ABSTRACT Oral delivery of peptide therapeutics remains a major challenge due to extensive enzymatic degradation, mucus entrapment, limited epithelial permeability, active efflux, and hepatic first‐pass metabolism, all of which restrict systemic exposure. Recent advances in nanocarrier engineering and AI‐driven design are beginning to overcome these barriers through synergistic innovations in medicinal chemistry, formulation science, and clinically validated enhancer platforms. Mechanistic insights into gastrointestinal physiology have enabled the rational development of technologies such as SNAC and TPE, supporting gastric or intestinal transcellular uptake and establishing the first clinically approved oral peptide formulations. Parallel progress in peptide engineering including cyclization, D‐amino acid substitution, lipidation, and transporter‐targeted conjugation enhances proteolytic stability and epithelial interaction, while next‐generation polymer–lipid hybrids, mucus‐penetrating carriers, bile‐acid–guided systems, and stimuli‐responsive biomaterials provide tunable protection and controlled release across the GI tract. Device‐assisted platforms such as LUMI, SOMA, and RaniPill further expand this toolkit by bypassing epithelial barriers altogether. These technological gains intersect with sustainable manufacturing and green bioprocessing considerations essential for scalable clinical translation, along with persistent global health challenges in LMIC settings. Collectively, these developments highlight a decisive shift toward clinically viable, patient‐centric oral peptide therapeutics and outline a forward SDG‐aligned trajectory in which computational design, eco‐efficient production, and advanced biomaterial systems converge to enable reliable systemic and local delivery via the oral route.
ABSTRACT Peptides and proteins serve as versatile building blocks for advanced nano‐assemblies. Precision chemistry offers a new approach that applies site‐selective modification, sequence‐defined synthesis, and dynamic covalent design to direct molecular interactions at the atomic level. This concept examines how these tools enhance control over assembly kinetics and morphology, enabling the creation of “smart” stimuli‐responsive systems. These precision chemistry tools and techniques produce high‐performance nanostructures, such as protein cages and engineered networks, tailored for drug delivery, immunotherapy, and regenerative medicine. Convergence of molecular precision and AI‐driven predictive modeling is essential for the next generation of adaptive, multifunctional biomaterials.
ABSTRACT Phage display remains a powerful high‐throughput selection platform for the in vitro evolution of biomolecules. Random peptide libraries have been successfully utilized to discover a diverse range of peptides with diagnostic and therapeutic applications. Nevertheless, intrinsic limitations—such as sparse sampling of the vast theoretical sequence space, compositional bias in the naïve library, and the unintended enrichment of nonspecific binders during biopanning—can skew selection outcomes. In the current review, we examine the dynamics of phage display selection through the conceptual frameworks of sequence space and fitness landscapes. Unlike some classical directed evolution approaches, the fitness landscape in phage display is not only shaped by the binding affinity of displayed peptides for the target but also immensely influenced by the biological nature of the bacteriophage itself. These properties can distort the landscape during iterative cycles of selection and amplification. Technical refinements in library design and construction, utilizing next‐generation sequencing (NGS) to identify enriched sequence clusters or recurring motifs in biopanning outputs, building smart, motif‐guided secondary libraries to narrow the search toward high‐fitness regions of peptide space, avoiding repetitious selection rounds, and applying sophisticated computational tools to decode large NGS datasets can significantly enhance the statistical chance of uncovering rare, high‐affinity, target‐specific peptides. Integrating these strategies into the phage display workflow enables researchers to more effectively explore the functional regions of sequence space and facilitates a more efficient, targeted navigation of the fitness landscape, reorienting phage display selection from a blind, largely random search into a guided, more informed journey.
Proteins are not only essential nutrients, but also the active components of various biopharmaceuticals, including hormones, immune-related molecules, and enzymes. The use of cryoprotectants is necessary to maintain the stability of protein drugs. Since most cryoprotectants require high concentrations to exhibit activity, there is a need for cryoprotectants that act at low concentrations. Although amino acids have been widely used as cryoprotectants, few studies have examined dipeptides and tripeptides as cryoprotective agents. This study identifies dipeptides and tripeptides with cryoprotective activity superior to that of amino acids. Screening experiments using lactate dehydrogenase (LDH), a cold-sensitive model enzyme, showed that the prolyltripeptide Pro-Leu-Leu effectively inhibited LDH cryoinactivation, and this activity was approximately 100-fold greater than that of proline, because the activity of 100 mM proline is roughly the same as that of 1 mM Pro-Leu-Leu. Pro-Leu-Leu also inhibited LDH denaturation induced by freezing and thawing (F/T) and prevented protein aggregation. Furthermore, Pro-Leu-Leu suppressed the cryoaggregation of human gamma globulin. Among prolyltripeptides, protective activity correlated weakly with hydrophobicity but did not correlate with their isoelectric point or net charge. Pro-Leu-Leu was among the most hydrophobic prolyltripeptides and exhibited the highest protective activity. These results suggest that Pro-Leu-Leu may limit the F/T-induced expansion of hydrophobic regions exposed on the protein surface by covering hydrophobic patches via nonspecific interactions, thereby preventing protein denaturation and aggregation. Prolyltripeptides such as Pro-Leu-Leu may therefore be useful as efficient cryoprotectants for protein pharmaceuticals.
Accurate prediction of protein secondary structure is essential for reliable tertiary structure prediction and peptide design. To address this, multiple algorithms have been proposed. The conformation of a residue within a polypeptide chain is strongly influenced by its immediate neighbors. The conformational tendencies of 20 residues in the presence of their first neighbors, constituting a total of 8000 tripeptides, were calculated. Using the propensity values of 8000 tripeptide variants, we propose an accurate method for secondary structure prediction. Various machine learning (ML) models were built using propensities, position-specific scoring matrices (PSSM) and amino acid binary features to predict three-state (Q3) and eight-state (Q8) secondary structures. The results suggest that the XGBoost ML model produced highly accurate predictions, achieving accuracies of 93% and 88% for Q3 and Q8 state, respectively for validation dataset CB513. A program for prediction called S8kPred is developed. This utility along with consensus secondary structure prediction is available online at https://www.s8kpred.in.
Peptide-based supramolecular hydrogels have emerged as a versatile class of biomaterials with promising applications in drug delivery, tissue engineering, and regenerative medicine. Among the various peptide-based gels, phenylalanine-containing short peptides have gained particular attention for their intrinsic ability to self-assemble through pi-pi stacking, hydrogen bonding, and hydrophobic interactions. In this paper, we have tuned the hydrogelation behavior of tripeptides containing a phenylalanine residue by modifying the length of the sidechain and the substituents on the phenyl ring. A parent tripeptide and its five different analogues containing modifications only of the phenylalanine residue were synthesized and all six peptides formed hydrogels. The structural organization and mechanical properties of the resulting hydrogels were examined using spectroscopic, rheological, and microscopic techniques. Distinct differences in gel strength and morphology were observed depending on the nature of the modification of the phenylalanine residue. The peptide containing a 4-nitrophenylalanine residue produced the most mechanically robust gel, whereas the analogous peptides containing a 4-methoxyphenylalanine and a bishomo-phenylalanine formed the weakest gel network. Understanding these structure-property relationships provides critical insights for the rational design of functional peptide-based materials and underscores the need for systematic exploration of chemical modifications in supramolecular hydrogel systems.
Existing spike protein-based COVID-19 vaccines have significantly prevented SARS-CoV-2 infection in most of the world's population. However, challenges remain regarding production costs, scalability, global efficacy, and safety. The current study aims to design a novel, conserved, and promiscuous B- and T-lymphocyte epitope-based universal COVID-19 vaccine derived from the spike protein. The designed monomeric self-assembling peptide vaccine (SAPV) constructs (with and without adjuvant/TAT/PADRE sequences) involving pentameric and trimeric coiled-coil motifs were evaluated for self-assembly through homo-oligomer modeling. The screened SAPV constructs (SAPVb3 and SAPVa5) were subsequently analyzed for their physicochemical properties and potential allergenicity, toxicity, subcellular localization, antigenicity, solubility, and antioxidative properties. Finally, the interaction, specificity, and stability analysis were performed through docking and simulation studies of the macromolecular complexes formed between the SAPV constructs and broadly neutralizing human monoclonal antibody (K501SP6) as well as toll-like receptors (TLR)-2/4. In the case of TLR-4, SAPVa5 demonstrated the strongest binding affinity (-1623.9 kcal/mol) compared to SAPVb3 (-1447.1 kcal/mol), control C3 (-1488.3 kcal/mol), and the reference immunogenic peptide of RBD (-954.4 kcal/mol). Moreover, immune simulations of SAPVb3/SAPVa5 and the self-assembling polypeptide nanoparticle vaccine (FMP014) yielded comparable results and elicited robust immune responses against SARS-CoV-2 variants. However, before conducting a clinical trial, SAPV constructs must be evaluated in a wet laboratory for immunogenicity and safety.
Collagen, the principal structural protein in mammals, adopts a right-handed triple helix composed of three left-handed polyproline-II helical polypeptide chains featuring repeating Gly-Xaa-Yaa tripeptide motifs. Positions Xaa and Yaa frequently contain charged amino acids, which can form salt bridges to stabilize the collagen triple-helical conformation and fibril formation. Over the past decades, researchers have exploited the charged amino acids to engineer salt bridges for constructing stable collagen-based biomaterials. However, the use of amino acid analogues to form salt bridges remains largely unexplored. Herein, we report for the first time the incorporation of charged N-gly residues into host-guest collagen mimetic peptides (CMPs) to establish salt bridges. Among these, the EGNap CMP exhibited the highest T-m value of 48.2 degrees C, marginally lower than that of the OGP sequence. Molecular modeling indicates that the N-gly can form salt bridges with the amino acid, but the distance of these salt bridges is greater than that of KGD. These findings establish a foundational strategy for leveraging N-gly-mediated electrostatic interactions in collagen engineering.
We demonstrate our results with the synthesis of inhibitors of Protein Arginine Deaminase 4 (PAD4), a key regulator of neutrophil elastase extracellular traps (NETs), as potential therapeutics for chronic obstructive pulmonary disease (COPD). Furthermore, we report an automated method for solid phase synthesis of macrocyclic stapled peptides with chloroacetamidine guanidine mimetic function as PAD4 inhibitors. This approach utilizes the 2,4,6-trimethylbenzenesulfenyl protective group for cysteine, followed by reaction with various halogen derivatives upon deprotection under mild conditions. Furthermore, derivatization of the amino group with ethyl chloroacetamidate hydrochloride after its deprotection, performed on solid phase, offers a new, efficient method for rapid and robust synthesis of stapled peptide derivatives. This process notably reduces time, energy, and solvent use compared to traditional in-solution methods.
Accelerated progression and global spread of antimicrobial resistance have significantly demanded the breakthrough of novel antimicrobials. Antimicrobial peptides (AMPs), such as defensins (from the genus Drosophila), bactenecin (peptides from bovine, caprine, and ovine), cathelicidins (peptides from birds, mammals, and reptiles), and buforins (derived from Bufo bufo gargarizans stomach), are preferred over conventional antibiotics due to their lower tendency to induce resistance, broad-spectrum antibiofilm responsiveness, and their potential to modulate the host immune response. Thus, they serve as a tool to address antimicrobial resistance. Functional and structural limitations, coupled with regulatory challenges, hinder the therapeutic and clinical translation of antimicrobial peptides. Moreover, several attempts have been made through numerous existing experimental and computational tools to streamline the preclinical or clinical design of AMPs as modern medicines. The current review summarizes the challenges, merits, and scope of AMPs against superbugs, highlights the enactment of potential AMPs as a promising advancement, the clinical trial status of approved AMPs, and outlines the necessities and priorities behind designing engrossed progressive strategies by considering the best existing tools.
Since birth, the human immune system continuously adapts to environmental exposures, developing robust immunological memory against a diverse range of antigens and allergens. Leveraging this pre-existing immunity, this study introduces a novel immunotherapeutic approach targeting the Nipah virus, a zoonotic pathogen with a high mortality rate and no approved vaccines or therapeutics to date. The NiV glycoprotein G (NiV-G), essential for viral attachment and fusion with host cells, was identified as a prime therapeutic target. This study proposes a peptide ligand complex (PLC) that constitutes high-affinity ligands of NiV-G joined with immunogenic peptides for stronger host innate immune response by an oxime linker using high-throughput virtual screening (HTVS) and molecular dynamics simulations, Bleomycin and Octreotide were selected as potent ligands for NiV-G. These were conjugated via an oxime linker to peptides derived from Escherichia coli outer membrane proteins (OMPs), which exploit the natural antibody memory against E. coli , constituting approximately 0.057% of total serum immunoglobulins in healthy individuals. The ligand component of PLC binds specifically to NiV-G, blocking viral entry, fusion, and preventing syncytia formation, an essential mechanism for cell-to-cell spread. Simultaneously, the peptide component activates immune effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), promoting the clearance of infected cells and free viral particles from circulation. Computational analyses confirmed the Safety, stability, and immunogenicity of the PLC components. This strategy presents a compelling alternative to conventional monoclonal antibody-based therapies, which are often constrained by high production costs, storage limitations, restricted accessibility, and susceptibility to antigenic drift. By harnessing pre-existing immune memory and redirecting it toward viral targets, the peptide ligand complex (PLC) offers a cost-effective and adaptable therapeutic platform. Beyond addressing the challenges posed by Nipah virus, this approach holds potential for combating other hard-to-treat infectious diseases and even cancer by transforming natural immunity into a powerful and versatile tool for targeted immunotherapy.
Signal transducer and activator of transcription 3 (STAT3) is emerging as a pivotal oncogenic factor and its overexpression has been associated to osteosarcoma (OS) development, progression, and poor prognosis. It may be a promising therapeutic target for OS treatment. Constitutive activation of STAT3 facilitates tumorigenesis by inducing aberrant cell growth, apoptosis evasion, immune suppression, and increased angiogenesis. In this study, we screened a panel of bacterial peptides from Serratia marcescens , namely, prodigiosin, serralysin, chitinase B, chitinase C, and L-methioninase, for their capability to inhibit the STAT3 activity. In silico pipeline was used to predict the anticancer potential of each candidate peptide, alongside its physicochemical properties and predicted toxicity and allergenic. The three-dimensional structures of each peptide were modeled through PEP-FOLD and validated via Ramachandran plot analysis with */PROCHECK. The estimation of binding strength and stability of the ensuing complexes was done by molecular docking with HADDOCK, followed by molecular dynamics simulations. The interactions were confirmed by binding free-energy analyses. The peptides showed strong and stable binding to the active domain of STAT3, along with favorable pharmacokinetic predictions. These findings indicate that the selected S. marcescens peptides may act as promising candidates for further development in peptide-based therapeutics against OS.
Cecropins are a class of antimicrobial peptides (AMPs) expressed by insects that have broad-spectrum antimicrobial activity and low cytotoxicity. However, cecropins are also long (40-45 amino acids) peptides, making their adaptation for therapeutic use challenging. Here, we present a series of 16 amino acid-long peptides derived from cecropin B from Drosophila melanogaster. The native truncated peptide is cationic and expected to form an amphipathic alpha-helix, but does not have antimicrobial activity. The incorporation of two Trp residues, depending on the position in the peptide backbone, results in antibacterial activity comparable to that of full-length cecropin B against the gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, and there is minimal improvement as additional Trp substitutions are made. The incorporation of three or four Trp residues results in antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis, which are not affected by the full-length peptide at tested concentrations. However, peptides containing three or four Trp residues also had increased levels of cytotoxicity and hemolysis. NMR studies revealed an intrinsically disordered peptide in water, with some residual secondary structure. Secondary structure analysis by circular dichroism spectroscopy found that the propensity for helicity increases with increasing numbers of Trp residues, although individual Trp interactions at the C-terminal side of the peptide seem to have the biggest role in peptide structure. Similar to the full-length cecropin peptides, the truncated AMPs cause membrane permeability, but differences in the rate of permeabilization and bacterial killing do not directly correlate with the minimum inhibitory concentration (MIC) for each peptide. We propose that the location of the Trp residues, as opposed to the number incorporated, determines the antibacterial activity, and that peptide activity depends on the membrane composition of the bacteria.
Parkinson's disease (PD) is one of the neurodegenerative diseases that most affects the population, especially those over 65 years old. It is characterized by motor difficulties such as resting tremors, muscle stiffness, slow movements, and lack of balance, significantly impacting the quality of life of these individuals. Experimental studies suggest that the protein alpha-synuclein (alpha-syn) causes PD by leading to the death of dopaminergic neurons, contributing to the worsening severity of this disease's symptoms. Considering the relevance of this protein in the development of PD, it could be an ideal target for the development of therapeutic vaccines for this condition. The aim of this study was to design a multi-epitope vaccine using peptide constructs derived from alpha-syn and bromelain as an adjuvant, based on immunoinformatics tools. A total of five B-cell epitope sequences and 15 TCD4+ cell (MHC-II) epitope sequences restricted to the most prevalent alleles were selected. The chosen epitopes were antigenic, non-allergenic, and non-toxic and were linked using AAY, KK, GPGPG, and EAAAK linkers to create vaccine constructs with enhanced immunogenicity, stability, and flexibility. The tertiary structures, model refinement of the initial structure, Ramachandran plots, and ProsaWeb predictions were assessed. Molecular docking and molecular dynamics simulation studies showed efficient binding of Vaccine 2 with the TLR2 and TLR4 receptors. In general, in silico approaches have demonstrated the potential of peptide-based vaccine constructs, designed from immunodominant epitopes of alpha-syn and bromelain, for the development of a multiepitope therapeutic vaccine against PD.
The rapid increase in multidrug-resistant microbial pathogens has emerged as a major global challenge, making it essential to develop and discover new, highly potent, broad-spectrum therapeutic agents. The heterocyclic framework has drawn considerable interest due to its wide range of biological activities, and the incorporation of a peptide can further enhance the selectivity. Herein, we report the synthesis of peptide-linked trifluoromethyl triazolo-pyrazine derivatives (7a-e and 10a-e), characterized using 1H NMR, 13C NMR, mass spectrometry, and IR spectroscopy. All the synthesized compounds' antibacterial and antifungal activities were evaluated using a minimum inhibitory concentration (MIC) assay. Biological efficacy results revealed that compounds 7a and 7b displayed MIC values (20-35 mu g/mL) that are comparable to or better than chloramphenicol (16-26 mu g/mL). Likewise, antifungal assays revealed that compound 10d exhibited activity (67-82 mu g/mL) notably superior to griseofulvin (130-160 mu g/mL). The obtained data clearly indicate that the synthesized compounds are potent against microbes with the inclusion of structural diversity of peptide-triazolopyrazine conjugates. Furthermore, molecular docking studies show strong binding interactions of the potent compounds with their targets, as evidenced by docking scores comparable to or surpassing the control compounds. These findings provide a promising foundation for the development of next-generation antimicrobial agents.
