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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.

Four bis-lactam [i, i+4]-stapled peptides with d- or l-α-methyl-thialysines were constructed on a model peptide sequence derived from p110α[E545K] and subjected to circular dichroism (CD) and proteolytic stability assessment, alongside the corresponding bis-lactam [i, i+4]-stapled peptide with l-thialysine. The % α-helicity values of these four stapled peptides were found to be largely comparable to each other yet greater than that of the stapled peptide with l-thialysine. An l-α-methyl-thialysine-stapled peptide built on a model peptide sequence derived from ribonuclease A (RNase A) was also found to exhibit a greater % α-helicity than its l-thialysine-stapled counterpart. Moreover, a greater proteolytic stability was demonstrated for the l-α-methyl-thialysine-stapled p110α[E545K] and RNase A peptides than that of their respective l-thialysine-stapled counterparts.

Neoantigen peptides hold great potential as vaccine candidates for tumor immunotherapy. However, due to the limitation of antigen cellular uptake and cross‐presentation, the progress with neoantigen peptide‐based vaccines has obviously lagged in clinical trials. Here, a stapling peptide‐based nano‐vaccine is developed, comprising a self‐assembly nanoparticle driven by the nucleic acid adjuvant‐antigen conjugate. This nano‐vaccine stimulates a strong tumor‐specific T cell response by activating antigen presentation and toll‐like receptor signaling pathways. By markedly improving the efficiency of antigen/adjuvant co‐delivery to the draining lymph nodes, the nano‐vaccine leads to 100% tumor prevention for up to 11 months and without tumor recurrence, heralding the generation of long‐term anti‐tumor memory. Moreover, the injection of nano‐vaccine with signal neoantigen eliminates the established MC‐38 tumor (a cell line of murine carcinoma of the colon without exogenous OVA protein expression) in 40% of the mice by inducing potent cytotoxic T lymphocyte infiltration in the tumor microenvironment without substantial systemic toxicity. These findings represent that stapling peptide‐based nano‐vaccine may serve as a facile, general, and safe strategy to stimulate a strong anti‐tumor immune response for the neoantigen peptide‐based personalized tumor immunotherapy. A sulfonium‐based stapling peptide carrier containing only nine amino acids is developed through computer‐aided screening for developing personalized nano‐vaccines. By cooperating with this carrier, a single neoantigen peptide could sufficiently trigger a robust and sustained tumor‐specific T cell response, further eliminating the established tumors in multiple tumor models by regulating the tumor infiltration microenvironment.

Adjuvants are non‐specific immune enhancers commonly used to improve the responsiveness and persistence of the immune system toward antigens. However, due to the undefined chemical structure, toxicity, non‐biodegradability, and lack of design technology in many existing adjuvants, it remains difficult to achieve substantive breakthroughs in the adjuvant research field. Here, a novel adjuvant development strategy based on stapling peptides is reported to overcome this challenge. The nano‐vaccine incorporating peptide adjuvant and recombinant HBsAg protein not only induced strong antibody titers that are equivalent to aluminum adjuvanted vaccines but also simultaneously activated T‐cell immune response. Similar results are also observed in herpes zoster vaccine and more complex influenza vaccine. The mechanism analysis demonstrates that antigen is efficiently carried into antigen‐presenting cells (APCs) by peptide, further promoting the secretion of cytokines and activation of APCs. In addition, by redesigning the adjuvant, it is found that the sulfonium centers, rather than the sequence of peptide played an important role in immune activation. This discovery may provide a new paradigm for the rational design of peptide‐based adjuvants. In brief, this study demonstrates that stapling peptides with sulfonium centers can provide a well‐defined, programmable, biocompatible, and effective adjuvant for multiple types of vaccines. A stapling peptide with two sulfonium centers is developed to be used as an excellent vaccine adjuvant candidate for the Hepatitis B vaccine, influenza vaccine, and herpes zoster vaccine. Just by changing the amino acids and ring types, it is easy to program peptide adjuvants to adapt to multiple vaccine subtypes and synergistically induce humoral and cellular immune activation.

Background The human epidermal growth factor receptor (HER) family of transmembrane tyrosine kinases is overexpressed and correlates with poor prognosis and decreased survival in many cancers. The receptor family has been therapeutically targeted, yet tyrosine kinase inhibitors (TKIs) do not inhibit kinase-independent functions and antibody-based targeting does not affect internalized receptors. We have previously demonstrated that a peptide mimicking the internal juxtamembrane domain of HER1 (EGFR; EJ1) promotes the formation of non-functional HER dimers that inhibit kinase-dependent and kinase-independent functions of HER1 (ERBB1/EGFR), HER2 (ERBB2) and HER3 (ERBB3). Despite inducing rapid HER-dependent cell death in vitro, EJ1 peptides are rapidly cleared in vivo, limiting their efficacy. Method To stabilize EJ1 activity, hydrocarbon staples (SAH) were added to the active peptide (SAH-EJ1), resulting in a 7.2-fold increase in efficacy and decreased in vivo clearance. Viability assays were performed across HER1 and HER2 expressing cell lines, therapeutic-resistant breast cancer cells, clinically relevant HER1-mutated lung cancer cells, and patient-derived glioblastoma cells, in all cases demonstrating improved efficacy over standard of care pan-HER therapeutics. Tumor burden studies were also performed in lung, glioblastoma, and inflammatory breast cancer mouse models, evaluating tumor growth and overall survival. Results When injected into mouse models of basal-like and inflammatory breast cancers, EGFRvIII-driven glioblastoma, and lung adenocarcinoma with Erlotinib resistance, tumor growth is inhibited and overall survival is extended. Studies evaluating the toxicity of SAH-EJ1 also demonstrate a broad therapeutic window. Conclusions Taken together, these data indicate that SAH-EJ1 may be an effective therapeutic for HER-driven cancers with the potential to eliminate triple negative inflammatory breast cancer.

SARS-CoV-2 is a novel virus with many unknowns. No vaccine or specific therapy is available yet to prevent and treat this deadly virus. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as the primary receptor to enter host cells and initiate the infection. The critical binding region of ACE2 is an ∼30-amino-acid (aa)-long helix. Here, we report the design of four stapled peptides based on the ACE2 helix, which is expected to bind to SARS-CoV-2 and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50 to 94% helicity). In contrast, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus-based single-cycle assay in HT1080/ACE2 cells and human lung cell line A549/ACE2, overexpressing ACE2. Three of the four stapled peptides showed potent antiviral activity in HT1080/ACE2 (50% inhibitory concentration [IC 50 ]: 1.9 to 4.1 μM) and A549/ACE2 (IC 50 : 2.2 to 2.8 μM) cells. The linear peptide NYBSP-C and the double-stapled peptide StRIP16, used as controls, showed no antiviral activity. Most significantly, none of the stapled peptides show any cytotoxicity at the highest dose tested. We also evaluated the antiviral activity of the peptides by infecting Vero E6 cells with the replication-competent authentic SARS-CoV-2 (US_WA-1/2020). NYBSP-1 was the most efficient, preventing the complete formation of cytopathic effects (CPEs) at an IC 100 of 17.2 μM. NYBSP-2 and NYBSP-4 also prevented the formation of the virus-induced CPE with an IC 100 of about 33 μM. We determined the proteolytic stability of one of the most active stapled peptides, NYBSP-4, in human plasma, which showed a half-life ( T 1/2 ) of >289 min. IMPORTANCE SARS-CoV-2 is a novel virus with many unknowns. No vaccine or specific therapy is available yet to prevent and treat this deadly virus. Therefore, there is an urgent need to develop novel therapeutics. Structural studies revealed critical interactions between the binding site helix of the ACE2 receptor and SARS-CoV-2 receptor-binding domain (RBD). Therefore, targeting the entry pathway of SARS-CoV-2 is ideal for both prevention and treatment as it blocks the first step of the viral life cycle. We report the design of four double-stapled peptides, three of which showed potent antiviral activity in HT1080/ACE2 cells and human lung carcinoma cells, A549/ACE2. Most significantly, the active stapled peptides with antiviral activity against SARS-CoV-2 showed high α-helicity (60 to 94%). The most active stapled peptide, NYBSP-4, showed substantial resistance to degradation by proteolytic enzymes in human plasma. The lead stapled peptides are expected to pave the way for further optimization of a clinical candidate.

Antimicrobial peptides (AMPs) are considered a promising therapeutic approach against multi-drug resistant microorganisms. Besides their advantages, there are limitations to be overcome so that these molecules can become market competitive. One of the biggest limitations is proteolytic susceptibility, which could be overcome by structural modifications such as cyclization, especially for helix-constraining strategies. Over the years, many helix stabilization techniques have arisen, such as lactam-bridging, triazole-based, N-alkylation and all-hydrocarbon stapling. All-hydrocarbon stapling takes advantage of modified amino acid residues and olefinic cross-linking to constrain peptide helices. Despite being a well-established strategy and presenting efficient stability results, there are different limitations especially related to toxicity. In this review, recent studies on stapled AMPs for antimicrobial usage are explored with the aim of understanding the future of these molecules as putative antimicrobial agents.

The sheer size and vast chemical space (i.e., diverse repertoire and spatial distribution of functional groups) underlie peptides’ ability to engage in specific interactions with targets of various structures. However, the inherent flexibility of the peptide chain negatively affects binding affinity and metabolic stability, thereby severely limiting the use of peptides as medicines. Imposing conformational constraints to the peptide chain offers to solve these problems but typically requires laborious structure optimization. Alternatively, libraries of constrained peptides with randomized modules can be screened for specific functions. Here, we present the properties of conformationally constrained peptides and review rigidification chemistries/strategies, as well as synthetic and enzymatic methods of producing macrocyclic peptides. Furthermore, we discuss the in vitro molecular evolution methods for the development of constrained peptides with pre-defined functions. Finally, we briefly present applications of selected constrained peptides to illustrate their exceptional properties as drug candidates, molecular recognition probes, and minimalist catalysts.

Background: The Fc region of immunoglobulin G (IgG) is a key target in therapeutic and analytical applications, such as antibody purification and site-specific bioconjugation. Although Protein A exhibits strong Fc-binding affinity, its large molecular weight and limited chemical flexibility pose challenges for use in compact or chemically defined systems. To address these limitations, we designed two α-helical peptides, SpA h1 and SpA h2, based on the Fc-binding helices of the Z34C domain from Staphylococcus aureus Protein A. Method: To enhance the structural stability and Fc-binding capability of these peptides, a lactam-based stapling strategy was employed by introducing lysine and glutamic acid residues at positions i and i + 4. Result: The resulting stapled peptides, (s)SpA h1 and (s)SpA h2, exhibited significantly improved α-helical content and IgG-binding performance, as demonstrated by circular dichroism (CD) spectroscopy and fluorescence-based IgG capture assays. Surface plasmon resonance (SPR) analysis confirmed specific, concentration-dependent interactions with the Fc region of human IgG, with (s)SpA h1 consistently showing the binding affinity and stability. Proteolytic resistance assays using α-chymotrypsin revealed that (s)SpA h1 maintained its structural integrity over time, exhibiting markedly enhanced resistance to enzymatic degradation compared to its linear counterpart. Furthermore, (s)SpA h1 exhibited strong Fc selectivity with minimal Fab affinity, confirming its suitability as a compact and Fc-specific binding ligand. Conclusions: These results confirm the successful design and development of structurally reinforced Fc-binding peptides that overcome the inherent limitations of short linear sequences through both high-affinity sequence optimization and lactam-based stapling. Among them, (s)SpA h1 demonstrates the most promising characteristics as a compact yet stable Fc-binding ligand, suitable for applications such as antibody purification and site-specific bioconjugation.

Human epidermal growth factor receptor (EGFR) is involved in strong association with malignant proliferation, which has been shown to play a central role in the development and progression of non-small cell lung cancer and other solid tumors. The tumor-suppressor protein MIG6 is a negative regulator of EGFR kinase activity by binding at the activation interface of asymmetric dimer of EGFR kinase domain to disrupt EGFR dimerization and then inactivate the kinase. The protein adopts two discrete fragments 1 and 2 to directly interact with EGFR. It is revealed that the MIG6 fragment 2 is intrinsically disordered in free unbound state, but would fold into a well-structured β-hairpin when binding to EGFR, thus characterized by a so-called coupled folding-upon-binding process, which can be regarded as a compromise between favorable direct readout and unfavorable indirect readout. Here, a 23-mer F2P peptide was derived from MIG6 fragment 2, trimmed into a 17-mer tF2P peptide that contains the binding hotspot region of the fragment 2, and then constrained with an ordered hairpin conformation in free unbound state by disulfide stapling, finally resulting in a rationally stapled/trimmed stF2P peptide that largely minimizes the unfavorable indirect readout effect upon its binding to EGFR kinase domain, with affinity improved considerably upon the trimming and stapling/trimming. These rationally designed β-hairpin peptides may be further exploited as potent anti-lung cancer agents to target the activation event of EGFR dimerization.