and enhanced bioavailability, making them attractive candidates for antiviral drug development (Schafmeister et al. 2000). For instance, applying this technique to a 36‐amino acid HIV fusion inhibitor resulted in a derivative with remarkable resistance to proteolytic degradation and notably improved pharmacokinetics, including enhanced oral absorption (Cong et al. 2023). Hydrocarbon stapling at i, i + 4 or i, i + 7 positions reinforces α‐helicity, proteolytic resistance and cell permeability by locking peptides into bioactive conformations, with staple placement optimised to avoid critical binding residues. Double stapling synergistically enhances protease resistance and pharmacokinetics by combining α‐helical stabilisation with explicit blockade of cleavage sites near each staple, enabling oral bioavailability and extended in vivo half‐life. Solvent‐exposed staple orientation (e.g., i, i + 7) maximises activity without disrupting binding interfaces, while preserving key hydrophobic residues (e.g., leucine) and polar residues maintains target affinity and specificity. Staple length and position dictate biological efficacy: longer linkers (e.g., 8‐atom staples) improve helicity and target engagement, whereas misplacement reduces activity despite structural stabilisation (Walensky and Bird 2014; Gomara et al. 2020). Incorporating an all‐hydrocarbon crosslink into natural peptide sequences can restore the shape and biological function of native α‐helices. This approach provides a versatile set of chemical tools that can be used to explore protein interactions and adjust interaction networks for possible therapeutic applications (Walensky and Bird 2014; Bird et al. 2011).

3.2. Lactam Stapling

Lactam stapling involves the formation of a covalent bond between the side chains of oppositely charged residues, typically lysine and glutamic acid or aspartic acid, resulting in an amide bond referred to as a lactam bridge (Sabale et al. 2021). It also calls for selectively protecting and deprotecting groups for the amino and carboxyl groups (Zhan et al. 2024). Lactam stapling is more straightforward than hydrocarbon stapling and yields a highly stable peptide (Taresh and Hutton 2022). Although both Asp‐Lys and Lys‐Asp lactam bridges improve helical stability, Lys‐Asp lactamization is preferred because of its improved helix‐inducing effect. It offers a flexible strategy for constraining peptides, especially in cases where hydrocarbon staples may not be suitable due to the peptide's sequence or structure. Two NHS ester‐containing functional cross‐linkers were attached to the amino groups of initialized Lys‐Lys residues, forming a bi‐lactam‐containing stapled peptide. Similarly, Glu‐Glu pairs can form bi‐lactam staples through bisamide formation (Mahesh et al. 2020; Patgiri et al. 2008; Fujimoto et al. 2004). Lactam stapling at the i, i + 4 or i, i + 7 positions effectively enhances α‐helicity, structural stability, protease resistance and cell permeability of peptides by constraining them into helical conformations. The staple is typically formed by creating an amide bond between the side chains of lysine and aspartic/glutamic acid residues, which are positioned on the same face of the helix for optimal cross‐linking. The length and position of the lactam linker, as well as the orientation of the amide bond, are critical parameters that influence the degree of helicity and biological activity of the stapled peptide. Avoiding staple placement at key polar or binding interface residues preserves target binding while maximising structural benefits, thus improving the peptide's pharmacological profile (Zhang et al. 2024).

3.3. Disulphide Bridging

Disulphide bridging or disulphide stapling, is a technique utilised to form bioconjugates with selective site‐specific attachments. This technique entails the introduction of cysteine residues at specific positions, facilitating the formation of disulphide bonds that enhance the stability of the peptide structure (Yu et al. 2023). However, disulphide bonds are redox‐sensitive and may be reduced under physiological conditions. This bridge is typically formed between sulphur atoms of two cysteine residues, creating the covalent link that stabilises the secondary and tertiary structure of the peptide, making them more resistant to enzymatic degradation and denaturation (Yu et al. 2023). A variety of reagents have been developed to facilitate the bridging of disulphide bonds, including next‐generation maleimides (NGMs) (Nunes et al. 2015; Schumacher et al. 2014), pyridazinediones (PDs) (Chudasama et al. 2011), bis‐sulfones (Walsh et al. 2019), divinylpyrimidines, divinyltriazines, arylenedipropiolonitriles, dichlorotetrazines and other compounds (Counsell et al. 2020; Koniev et al. 2018; Stieger et al. 2021). Disulphide stapling offers enhanced bioactivity by stabilising active conformations, improving binding affinity and effectiveness at lower doses. The stapling increases cell permeability by incorporating hydrophobicity and rigidity, enabling better cellular uptake. Disulphide stapling at i, i + 4 or i, i + 7 positions enhances α‐helicity, protease resistance and cellular permeability by forming reversible macrocyclic bridges between cysteine residues, with optimal staple length (18–48‐membered rings) maximising structural stability. Dimerization via disulphide bonds creates antiparallel, leucine‐rich hydrophobic interfaces (e.g., LxxLL motifs) that resist proteolysis and enable higher‐order oligomerization, driven by residues like Leu6/Leu10 for knobs‐into‐holes packing. Reversible stapling allows controlled release of native peptides under reducing conditions (e.g., TCEP), facilitating targeted delivery while maintaining bioactivity, critical for prodrug applications and spatiotemporal regulation. Staple placement away from binding interfaces preserves target affinity, whereas solvent‐exposed orientations minimise disruption of key polar/hydrophobic residues essential for biological function (Yao et al. 2022).

3.4. Triazole‐Based Stapling

This technique, also known as Cu(I)‐catalysed azide‐alkyne cycloaddition (CuAAC) uses click chemistry to create a triazole ring between two azide and alkyne‐containing amino acid residues (Gaikwad et al. 2024). The triazole linkage mimics the stability offered by hydrocarbon stapling while being more biocompatible and versatile in chemical design. One of the major advantages of this technique is its remarkable tolerance for diverse functional groups, allowing peptides with unprotected functional groups to serve as linear precursors for triazole staple formation. Optimal linker configuration combines L‐Nle (εN ~3~) at position i and D‐propargylglycine (D‐Pra) at i + 4, forming an 8‐atom triazole staple that maximises α‐helicity (up to 90%) and binding affinity while minimising backbone distortion. Double stapling with reversed orientations (e.g., azide/alkyne at i/i + 4 and alkyne/azide at i + 7/i + 11) achieves > 90% helicity and enhanced proteolytic stability, avoiding mixed‐stapling artefacts. Solvent‐exposed placement is critical: staples must avoid arginine residues and destabilising motifs (e.g., threonine), with proximity to destabilising residues necessitating strategic positioning to maintain affinity. Triazole bridges provide metabolically stable, redox‐insensitive constraints that mimic disulphide bonds, enhancing proteolytic resistance and enabling cell permeability without redox sensitivity (Kawamoto et al. 2012). Its high biocompatibility, efficiency and mild reaction conditions make CuAAC a preferred method for peptide stapling. This strategy is widely utilised for its ability to create stable, bioorthogonal linkages, enhancing peptide stability and functionality in biological systems (Barrow et al. 2019).

3.5. Other Strategies

The initial peptide side‐chain cyclization method primarily involves the reaction between natural amino acids, such as lysine, which forms an amide bond with the side chains of glutamic acid or aspartic acid. This approach leverages the inherent reactivity of amino acid side chains to induce conformational constraints within the peptide. Such cyclization improves the structural stability of peptides and improves their resistance to enzymatic degradation (Li et al. 2024). Moreover, this method serves as a foundation for developing more advanced cyclization strategies, including thioether or disulphide bond formation, to further enhance peptide properties (Felix et al. 1988). Various stapling techniques are designed to use the special qualities of amino acid side chains, allowing for accurate and targeted alterations. Each technique offers unique benefits depending on the intended use, such as increasing medicinal efficacy, boosting binding specificity or focusing on intracellular or extracellular settings. Table 2 emphasises the key distinctions between some popular stapling techniques, highlighting their chemical characteristics. A comprehensive summary of various stapling strategies of peptides detailing the amino acid combination and their corresponding key features is given in Table 3.

TABLE 2.

Optimising parameters for Helix stabilisation.

Staple type	Chirality of amino acid	Chirality of amino acid at i + n	Staple length	Ideal stapling pattern	Ideal linker combination
i, i + 3	R	S	6–8	R3 + S5/R5 + S3	3‐carbon linker (R) + 5‐carbon linker (S)
i, i + 4	S	S	8	S5 + S5	Two 5‐carbon linkers (S chirality)
i, i + 7	R	S	11	R8 + S5/R5 + S8	8‐carbon (R) + 5‐carbon (S)/vice versa
i, i + 4 + 4	R	R	6–8	S5 + B5 + R5	5‐carbon (S) + bridged 5‐carbon (B) + 5‐carbon (R)
i, i + 4 + 7	S	S	6–8	S5 + B5 + S8	5‐carbon (S) + bridged 5‐carbon (B) + 8‐carbon (S)

Note: B, bridge carbon; i, position of an amino acid residue in the peptide sequence; Optimal, best combination of chemical modification and staple placement; R, Rectus (amino acid with right‐handed chirality); S, Sinister (amino acid with left‐handed chirality); Staple length, the number of amino acid residues separating the two attachment points. The R/S notation is used to name amino acids based on the Cahn‐Ingold‐Prelog rules. The Shorter staples (length 6/8) are often used for tighter constraints, while longer (Kainov et al. 2025) ones provide more flexibility.

TABLE 3.

Stapling strategies in peptide design: amino acid combinations and key features.

Sr. No.	Incorporation	Stapling strategy	Key feature	References
1.	Two identical natural amino acids	Cys‐Cys	Forms disulphide bonds, is reversible under reductive conditions, and is helpful for intracellular delivery.	(Zhan et al. 2024)
		Glu‐Glu	Involves carboxyl groups for lactam bridge formation; highly stable under physiological conditions.	(Phelan et al. 1997)
		Lys‐Lys	Uses cross‐linkers like glutaraldehyde and enhances stability with minimal structural disruption.	(Fujimoto et al. 2004)
		Met‐Met	Relies on thioether formation, is stable and non‐reversible & is helpful for extracellular targets.	(Wang et al. 2022)
		Trp‐Trp	Uses π‐π stacking or photo‐crosslinking; enhances aromatic interaction and binding specificity.	(Makwana and Mahalakshmi 2015)
		Tyr‐Tyr	Functionalization through reactions such as crosslinking using oxidative coupling, more hydrophilic properties and hydrogen‐bonding capabilities	(Zhang et al. 2023)
2.	Two different natural amino acids	Cys‐Lys	Combines thiol and amine groups and offers flexibility in designing hydrophilic or lipophilic peptides.	(Brunel and Dawson 2005)
		Cys‐Met Stapling	Uses thiol and thioether linkages; highly stable and resistant to enzymatic degradation.	(Wang et al. 2022)
		Cys‐Trp/Tyr	Integrates aromatic and thiol interactions; enhances hydrophobicity and target binding.	(Ohkawachi et al. 2023)
		Lys‐Asp	Combines amine and carboxyl groups to form lactams; provides structural rigidity and chemical diversity.	(Felix et al. 1988)
		Lys‐Tyr/Arg	Utilises amine and hydroxyl or guanidinium groups for diverse linkages; enhances hydrophilic and amphipathic properties.	(Li et al. 2021)
3.	Non‐natural amino acids	Mpc‐Mpc stapling	Uses methylene‐phenyl‐methylene crosslinks, provides extreme rigidity and is suitable for precise structural constraints.	(Kusebauch et al. 2006)
		Sec‐Sec stapling	Forms diselenide bonds; reversible like disulphides but more redox‐sensitive, enabling fine‐tuned stability.	(Halmagyi et al. 1990)
		SPAAC	The bio‐orthogonal approach forms triazole staples, which are highly specific and stable in living systems.	(Macias‐Contreras et al. 2020)

4. Interplay Between Stapled Peptide and Various Human Neurotropic Viruses

The stapling peptide helices technique has proven highly effective in creating stable, protease‐resistant mimics of antigenic structures with remarkable fidelity. This innovative approach holds significant potential in advancing vaccine development and controlling infectious diseases. By enhancing peptides' structural integrity and biological activity, stapling can improve their ability to mimic vital antigenic sites, which are crucial for triggering robust immune responses. Various stapled peptides targeting neurotropic viruses to mitigate viral infection are mentioned in Table 4, indicating their potential as effective therapeutic agents.

TABLE 4.

Application of stapled peptides in the treatment of neurotropic viral infection.

Source	Peptide name (Template)	Target	Type and position of staple	Experimental setup	References
SARS‐CoV‐2	(IEEQAKT X LDK X NHE X EDL X YQSSL)	RBD	Aliphatic stapling i, i + 4 Helicity: ~60%	In silico peptide modelling: CHARMM36, GROMACS, VMD	(Choudhury et al. 2022)
	(IEEQAKT X LDK X NHE X EDL X YQSSL)	RBD	Lactam stapling at i, i + 4	In silico peptide modelling: CHARMM36, GROMACS, VMD	(Choudhury et al. 2022)
	(SLDQINVTFLDLEYEMKKLE E 7 AIK E 7 KLEESYIDLKEL)	Spike protein	i, i + 4 position side‐chain cross‐linking Helicity: 59.2%	Cyclobutane‐based conformationally constrained amino acids	(Chen et al. 2023)
	ACE2_19–45 (STI X EQA X LFLDKFNHEAEDL X YQS X L)	SARS‐CoV‐2: Spike glycoprotein	i, i + 4 hydrocarbon stapling Helicity: 31.1%	Solid phase peptide synthesis In vitro: A549, live SARS‐CoV‐2 virus, spike protein RBD (Spike319‐541)‐In suspension HEK‐293F cells	(Jiang et al. 2024)
	Ac‐NH‐HE‐[Lys‐EDL‐Asp]‐YQ‐CO‐NH2	SARC‐COV: Spike RBD/hACE2	9‐mer Lactam Stapled at i, i + 4	Amino acid: Lys‐Asp In vitro: HEK293 (ATCC, CRL 11268), HEK‐293 T‐hACE2, (NR‐52511) Lentivirus vector (NR‐52516)	(Ferková et al. 2023)
	NYBSP‐4 (Ac‐TIEEQ‐ Z ‐KTFLDK‐ X ‐NHEAEDLFYQ‐ X ‐SLA‐ X ‐WN‐NH2) X = s‐2‐(4‐pentenyl) alanine Z = −(R)‐2‐(7‐octenyl) alanine	SARS‐COV‐2: ARS‐CoV‐2 RBD	i/i + 4 and i/i + 7 double‐ hydrocarbon stapling Helicity: 80%	In vitro: human lung carcinoma cells (A549‐NR‐53522), NIAID, NIH. HeLa, A549, HT1080 (human fibrosarcoma) and HEK293T/17 cells Plasmid: pNL4‐3DEnv‐NanoLuc, pSARS‐CoV‐2‐SD19, NL4‐3. Luc.R‐E‐DNA Envelope expression vector: HEF‐VSV‐G	(Curreli et al. 2020)
	hACE2221‐55A36K‐F40E (IEEQAKTFLDKFNHE K EDL E YQSSLASWNYNTNIT)	SARS‐CoV‐2: Spike protein RBD	Lactam‐based i,i + 4 stapling Helicity: 52%	Solid‐phase peptide synthesis.	(Maas et al. 2021)
	SCH2‐1‐20 (Ac‐DISGINASVVNIQKEIDRL S 5 EVA S 5 NLNEIDLQEL‐NH2)	SARS‐COV‐2: 6‐HB (HR1–HR2 complex)	Hydrocarbon stapling at i,i + 4 at peptide backbone Helicity: 47.2%	Solid‐phase peptide synthesis. In vitro: SARS‐CoV‐2 Virus (PubMed No: MT627325), 293 T cells. Analysis: HPLC, mass spectroscopy, & CD. Laboratory: ABSL‐3	(Zheng et al. 2021)
HIV‐1	(QDNIRA S 5 lef S 5 KNWAWWKLFAKARPLLV)	EFHD2 adaptor protein	Hydrocarbon stapling	Solid‐phase peptide synthesis. In vitro: RAW264.7 macrophage cell line, primary BMDMs (6–12‐week‐old male 90 mice), virus: AIDS In Vivo: wild type 8‐week‐old control mice (2 NASH model, NASH‐HCC model), Lysm‐Cre mice (Jax strain #: 004781), Lck‐Cre mice (Jax 37 strain #: 003802), human liver tissues from two cohorts of patients	(Fu et al. 2024)
HIV‐1	RQ‐01: 600TCHILGPDCAIEPHDWT 8 NITDKI X QIIHDFV(K*)631	Heptad‐Repeat‐1 domains	i, i + 7 STAPLE hydrocarbon stapling.	Fmoc solid phase synthesis. In vitro: vero cell passage 2, HeLa cells, virus: SARS‐CoV‐2 WA‐01 isolate [NR‐52281, Lot 700,033,175] In vivo: 48 Syrian (Golden) hamsters (n = 8 per arm, n = 4 per sex) (strain code 049)	(Bird et al. 2024)
	D26: Ac‐G * EAL * YL * NLL * YW * NH2 * ‐ non‐natural (S)‐2‐(4′‐pentenyl)‐ alanine residues	HIV‐1: Inactivate cell‐free HIV‐1 Virions	Hydrocarbon double‐stapled at i and i + 4 positions Helicity: 90%	Solid‐phase peptide synthesis. In vitro: HIV [Strain IIIB (X4) and Bal (R5)], clinical isolate 89BZ_167, MT‐2 cells (for X4), CEMx 174 5.25 M7 cells (for R5) In vivo: Sprague–Dawley rats (BALB/c mice) (safety assessment)	(Wang et al. 2024)
	NYAD‐1: H‐ ITF X DLL X YY X GP‐NH2 X: (S)‐Fmoc‐2‐(20 ‐pentenyl) alanine	HIV‐1: Gag polyprotein	i, i + 4 hydrocarbon stapling Helicity: ∼80%	Fmoc solid‐phase synthesis. In‐vito: Sup‐T1 cells, primary HIV‐1 strains, U87‐T4‐CXCR4 cells, 293 T cells, PBMC (processed from blood) Expression vector: pET28a v, pET14b vector	(Zhang et al. 2008)
	SC34EK‐1a: Ac‐WZEWDKKIEEYTKKIEE L* IKK S* QEQQEKNEKELK‐NH2	HIV‐1: Viral entry	i, i + 4 hydrocarbon stapling (Leui, Seri+4 and Lysi, Leui+4) Helicity: 13.7%	Solid‐phase peptide synthesis. In vitro: HIV inoculum (HIVIIB, X4‐tropic), chymotrypsin digestion	(Wu et al. 2021)
	StP1‐E1P47: WILEYLWKVPF D FW K GV	HIV‐1: gp41 coiled‐coil pocket	Lactum stapling at Glu‐4 and Lys‐8 Helicity: ~85%	Solid‐phase peptide synthesis. Characterisation: CD, NMR, molecular modelling Stability study: human blood serum Ex vivo: human colorectal tissue specimens (HIV negative), virus. HIV‐1BaL	(Gomara et al. 2020)
	hCS6ERE: IEELI ^ A AQ ^ QQRK NEEALRE L ^: Position of Homocysteine Residue Reacting to Form Staples	HIV‐1 gp41 Hexameric coiled‐coil fusion complex	Hydrocarbon stapling at i, i + 4; and One helical turn Helicity: 49%	Solid‐phase Fmoc synthesis. In vitro: HL2/3 cells (effector), TZM‐bl cells Virus: HIV‐1 X4 strain IIIB, HIV‐1 R5 train Bal and T20‐ and T2635‐Resistant Strains. In vivo: rat plasma and tissue homogenate	(Meng et al. 2019)
	NYAD‐67: ISF‐ R8 ‐EWLQAY‐ S5 ‐EDE	HIV‐1: Capsid	i, i + 7‐hydrocarbon‐stapled	In vitro: TZM‐bl cells, HeLa cells, HEK 293 T/17 cells Virus: HIV‐1 CRF07_BC and CRF01_AE isolates Expression vector: pSG3ΔEnv Plasmid	(Wang et al. 2017)
	SAH‐MPER: ELDK X ASL X NWFNITNWLWYIK	HIV‐1: gp41 Juxtamembrane fusion	Double‐stapling at i, i + 4 and, i, i + 3 hydrocarbon stapling	Automated peptide synthesiser with Rink amide AM LL resin
	17LTFR8EYWAQLS5AAAAA33	MDM2 and MDMX	i to i + 7 hydrocarbon stapling	Solid‐phase peptide synthesis. Isolation: Reverse Phase‐HPLC	(Guerlavais et al. 2023)
	NLH16: TAYF X LIL X GRW	HIV‐1 IN	α,α‐disubstituted olefinic amino acids at i and i + 4 positions Helicity: 40.5%	Fmoc‐based peptide synthesis. In vitro: MT‐4 cells.	(Long et al. 2013)
HSV	B‐5 s (Ac‐FLGW L FKV A SKVL‐NH2)	HSV, retrovirus: viral envelope	Hydrocarbon stapling at positions 5 and 9	Fmoc peptide synthesis. In vitro: Huh7.5 and 293 T cells, Virus: Wild‐type HSV‐1 (ATCC VR‐1493) Bacterial Strains: Pseudomonas aeruginosa PA14, Escherichia coli EDL933, Klebsiella pneumoniae KP1, Staphylococcus aureus Newman, Staphylococcus aureus SA3, Vibrio cholerae N16961	(Kim et al. 2021)
	P3: QAKTFLDKFNHE‐ NHCHO ‐EDL‐ NHCHO ‐YQ	RBD	Triazolyl bridge stapling at position 36 and 40	MW‐assisted solid‐phase peptide synthesis In vitro: Vero E6, Virus: ARS‐CoV‐2 viral stock	(Quagliata et al. 2023)
RSV	4ca: E + IN X SL X FIR X SDELLHNV X: S‐Pentenyl alanine +: Pentenyl alanine	HR2 and HR1 domain	Two ‘i, i + 4’ staples and with ‘i, i + 3’ and ‘i, i + 4’ hydrocarbon stapling Helicity: > 100%	Solid‐phase peptide synthesis. In vitro: A549 (ATCC CCL‐185) cells, HEp‐2 (ATCC CCL‐23) cells. Expression vector: pET‐15b, Host: Escherichia coli BL21(DE3). In vivo: female BALB/c mice (8 weeks old), 4 mice per group.	(Gaillard et al. 2017)
	sP9(235–239): Ac‐DN[ X LSL X ]DF‐COOH	Capsid protein (N‐protein)	Hydrocarbon stapling at i, i + 4 position Helicity: 61.2%	Solid‐phase peptide synthesis.	(Yan et al. 2023)
Human orthopneumovirus	hC‐peptide: Ac‐DN X LSL X DF‐COOH	Viral fusogenic glycoprotein (F‐protein)	Hydrocarbon bridge spanning two [i, i + 4] residues of C‐Peptide at Positions 235,239	Fmoc solid phase synthesis. In silico: PepFold3, HPepDock2, Rosetta FlexPepDock server, AMBER ff14SB force field	(Shen, Chen, et al. 2023)
Ebola Virus	EBOV‐6 (610–633): IEPHDWTKNI K DKI DK IIH D FVDKTLPDQG‐C	EBOV‐GP2	Lactamization	Custom peptide synthesis. In vitro: HeLa (CCL‐2), U2OS (HTB‐96), HEK 293 T (CRL‐11268) and BHK 21[C‐13] (CCL‐10) Virus: CHIKV strain AF15561 In vivo: BHK‐21 Syrian golden hamster, 6–12‐week‐old BALB/C mice (n = 10 per group) Expression vector: EBOV‐May GP1,2‐(Δ309–489), pQCXIX vector (Clontech)	(Pessi et al. 2019)

Note: The blue letters indicate the positions of the amino acids involved in stapling.

Abbreviations: CHIKV, Chikungunya virus; EBOV, Ebola virus; HIV‐1, human immuno‐deficiency virus; HR 1&2, heptad repeat 1&2; HSV, herpes simplex virus; RBD, receptor binding domain; RSV, respiratory syncytial virus.

Stapled peptides have shown promise in combating various infectious diseases, including HIV and hepatitis C, due to their improved stability, cellular permeability and resistance to enzymatic degradation. These characteristics make them ideal candidates for therapeutic interventions targeting pathogens with complex lifecycles or high mutation rates. Additionally, the ability to design stapled peptides with tailored functionalities opens new avenues for developing vaccines and therapeutics against emerging and re‐emerging infectious diseases compared to traditional antiviral drugs. Integrating stapled peptides into vaccine platforms can also address challenges such as antigen instability and poor immunogenicity, which are common obstacles in traditional vaccine development. As research progresses, stapled peptides will likely play an increasingly pivotal role in therapeutic and prophylactic strategies for managing infectious diseases. Stapled peptides were developed to treat various neurotropic viral infections, as discussed in (Figure 4).

FIGURE 4.

Overview of stapled peptides developed for treating various viral diseases, highlighting the targeted viruses and corresponding peptide designs.

4.1. Human Immunodeficiency Virus (HIV)

HIV remains a major global health threat, and the development of effective therapeutic agents is a priority. HIV‐1 viral capsid proteins comprise two essential structural domains, the N‐terminal (NTD) and C‐terminal (CTD), crucial for assembling infectious particles and encapsulating genetic material and replication enzymes. Stapled peptides have been designed to target essential HIV proteins, such as the envelope glycoprotein and the helix–helix interfaces of gp41 (Bird, Irimia, et al. 2014). The researchers explored peptides focusing on HIV‐1, which has been challenging to target with vaccines. They developed stapled α‐helices of the membrane‐proximal external region (SAH‐MPER) of the gp41 protein. These stapled peptides, named doubly stapled SAH‐gp41 (662–683) showed high resistance to protease degradation and bound with high affinity to broadly neutralising antibodies 4E10 and 10E8, effectively mimicking the native MPER epitope. This indicates that stapled peptides may facilitate the development of chemically stabilised antigens for HIV‐1 vaccines (Bird, Irimia, et al. 2014). In 2010, Bird and team addressed the limitations of long therapeutic peptides, such as poor stability and bioavailability, by introducing hydrocarbon double‐stapling. This technique enhanced protease resistance, improved pharmacokinetics, including oral absorption and optimised antiviral activity in HIV‐1 fusion inhibitors, with potential applications to other peptide therapeutics like exenatide (Bird et al. 2010). Researchers Zhang and team have used hydrocarbon stapling to stabilise peptides from the α‐helical region of the dimer interface, enhancing their cell penetration and ability to disrupt CTD dimer formation. As per the report, the stapled peptides showed inhibition of HIV‐1 particle formation and showed potent antiviral activity against diverse viral strains, including those resistant to reverse transcriptase and protease inhibitors (Zhang et al. 2011).

The capsid domain of the HIV‐1 Gag polyprotein plays a key role in viral assembly, making it a promising target for anti‐AIDS therapies (Bird, Irimia, et al. 2014). Zhang and team developed a cell‐penetrating peptide, NYAD‐1, by applying hydrocarbon stapling to a 12‐mer alpha‐helical peptide, capsid assembly inhibitor (CAI), known to disrupt capsid assembly. NYAD‐1 was engineered by substituting two natural amino acids at positions i and i + 4 with the non‐natural amino acid (S)‐Fmoc‐2‐(2′‐pentenyl) alanine, enhancing its alpha‐helicity and cell‐penetrating properties. NYAD‐1 demonstrated low micromolar potency against HIV‐1 in cell culture, showing potential for further development as an antiviral drug (Tzotzos 2022; Zhang et al. 2008).

A team of scientists rationally designed i, i + 7 hydrocarbon‐stapled peptides targeting HIV‐1 assembly and entry, showing antiviral activity against major HIV‐1 subtypes in China. The study tested three peptides, NYAD‐36, NYAD‐67 and NYAD‐66, against primary CRF07_BC and CRF01_AE isolates in PBMCs and Env‐pseudotyped viruses in TZM‐bl cells. As per the report, all peptides effectively inhibited viral replication with IC50 in the low micromolar range, though NYAD‐36 and NYAD‐67 exhibited superior potency compared to NYAD‐66 (Wang et al. 2017).

4.2. Herpes Simplex Virus (HSV)

Herpesviruses, including HSV, are persistent and widespread pathogens, and stapled peptides have shown promising antiviral activity against various human neurotropic viruses, and their efficacy is closely linked to specific structural features. The placement and type of staple significantly influence the peptide's secondary structure, proteolytic stability and binding affinity to viral targets. Current research focuses on optimising these stapled peptides for topical applications to treat HSV infections. Research on HSV has demonstrated that stapled peptides derived from viral fusion proteins can inhibit viral replication by interfering with the fusion process. Enhancing membrane permeability, these stapled peptides show increased potency in preventing HSV entry into host cells (Guan et al. 2021). In 2022, Hanchan Guan and team developed hydrocarbon‐stapled peptides derived from the HSV‐1 polymerase C‐terminus structure to inhibit the processivity factor critical for HSV‐1 DNA synthesis. The stapled peptides maintained their alpha‐helical structure, allowing them to inhibit viral DNA synthesis and prevent HSV‐1 infection. An optimised peptide with the N‐terminus substituted by two hydrophobic valine residues exhibited enhanced stability and specificity. This optimised peptide selectively inhibited HSV‐1 in human primary corneal epithelial cells without affecting unrelated viruses, suggesting its potential as a topical therapeutic for ocular Herpes Keratitis (Guan et al. 2021). These findings underscore a clear structure–activity relationship: the antiviral efficacy of stapled peptides against neurotropic viruses is highly dependent on rational staple design (placement and type), sequence optimisation (e.g., hydrophobic substitutions) and the preservation of secondary structure. Such insights provide a valuable framework for the future development of potent, selective and stable antiviral peptides for clinical applications.

4.3. SARS‐COV

SARS‐CoV‐2, a well‐known virus causing COVID‐19, has brought attention to the pressing need for novel antiviral treatments that target viral entrance and replication. The capacity of peptide‐based therapies to interfere with protein–protein interactions essential to viral processes has drawn interest (V'kovski et al. 2021). Peptide stapling techniques present a viable approach to improve these antiviral peptides' stability, bioavailability and efficacy and open the door to more potent therapies against SARS‐CoV‐2 and other new viral threats.

The researchers developed a novel class of stapled lipopeptides that combine lipidation and hydrocarbon stapling to improve the stability, pharmacology and antiviral efficacy of fusion‐inhibitor peptides. They designed a lead compound, RQ‐01, which targets the highly conserved six‐helix bundle (6‐HB) fusion mechanism used by SARS‐CoV‐2 and other viruses for cell entry. RQ‐01 showed high stability and nanomolar potency across multiple SARS‐CoV‐2 strains, with improved solubility and antiviral breadth over previous peptides. The lipopeptide can be synthesised efficiently on resin, including the lipidation step, simplifying production and enabling rapid iteration for lead optimisation (Bird et al. 2024). In 2023, a study claimed to develop a highly selective biosensor for rapid SARS‐CoV‐2 detection by functionalising a conductive polymer with a lactam‐stapled hACE‐2 peptide. With 95% sensitivity, 100% specificity and a 40 TCID50/mL detection limit, it outperformed commercial rapid antigen tests and delivered results in under a minute. The biosensor's potential for miniaturisation enhances portability, reduces power consumption and supports multiplexed detection, making it ideal for medical diagnostics and environmental monitoring (Meshesha et al. 2023).

RQ‐01, nasally administered stapled lipopeptide, protected hamsters from SARS‐COV‐2 induced damage, showing promise as pre‐ and post‐exposure prophylaxis. Its stability supports use in vaccine delays and for immunocompromised patients. This platform could also target other viruses and diseases like diabetes and cancer.

4.4. Respiratory Syncytial Virus (RSV)

The fusion (F) protein of respiratory syncytial virus (RSV) is a key glycoprotein integral to viral entry and pathogenesis. It facilitates the fusion of the viral envelope with the host cell membrane, enabling the release of the viral genome into the host cytoplasm (Patel et al. 2019). Structurally, the F protein transitions from a metastable prefusion conformation to a stable post‐fusion state during the infection process, a critical step for viral infectivity. This protein is a prime target for vaccine development and therapeutic interventions due to its highly conserved nature and pivotal role in mediating host‐cell interactions (Patel et al. 2019). Peptide T118, derived from the CHR region of the RSV fusion (F) protein, exhibits potent nanomolar inhibitory activity against RSV infectivity and syncytium formation assays (Lawless‐Delmedico et al. 2000). Despite its efficacy, the linear structure of T118 shows low α‐helicity and predominantly adopts random coil conformations in solution, which adversely impacts its inhibitory potential (Bird, Boyapalle, et al. 2014).

In 2017, Gaillard et al. identified novel double‐stapled peptides targeting the HR2 domain of the RSV F protein, disrupting the six‐helix bundle (6HB) formation required for viral entry. Peptide 4 emerged as a promising candidate, tolerating chemical modifications and effectively inhibiting RSV infection in mice via intranasal delivery. While inhalable therapy shows potential, challenges in paediatric formulations persist. Ongoing trials, such as ALX171 in infants, may guide future therapeutic strategies, including injectable long‐acting formulations (Gaillard et al. 2017). In 2023, an in silico study highlighted peptide‐mediated protein–protein interaction (PmPPI) as a therapeutic strategy against hRSV, focusing on binding the P‐protein's C‐terminal tail to the N‐protein globular domain. Computational and experimental analyses revealed that the intrinsically disordered C‐terminal tail adopts a helical structure upon binding, incurring an entropy penalty (Blocquel et al. 2012; Chandra et al. 2023). Chemically stapled P‐peptides, derived from the P‐protein's C‐terminus, successfully constrained their disordered conformation into an ordered helical form, enhancing binding affinity by reducing the entropy penalty (Yan et al. 2023).

4.5. Ebola Virus

Ebola virus (EBOV) causes severe haemorrhagic fever with high mortality rates (Jacob et al. 2020). Its entry into host cells is mediated by the envelope glycoprotein (GP), which is cleaved into two subunits: GP1 for receptor binding and GP2, which drives membrane fusion. Targeting GP2 is a promising strategy to prevent EBOV infection, as its conformational changes during fusion are essential for viral entry. Inhibiting GP2's ability to facilitate membrane fusion would block the virus from entering host cells, potentially preventing infection. This makes GP2 a critical target for therapeutic development in combating EBOV (Banerjee et al. 2023). Pessi et al. tested peptides targeting the C‐terminal heptad‐repeat (HR2) domain of EBOV GP2 to inhibit fusion and viral entry in 2023. The peptides were cholesterol‐conjugated to enhance serum half‐life and stabilised to improve potency and were found to be active in vitro against both EBOV and Marburg virus (MARV). Lead peptide EBOV‐7 showed low micromolar EC50 values and successfully protected mice from lethal EBOV infection in a mouse model, suggesting their potential as novel therapeutics against EBOV (Pessi et al. 2019).

4.6. Current Challenges and Future Directions

Stapled peptides have emerged as a transformative platform in therapeutic design, bridging the gap between small molecules and biologics. The ability of this approach to enhance peptide stability, bioactivity and cell penetration holds significant promise for applications in virology, oncology and chronic diseases. However, these advancements present challenges as well. The intricate chemical changes required for stapling limit large‐scale manufacture and make synthesis expensive. Furthermore, the adoption of non‐natural alterations may cause immunogenicity issues. Because stapled peptides frequently have low oral bioavailability, parenteral treatment is required for efficient distribution. More in vivo studies are required to assess pharmacokinetics, toxicity and long‐term safety, even when in vitro studies show their effectiveness.

The current achievements in the fields have inspired scientists to focus on reducing and improving the scalability of the approach, enabling broader accessibility. Innovations to enhance oral bioavailability are crucial to expanding their therapeutic utility. Combining stapled peptides with other antiviral agents or advanced delivery systems could further amplify their therapeutic potential. Staples peptide represents a versatile and promising tool for addressing unmet medical needs, particularly in antiviral therapy. Continued research and innovation in this field could pave the way for groundbreaking applications.

5. Conclusion

Peptide stapling represents a significant advancement in the development of antiviral therapeutics. Stabilising the α‐helical structure of peptides can improve their therapeutic potential by increasing stability, bioavailability and cellular permeability. Enhancing the stability, permeability and efficacy of antiviral peptides through stapling may address the limitations associated with linear peptides. As research into stapled peptides continues, this technology may be critical in developing next‐generation antiviral therapies.

Despite these advantages, designing and synthesising stapled peptides remains resource‐intensive, requiring significant cost, labour and time investment. Further exploration and optimisation of the design and cost of staple peptides will enhance accessibility and scalability for widespread therapeutic applications.

Author Contributions

Sanskruti Patil: investigation, visualization, data curation, formal analysis, writing – original draft. Rakesh Rahangdale: investigation, writing – review and editing, methodology, data curation. Mukesh Pasupuleti: writing – review and editing, methodology. Puttur Santhoshkumar: writing – review and editing, validation, data curation. Raghu Chandrashekar Hariharapura: conceptualization, writing – review and editing, project administration, supervision, resources.

Conflicts of Interest

The authors declare no conflicts of interest.

Patil, S. , Rahangdale R., Pasupuleti M., Santhoshkumar P., and Hariharapura R. C.. 2025. “Unleashing the Antiviral Potential of Stapled Peptides: A New Frontier in Combating Human Neurotropic Viral Infections.” Microbial Biotechnology 18, no. 9: e70221. 10.1111/1751-7915.70221.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.